Thesis Susanne Roosing by Susanne Roosing

Susanne Roosing 1

Cone dystrophies: enlightening the genetic spectrum

Susanne Roosing

Research described in this thesis was financially supported by the Foundation Fighting Blindness USA, the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, the Gelderse Blinden Stichting, the Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Retina Nederland, the Stichting Blinden-Penning, the Stichting Macula Degeneratie fonds, the Rotterdamse Stichting Blindenbelangen. Publication of this thesis was financially supported by the Department of Human Genetics, Radboud University Medical Center; Stichting A.F. Deutman Oogheelkunde Researchfonds, Alcon Nederland B.V., AMO abbott Groningen, Bayer B.V., Carl Zeiss, ChipSoft B.V., Landelijke Stichting voor Blinden en Slechtzienden, Koninklijke Visio, Laservision Instruments B.V., Novartis Pharma B.V., Oculenti Contactlenspraktijken, Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting voor O Â oglijders, Ursapharm and Grieks Italiaans Restaurant Mr. Jacks.

ÂŠ Susanne Roosing, New York, USA Design: Esther Heijmans-Roosing Cover design: Esther Heijmans-Roosing Printed by: Proefschriftmaken.nl || Uitgeverij BOXPress ISBN: 978-90-8891-937-4

Cone dystrophies: enlightening the genetic spectrum Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. Th. L.M. Engelen, volgens besluit van het college van decanen in het openbaar te verdedigen op woensdag 8 oktober 2014 om 14.30 uur precies

door Susanne Roosing geboren op 3 Juli 1984 te Geldrop

Promotores Prof. dr. F.P.M. Cremers Prof. dr. C.B. Hoyng Prof. dr. C.C.W. Klaver (Erasmus MC)

Copromotor Dr. A.I. den Hollander

Manuscriptcommissie Prof. dr. J.G.J. Hoenderop (Voorzitter) Dr. C.J.F. Boon (Leids Universitair Medisch Centrum) Dr. M. Michaelides (University College London, UK)

Paranimphs Galuh Astuti Alex Garanto

Table of contents List of abbreviations Chapter 1 Introduction 1.1 General introduction 1.2 Genomics technologies and disease identification strategies 1.3 Causes and consequences of inherited cone disorders 1.4 Outline and aim of this thesis

8 11 13 23 29 97

Chapter 2 Comprehensive analysis of the achromatopsia genes CNGA3 and CNGB3 in progressive cone dystrophy

101

Chapter 3 Novel genotype-phenotype correlations for cone dysfunction 3.1 Maternal uniparental isodisomy of chromosome 6 reveals a TULP1 mutation as a novel cause of cone dysfunction 3.2 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

113 115

Chapter 4 Novel mutated genes involved in cone dysfunction 4.1 Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders 4.2 Mutations in RAB28, encoding a farnesylated small GTPase, are associateed with autosomal recessive cone-rod dystrophy 4.3 Mutations in MFSD8, encoding a lysosomal membrane protein, are associated with non-syndromic cone dysfunction

135 153 155 171 189

Chapter 5 General discussion 5.1 Novel disease mechanisms in cone dysfunction 5.2 Considerations in future cone dysfunction disease gene identification studies 5.3 Evolving genomic technologies 5.4 Prenylation defects in inherited retinal diseases

211 213 219 225 233

Summary

259

Samenvatting

263

Eenvoudiger gezegd 267 List of publications

271

Acknowledgements

275

Portfolio

281

About the author

285

Abbreviations aa Amino acid AAV Adeno associated virus ACHM Achromatopsia AD Autosomal dominant AF Autofluorescence AR Autosomal recessive BCM Blue cone monochromatism BCVA Best corrected Snellen visual acuity bp Base pair CC Connecting cilium CD Cone dysfunction CF Counting fingers cGMP Cyclic guanosine monophosphate cM Centimorgan CNG-channel Cyclic nucleotide gated channel CNTF Ciliary neutrophic factor COD Cone dystrophy CRD Cone-rod dystrophy D Diopters DNA Deoxyribonucleic acid DGGE Denaturing Gradient Gel Electrophoresis EOG Electrooculography ERG Electroretinogram ESC Embryonic stem cells ESE Exonic splice enhancer EVS Exome variant server FAF Fundus Autofluorescence GCL Ganglion cell layer GDP Guanosine diphosphate GTP Guanosine triphosphate IBD Identity by descent INL Inner nuclear layer IPL Inner plexiform layer IRD Inherited retinal dystrophies IS Inner segments ISCEV International Society for Clinical Electrophysiology of Vision JBTS Joubert syndrome kDa Kilodalton LCA Leber congenital amaurosis LE Left eye LOD Logarithm of odds

MAF Minor allele frequency Mb Megabases MD Macular dystrophy mERG Multifocal electroretinogram MIM Mendelian inheritance in man MKS Meckel-GrĂźber syndrome MRI Magnetic resonance imaging mRNA Messenger ribonucleic acid NAS Nonsense-associated altered splicing NCL Neuronal ceroid lipofuscinosis NGS Next generation sequencing NMD Nonsense mediated decay OCT Optical coherence tomography OMIM Online Mendelian inheritance in men ONL Outer nuclear layer OPL Outer plexiform layer OS Outer segments PCR Polymerase chain reaction PDE Phosphodiesterase PDI Protein disulphide isomerase PTC Premature termination codon PolyPhen Polymorphism phenotyping rAAV Recombinant adeno associated virus RdCVF Rod derived cone viability factor RE Right eye RP Retinitis pigmentosa RT-PCR Reverse transcriptase polymerase chain reaction RPE Retinal pigment epithelium SE Spherical equivalent SIFT Sorting intolerant from tolerant SNP Single nucleotide polymorphism SLSN Senior LĂ¸ken SD-OCT Spectral domain optical coherence tomography STGD Stargardt disease VA Visual acuity vLINCL Variant late infantile neuronal ceroid lipofuscinosis WES Whole exome sequencing WGS Whole genome sequencing XL X-linked wt Wildtype

Chapter 1 Introduction

1.1 Chapter 1.1 General introduction

Seeing is believing. Vision is an intriguing phenomenon in life, enabled by highly organized processes in the eye. Visual perception is one of our primary recourses of retrieving information from the outside world. Visual perception has been investigated since the dawn of mankind, however, the actual processes required for perceiving a colorful image remain brain-teasing.

The eye and the general process of vision

The visual process is a sophisticated system that is able to identify and discriminate among stimuli varying from chromatic or achromatic, motion or motionless and designed or unpatterned. The human eye is composed of three main structures. The outer layer maintains the shape of the eye, and serves as a protector of the inner layers. It is formed by connective tissue structures, the sclera and the cornea. The middle layer is formed by the choroid, ciliary body and iris. The choroid is important for blood supply and nutrition of the eye. The iris enables further accommodation of the incoming light. The inner layer, the retina, provides the final step in translating light to a sharp image and serves as the sensory part of the visual system (Figure 1).

Figure 1 Anatomy of the human eye (Figure adapted from: http://www.drpion.be/bouw-van-het-oog.htm) A beam of light reflected by an image enters the eye though the cornea, and for further visual processing needs to be focused on the retina. The light passes through the anterior chamber and pupil, a round opening central of the colored iris. A protective mechanism of the eye against overexposure of light is the adjustment of pupil size. High intensities of light constrict the pupil diameter, whereas in limited light intensities the pupil expands enabling more light to enter the eye. The subsequent structure is the transparent crystalline lens, which together with the cornea refracts the beam of light entering the eye to grant a sharp focus onto the retina.

15 General introduction

1.1

The retina, the light-sensitive layer lining the inner-eye, contains the rod and cone photoreceptors and exerts three functions. Firstly, the retina serves as a converter of the optical image to an electrical signal which is facilitated by these photoreceptors. The outer region of the retina provides peripheral vision and is mainly populated by rod photoreceptors, whereas the central region (called the macula) contains mostly cone photoreceptor cells and is necessary for detailed color vision. The most centered region (one square millimeter) of the macula is called the fovea. The 0.35 mm diameter centre of fovea, the foveola, exclusively contains cone photoreceptor cells. The second function of the retina is to send the electrical signal generated by the photoreceptor cells via the optic nerve to the brain where it is translated to an image which we know as vision. A third function of the retina is to create signals through specialized ganglion cells. Information on the 24-hour light/dark cycle is processed via the optic nerve to the suprachiasmatic nucleus. This brain structure contains ~20,000 neurons and regulates the bodyâ&#x20AC;&#x2122;s biological â&#x20AC;&#x2DC;clockâ&#x20AC;&#x2122;, which influences changes in mental and physical characteristics during the day, i.e. the circadian rhythms.

Anatomy of the retina-RPE

The retina is a complex tissue composed of two major layers; the neural retina and the retinal pigment epithelium (RPE, figure 2). The RPE is a highly pigmented monolayer which acts as a blood-retina barrier, allowing exchange of nutrients, growth factors and waste products between the subretinal space and the choriocapillaris. The pigmentation of the RPE provides absorption of excess light focused on the retina after entering the eye. The RPE also functions in retinal metabolism by regulating photoreceptor excitability, the visual cycle and phagocytosis of shed photoreceptor outer segments,1-3 and maintains structural integrity. The RPE also regulates the secretion of immunosuppressive agents or factors and thereby co-ordinates the immune response potential.

Figure 2 Cellular organisation of the retina Schematic representation of the various cell types within the two retinal layers (Figure derived by and published with permission of J.L Wilkinson-Berka4).

Besides the RPE, the retina is composed of the neural (or sensory) retina, which is connected to the central nervous system (Figure 2). 5 and the references therein The incoming light first hits the photoreceptors, which transfer the energy of an absorbed photon into an electric signal which is transmitted via the synaptic terminal to the second layer of bipolar horizontal and amacrine cells. These second order neurons are responsible for the first stage of processing the image by connecting the input from multiple photoreceptors. These signals are then transported towards the third layer, composed of ganglion cells, which communicate the neuronal signal through the optic nerve to the lateral geniculate body, where the signal crosses a synaps and travels via the optic radiation to the striate cortex and extrastriate associated cortices for final visual processing.

The macula lutea

The posterior retina contains the macula, which is recognizable as a yellowish region on the temporal side slightly below the optic disc and covers a diameter of 5.5 mm (Figure 3). The color of the macular region is generated by two xanthophyll pigments, lutein and zeaxanthin, which serve as antioxidants and protection against sunlight for the eye.6-9 The central region of the macula, called the fovea, is characterized by a ~1.5 mm-wide depression in the inner retina surface. This region of the macula provides acute high-resolution vision in the human eye. The fovea is not composed of layers like the other regions of the retina, does not contain rods, is capillary free, and is exclusively composed of cone photoreceptor cells. The importance of the fovea is striking, as approximately 50% of the nerve fibers in the optic nerve acquire their information from the fovea.

Figure 3 Position of the fovea in the posterior pole of the human eye (Figure adapted and published with permission from http://www.positscience.com/media-gallery/ detail/160/62)

17 General introduction

1.1

Photoreceptors

The human retina contains two types of photoreceptor cells, the rods and cones. Scotopic vision, at lower light intensities, involves perceiving contrast and brightness of light, as well as peripheral vision and detection of motion is provided by the highly sensitive rods. The less sensitive cones enable photopic vision at higher intensities of light, are responsible for color vision and seeing details at high spatial acuity. A healthy human retina contains close to 3 million cones, which presents ~5% of all photoreceptors, while the remaining ~95% is composed of approximately 60 million rods. Rods are absent in the fovea, are highly concentrated near the macula, and are the dominating photoreceptor cell in the mid-peripheral and peripheral retina. Cone photoreceptor cells are highly concentrated in the fovea (~160,000/mm2), but these numbers decrease rapidly towards the periphery. Rods can detect a single photon and provide sight in dimmed light, which makes humans capable of seeing objects by focusing slightly besides the object. The object could be missed by the cones, as they need approximately 100 photons to produce the same response due to their low sensitivity in dimmed light.

Figure 4 Cone and rod cell distribution in the retina (Figure derived from http://www.olympusmicro.com/primer/lightandcolor/images/humanvisionfigure4.jpg) Whilst cones have the necessity of relatively bright light, they provide us the sharpest images and give humans the ability to see colors. Color vision is created by three types of cones: 1) long wavelength, L-cones, are sensitive for the yellow spectrum (but are called red cones) from 565-580 nm; 2) medium wavelength, M-cones, absorb green light from 534-545 nm and 3) short wavelength, S-cones, respond to blue (violet) wavelengths with a peak absorption at 440 nm. These different absorption signals from different cones are converted by the brain into a color image with all possible colors. Cones are shorter with a wider base and have cone-shaped outer segments, whereas rods are longer and are cylindrical-shaped. The photoreceptor cells are both composed of a synaptic terminus, an inner segment and an outer segment (Figure 5).

1.1

Figure 5 Schematic representation of the rod and cone photoreceptor cell The synaptic terminus acts as a medium by forming synapses to transmit the generated electrical membrane potential to the bipolar cells. The inner and outer segments are linked via the connecting cilium. The outer segments contain the light absorbing proteins and processes, whereas the inner segments host various organelles such as the mitochondria, ribosomes and rough endoplasmic reticulum. The body of the photoreceptor cell contains the cell nucleus. The outer segments of rods consist of tightly packed discs surrounded by a plasma membrane, which create the surface area for the photosensitive pigment rhodopsin. Cones are not built up of separate discs, but the conical shaped outer segments have invaginations of their cell membranes, which form a stack of continuous membranes. The photopigments for cones, opsins, are embedded and synthesized and regenerated by the replacement of the all-trans-retinal by 11-cis-retinal by the RPE. Photoreceptor cells do not divide anymore, their membranous discs decay, and in the case of rods they are detached at the end of the outer segment, taken up by RPE cells, absorbed by phagosomes and recycled. Approximately 10%, equal to ~100 rod discs, are recycled in this way every night in humans. Cone outer segments are not detached for recycling, but apical processes of the RPE phagocytize parts of cone outer segments. Different than rods, this occurs in the light.

19 General introduction

Color vision

Organisms can perceive colors or have color vision by the ability to distinguish the different wavelengths of light. Color vision remains an intriguing phenomenon and is created by billions of interacting neurons in the brain. How the brain handles these properties of light and translates them into color vision remains largely a mystery. An important concept in color vision is that a cone only responds to the energy it absorbs (Maxwell 1872). If the energy absorbed by the cone is the same for different wavelengths, they would produce identical responses. This univariant response may give problems when an object reflects a similar amount of energy as the background. Hence, the contrast in wavelength allows one to distinguish objects, even when energy contrast is minimal or absent. Thus, color vision combines the energy and wavelengths of light to detect objects.

Figure 6 Absorption spectra of cones and major colors produced by trichromatic color vision in humans (Figure derived from http://www1.appstate.edu/~kms/classes/psy3203/Color/absorption_spectrum.jpg) To differentiate between objects based on spectral reflectance, two or more cones are needed. Human color vision is trichromatic, meaning that three classes of cones contain pigments that absorb different light wavelength sensitivities (Figure 6). The visual spectrum of human vision is dependent on the ability of light penetration in the eye and absorbance by the photoreceptors. The anterior segment of the eye absorbs ultra-violet light and therefore seldom reaches the photoreceptors, whereas infra-red light is able to penetrate the eye, but the energy may be too small to activate opsins. This trichromatic cone system contributes to the large variety of major colors humans can appreciate. Vision of colors varies among species. Other than trichromatic humans or most related primates, most mammals have dichromacy showing a red-shift in the absorption spectrum, which likely enables these usually nocturnal animals to distinguish colors in dim light. Marine mammals are monochromats with a single cone type as they are adapted for low-light vision. Invertebrates like (honey- and bumble) bees, wasps and sawflies are also trichromatic and cannot visualize the color red, but instead they are sensitive to ultraviolet. Tetrachromatic vision by four cone types, or pentrachromatic vision by five types of cone is present in birds and reptiles, respectively. Butterflies are also thought to be pentachromatic, but the most complex color vision is found in the mantin shrimp which has 12 different spectral cone receptors.

1.1

21 General introduction

1.2 Chapter 1.2 Genomics technologies and disease identification strategies

The studies in this thesis have been facilitated by the ongoing developments of technologies in genomics. To discover gene defects in CD knowledge on the genetic variation in the population is crucial. In this paragraph genetic variation and the most widely used technologies in this thesis are explained. These techniques have paved the way to identify a spectrum of genetic defects underlying CDs that are comprehensively described in chapter 1.3.

Genetic variation

Although the deoxyribonucleic acid (DNA) of two individuals is more than 99.9% identical, there remain differences both in composition and structure of the DNA between individuals.10 A nucleotide that varies in more than 1% in the normal population is called a polymorphism. If this variation affects only one nucleotide it is referred to as a single nucleotide polymorphism (SNP), while highly variable repeated sequences of 1-6 basepairs (bp) are called microsatellites.11 SNPs are the most common genetic variations among persons and cover approximately 0.1% of the genome, with an estimated occurrence of one SNP in every 100-300 bp. The frequency of a specific SNP or allele in a population is called the allele frequency. Current estimates are that there are approximately 15 million SNPs with a minor allele frequency (MAF) of >1%.10 A diploid genome has 6 million differences with any other individual, of which at least 20,000 occur in the coding parts (the exons) of genes.10 When a variant is only on one copy of the chromosomes it is called heterozygous, and when the same variant occurs in both chromosomes it is referred to as homozygous. Variants that cause disease, and/or affect the function of a gene, are called mutations.

Homozygosity mapping

In autosomal recessive disease, an individual needs to carry mutations on both chromosomes in order to be affected. When identical mutations are observed these are called homozygous mutations, whereas two different mutations in the same gene are called compound heterozygous. The latter are mostly found in patients of outbred populations, whereas homozygous mutations are often found in patients originating from isolated populations due to geographic restrictions, religion, cultural or language reasons. Homozygosity mapping is a method used to detect homozygous mutations and is based on the following principle. In reproductive cells, during meiosis, a process called recombination occurs, which implies the exchange of two chromosomes at random positions. not only a mutation is passed on to the next generation, but also the surrounding region of the chromosome. A combination of polymorphisms on a merged chromosome is called a haplotype, and its length and position is determined by recombinations between the original parental haplotypes. A haplotype containing a mutation will decrease in size through generations, and can occur in offspring as an identical-bydescent (i.e. homozygous) region. In isolated populations or in families in which the parents are consanguineous, the homozygous regions containing the mutation generally span 20-40 cM of the genomic DNA (Figure 1A).12 When the ancestor who carried the mutation lived several generations ago, the homozygous region containing the mutation in a patient will be much smaller (1 - 7 cM) due to the higher number of recombinations (Figure 1B). Genomewide genotyping arrays can be used to detect these homozygous regions by assessing hundreds of thousands of SNPs in a personâ&#x20AC;&#x2122;s DNA. When identical SNPs on both chromosomes are found, the SNP is called homozygous. A stretch of homozygous SNPs, composing a homozygous region, potentially originates from a common ancestor. Homozygous regions can also occur by chance as there may be limited haplotype diversity, little historic recombinations (i.e. haplotype blocks) or regions near the centromere of chromosomes which present a lower number of recombinations.13 Despite this drawback, homozygosity mapping has been proven to be an effective tool for the identification of the

25 Genomics technologies and disease identification strategies

1.2

disease region, and in combination with a positional candidate gene approach has been widely used to elucidate the genetic defect in individuals who suffer from an autosomal recessive disease.14-18

Next generation sequencing

DNA sequencing technology has evolved tremendously in the last 4 decades. In 1977, the enzymatic dideoxy Sanger sequencing technique and the Gilbert chemical degradation method were described.19; 20 Since the early 1990s, semi-automated Sanger biochemistry has been implemented to perform DNA sequence analysis.19-21 Although this technique covers the optimal resolution for genetic variant detection, its downside is that the scale generally allows only 96 to 384 reactions in parallel, which makes this a time-intensive and expensive approach at a cost of roughly $500 per Mega base (Mb) of DNA. Giant leaps were made in technology in the last years by the development of next generation sequencing (NGS), which is capable of sequencing millions of DNA fragments in a highly parallel fashion. Whole exome sequencing (WES) targets ~50 Mb of human coding regions, together called the exome, and covers approximately 1% of the entire genome. After the first successful application of exome sequencing in Miller syndrome 22 and Kabuki syndrome23 only a few years ago, its power has grown by the identification of large numbers of novel genes associated with rare as well as common disorders, 22 and the costs of performing exome sequencing are still decreasing. Currently, NGS technology has expanded to cover the entire genome sequence, referred to as whole genome sequencing (WGS), in which not only the coding variant but also all intronic variants of a genome can be determined. Great interpretational challenges are faced to pinpoint gene defects to intronic variants as they may cause regulatory or translational defects, but do not have a clear effect on the protein itself. Functional studies on these intronic variants are needed to provide the knowledge to make great use of this technology.

1.2

Homozygous region 20-40 cM

Year

Ancestor

1650 1750

1800

1900

Patient

2014 Homozygous region 1-7 cM

Figure 1 Schematic representation of the inheritance of ancestral chromosome that harbors a mutation The chromosomal region carrying the culprit mutation and originating from the original ancestral chromosome in the great grandparent reduces in size due to recombination between homologues chromosomes with every generation. A) In consanguineous families, a recent common ancestor likely spreads the mutation. Because the limited number of generations between the ancestor and the affected individual, a large fraction of the ancestral haplotype is present in both copies of the affected individual, which results in a relatively large homozygous region.12 B) When the common ancestor with a heterozygous mutation lived many generations ago, only a small fraction of this haplotype will be present on both chromosomes of a patient. Therefore the homozygous region of this affected individual is relatively small. (Figure derived by and published with permission of K.L. Littink.)

27 Genomics technologies and disease identification strategies

Susanne Roosing, Alberta A.H.J. Thiadens, Carel B. Hoyng, Caroline C.W. Klaver, Anneke I. den Hollander, Frans P.M. Cremers

Prog Retin Eye Res. In press 28

1.3 Chapter 1.3 Causes and consequences of inherited cone disorders

Abstract

Hereditary cone disorders (CDs) are characterized by defects of the cone photoreceptors or retinal pigment epithelium, and include achromatopsia (ACHM), cone dystrophy (COD), conerod dystrophy (CRD), color vision impairment, Stargardt disease (STGD) and other maculopathies. Forty-two genes have been implicated in non-syndromic inherited CDs. Mutations in the 5 genes implicated in ACHM explain ~93% of the cases. On the contrary, only 21% of CRDs (17 genes) and 25% of CODs (8 genes) have been elucidated. The fact that the large majority of COD and CRD-associated genes are yet to be discovered hints towards the existence of unknown cone-specific or cone-sensitive processes. The ACHM-associated genes encode proteins that fulfill crucial roles in the cone phototransduction cascade, which is the most frequently compromised (10 genes) process in CDs. Another 7 CD-associated proteins are required for transport processes towards or through the connecting cilium. The remaining CD-associated proteins are involved in cell membrane morphogenesis and maintenance, synaptic transduction, and the retinoid cycle. Further novel genes are likely to be identified in the near future by combining large-scale DNA sequencing and transcriptomics technologies. For 31 of 42 CD-associated genes, mammalian models are available, 14 of which have successfully been used for gene augmentation studies. However, gene augmentation for CDs should ideally be developed in large mammalian models with cone-rich areas, which are currently available for only 11 CD genes. Future research will aim to elucidate the remaining causative genes, identify the molecular mechanisms of CD, and develop novel therapies aimed at preventing vision loss in individuals with CD in the future.

1. Introduction

Cone disorders (CDs) are a group of inherited diseases of the cone, or cone and rod photoreceptors, or retinal pigment epithelium (RPE), that are associated with various forms of stationary or progressive visual impairment. Linkage analysis, homozygosity mapping, and more recently whole exome sequencing (WES), facilitated the identification of genes mutated in patients with CDs such as achromatopsia (ACHM), cone dystrophy (COD), cone-rod dystrophy (CRD), and other cone-related disorders. CDs can follow all modes of Mendelian inheritance, i.e. autosomal recessive (AR), autosomal dominant (AD) and X-linked (XL), and can present as non-syndromic and syndromic forms. Thus far, reviews were focused on stationary cone disorders24 as well as the genetic spectrum of cone disorders.25 Here, we provide an overview of the expanded genetic repertoire of inherited cone disorders and novel genotype-phenotype correlations. Despite the high clinical and genetic heterogeneity of CDs the genetic defects underlying 21-93% of the cases have been identified. The proteins encoded by CD-associated genes are involved in a variety of processes, including the cone phototransduction cascade, visual cycles, photoreceptor development, ciliary transport, disk membrane morphogenesis and synaptic transport. Studies in mammalian models show that gene replacement therapies, due to the accessibility and immune-privileged nature of the eye, provide an opportunity to correct the visual impairment. In this review we present a comprehensive overview of the genes and associated disease mechanisms of non-syndromic CDs, touch upon the genetic causes of syndromic CDs, and evaluate therapeutic strategies aiming to prevent vision loss in CD in the future.

2. Phenotypic and genetic characteristics of cone disorders

Hereditary CDs constitute a large clinically heterogeneous group of diseases in which the cone photoreceptors or RPE are primarily affected. Cone photoreceptor cells account for only 5% of the total number of photoreceptors, the other 95% consisting of rods. In humans, the retina contains 3-6 million cone photoreceptors concentrated in the fovea enabling detailed vision, spatial resolution, reading, facial recognition and color vision. These important cone functions explain why CDs have such a disabling impact on the daily living tasks of those affected, further compounded by the early onset of disease and severe visual outcome of many of these disorders. We analyzed all publicly available literature on CDs and, to estimate the contribution of specific genes, only included studies with clearly defined patient cohorts (Supplementary Tables 1-5). Using this approach, we estimated that AR/isolated (I) CDs are found in 76.7% of cases, AD inheritance in 21.6% of cases and X-linked inheritance in the remaining 1.4% (Figure 1). In this chapter we provide an overview of the clinical and genetic characteristics of different forms of non-syndromic and syndromic CDs. Figure 1 Frequency of clinical subtypes and inheritance patterns of genetically heterogeneous cone disorders Estimate of the frequencies of clinical subtypes and modes of inheritance of the genetically heterogeneous forms of CD, i.e. achromatopsia (ACHM), which invariably is inherited in an AR manner, cone dystrophy (COD), and cone-rod dystrophy (CRD). The majority of the AR COD and AR CRD cases are isolated (I) cases in which a recessive mode of inheritance is most likely. In most studies in which AD forms of COD and CRD were analyzed, no clear distinction was made between these disease entities, and therefore they are grouped together.

31 Causes and consequences of inherited cone disorders

1.3

2.1. Clinical methods

Ophthalmic examination and diagnostic testing is essential to differentiate cone-rod from rodcone (i.e. RP) disorders. Although a number of tests are helpful in the diagnostic process, color vision testing of all three color axes such as Hardy Rand and Rittler tests, full-field ERG, and Goldmann perimetry, have been proven to be the most useful (Thiadens et al., 2013). With SD-OCT the morphology of the cone photoreceptor cells can be visualized. A single-section scan to obtain a longitudinal section across the center of the macula, and a volume scan should be performed to ensure capturing the center of the fovea. Retinal thickness measurements in the fovea including the outer nuclear layer, inner and outer segments of the cone cells, and RPE. For future gene therapy purposes, SD-OCT can be considered as a useful diagnostic tool for visualizing the severity of cone photoreceptor loss. In Table 1, we summarized the ophthalmic symptoms and characteristics for ACHM, oligocone trichromacy (OT), blue cone monochromatism (BCM), COD, CRD and Stargardt disease (STGD). Further details of these and other CDs can be found below.

2.2. Clinical characteristics of non-syndromic cone disorders 2.2.1. Achromatopsia

Patients with achromatopsia (ACHM, figures 2A and 2B, table 1) present shortly after birth with significantly reduced visual acuity, severe photophobia, a congenital pendular nystagmus, and color vision defects in the protan, deutan and tritan color axes. High refractive errors are common in these patients. The macula can have a variable appearance ranging from no abnormalities to atrophic lesions. Full-field electroretinography (ERG) demonstrates absent or residual cone responses with normal rod responses.26,27 The first signs of pathology on SD-OCT are loss of inner- and outer cone segments with disruption of the ciliary layer, which may be followed by the appearance of an optical empty cavity with cell loss in the cone photoreceptor layer, at least in some studies. The end stage is generally characterized by a complete loss of photoreceptor and atrophy of the RPE in the fovea. This cascade of events takes place in the second decade and progresses with age.28,29 For future gene therapy purposes, SD-OCT can be considered as a useful diagnostic tool for visualizing the severity of cone photoreceptor loss.

The estimated prevalence of ACHM is 1:40,000 individuals and it is exclusively inherited in an AR manner. The genetic etiology of ACHM has been almost completely unraveled. Mutations in the five genes associated with ACHM (Figure 3A) explain 92,8% of cases in the Caucasian population. All causal genes encode essential proteins in the cone phototransduction cascade (see paragraph 3).26,27 Importantly, the limited number of genes involved in ACHM renders this disease a suitable candidate for future gene-based therapies. Nevertheless these therapies can only succeed if viable cone photoreceptor cells are still present in the retina. 30-33

2.2.2. Cone dystrophy

Patients with cone dystrophy (COD, figures 2C and 2D, table 1) have normal cone function initially, but present with visual loss and color vision disturbances in the first or second decades of life. Complaints of photophobia or hemeralopia may also exist. In X-linked (XL) COD cases with a mutation in the ORF15 region of RPGR, hemeralopia has been reported as a first symptom preceding visual loss in more than a quarter of the patients. 34 These RPGR-associated COD patients typically have high myopic refractive errors of more than 6 diopters. 34 Because the cone function is initially normal in COD nystagmus is usually not observed. Macular abnormalities may be present ranging from no abnormalities to a bullâ&#x20AC;&#x2122;s eye maculopathy or RPE atrophy. The optic nerve may have variable degrees of temporal pallor. Goldmann perimetry shows a reduced central sensitivity or (relative) scotoma with intact peripheral visual fields.35

Table 1 Clinincal characteristics to differentiate the major inherited cone disorders ACHM

BCM

COD

CRD

STGD

Vision

0.05-0.20

0.05-0.40

~0.10

0.05-0.40

0.1-0.5

Nystagmus

+/-

Color vision

Disturbed in protan/deutan/ tritan color axes

Not disturbed

Disturbed in protan and deutan color axes

Disturbed in protan/deutan/ tritan color axes

Disturbed in protan/deutan color axes

Photophobia +

+/-

Hemeralopia -

+/-

Nyctalopia

+/-

Hypermetropia >+2D

+; ~30%

Myopia <-2D

+; ~25%

+; ~20% even more than >-6D

Retinal appearance

Normal to bull’s eye/ atrophic macular lesions

Normal to macular atrophy and RPE atrophy

Normal to bull’s eye/ atrophic macular lesions, temporal pallor of optic disc

Normal to bull’s eye/ atrophic lesions, peripheral RPE atrophy, vascular attenuation

Bull’s eye, mostly surrounded by flecks. In rare cases flecks spread up to the arcades

SD-OCT

Normalabsence of central cone PRL

Thinner central Absence of retina, no central cone absence of PRL cone PRL

Normalabsence of central cone PRL

Normalabsence of central cone PRL, central retinal thinning

Thinning or absence of central cone PRL

FAF

Normal to absent FAF in the macula

70% dark choroid

Goldmann perimetry

Reduced central Reduced central sensitivity to sensitivity (relative) central scotoma

Reduced central sensitivity to (relative) central scotoma

Reduced central Reduced central sensitivity to sensitivity (relative) central scotoma, with variable peripheral involvement

mfERG

Reduced/non detectable macular responses

Reduced

ffERG

Reduced/absent cone responses. Normal rod responses

Normal to slightly reduced cone responses. Normal rod responses

Reduced/absent cone responses with a preservation of the blue cones

Reduced/absent cone responses. Initial normal rod responses

Cone responses are absent or more severely reduced than rod responses

Reduced cone responses

+, present; -, absent; ACHM, achromatopsia; AD, autosomal dominant; AR, autosomal recessive; BCM, blue cone monochromatism; COD, cone dystrophy; CRD, cone-rod dystrophy; D, Diopters; FAF, fundus autofluorescense; ffERG, full-field electroretinogram; mfERG, multifocal electroretinogram; NA, not analyzed; ND, not described; OT, oligocone trichromacy; PRL,photoreceptor layer; SD-OCT, spectral domain-optical coherence tomography; STGD, Stargardt disease; XL, X-linked.

33 Causes and consequences of inherited cone disorders

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Figure 2 Fundus and autofluorescence images of cone disoders Fundus and autofluorescence images of patients with a cone disorder (CD), in order of description in the article from left to right and up and down (A-R). A/B) Achromatopsia. A) The central macula shows mottling of the RPE, which corresponds with B) the small region of absent fundus autofluorescence (FAF) in the center due to the RPE alterations. C/D) Cone dystrophy. C) The optic disc shows temporal pallor of the optic disc; the macula presents with a bullâ&#x20AC;&#x2122;s eye maculopathy with mottling of the RPE (color photograph). D) The fovea shows a small central region with absent FAF caused by RPE atrophy, surrounded by discrete hyperfluorescent lesions (FAF image). E/F) Cone-rod dystrophy. E) The central macula shows an area of diffuse RPE degeneration (color photograph). F) The lesion corresponds to absence of FAF in

the center, surrounded by a discrete lesion of higher FAF intensity (FAF image). G/H) Stargardt disease. G) The posterior pole shows multiple yellow fish-tailed flecks, some pigment clumping, and bull’s eye maculopathy with RPE atrophy (color photograph). H) The central macula shows decreased FAF, whereas the flecks exhibit increased FAF on a background of irregular FAF (FAF image); I/J) Best vitelliform macula dystrophy. I) The macula has an egg-yolk appearance corresponding to the vitelliform stage of Best (color photograph). J) The FAF of the lesion is intensely increased (FAF image); K/L) Central areolar choroidal dystrophy. K) In the macula a sharply demarcated lesion with RPE atrophy is visible, surrounded by a few small round white lesions (color photograph). L) The atrophic area causes a decreased FAF bordered by a small band of increased FAF (FAF image). M/N) Butterfly-shaped macular dystrophy. M) The macula shows an irregular yellow pigmented lesion with some RPE degeneration (color photograph). N) The central area has decreased FAF while the yellow pigment show increased FAF resembling a ‘pattern’ (FAF image). O/P) Adult-onset foveomacular vitelliform dystrophy. O) The macula reveals an oval yellow subfoveal lesion with central pigment (color photograph). P) The lesion shows an intensely increased FAF; the border of the lesion shows a band of decreased FAF (FAF image). Q/R) Cystoid macular dystrophy. Q) The macula shows focal thickening of the retina in the center (color photograph). R) The pooling of fluid in the macula gives a classical flower-petal pattern of FAF (FAF image). Color vision is generally affected in all three axes. Reduced cone responses with preserved rod responses on ERG are an important clinical hallmark for the diagnosis of COD, although the vast majority of patients with COD develop rod involvement over time complicating the diagnosis. The mean onset of disease is at adolescence but the course of the disease may vary. Visual acuity generally worsens to legal blindness before 50 years of age.34 COD has an estimated prevalence of 1:30,000-40,000.24 Although all Mendelian inheritance patterns have been reported the AR form is by far the most common. The genetic causes of the autosomal dominant (AD) and XL forms have been solved to a large extent, but 75% of AR forms remain to be elucidated (see Figure 3B). This is remarkable and hints towards the existence of completely unknown and uncharacterized cone-specific or cone-sensitive processes. Some of the genes associated with AR forms have a characteristic phenotypic appearance, like COD patients with mutations in the KCNV2 gene. Mutations in this gene show supra-normal rod responses on ERG. Besides these remarkable ERG findings, most young patients also have a relatively late onset in loss of visual acuity. 36 The rod responses on ERG do not appear to coincide with a gain of rod visual acuity, on the contrary, studies showed loss of acuity for rod as well as cone cells in patients carrying KCNV2 mutations. 36-38 The clinical course of ABCA4associated COD has also been studied in detail. This gene encodes a transmembrane protein located in rod and cone outer segments. AR-COD patients with mutations in this gene show an earlier onset, a rapid decline of visual acuity, and a significantly worse visual outcome than patients without ABCA4 mutations. 34

2.2.3. Cone-rod dystrophy

Cone-rod dystrophy (CRD, figures 2E and 2F, table 1) can be distinguished from COD by early rod involvement or concomitant loss of both cones and rods on ERG. Symptoms resemble those of COD, with complaints of visual acuity and central vision, sometimes photophobia, and color vision disturbances. However, patients may also experience nyctalopia due to rod degeneration. With an onset in childhood and a rapid decline of the visual function to legal blindness before the age of 40, the course of CRD seems more severe than COD.34 The macular appearance resembles that of COD, but patients may also develop retinal vascular attenuation and peripheral pigment deposits. Visual fields show a (relative) central scotoma with variable

35 Causes and consequences of inherited cone disorders

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degrees of peripheral involvement. CRD is usually non-syndromic but it can manifest as part of a syndrome (see 2.3). As in COD the prevalence of CRD is 1:30,000-40,000 and can be inherited in an AR, AD or XL manner. 39 Currently, 17 genes and some additional loci are known to be involved in AR CRD explaining approximately 22% of the cases (Figure 3C). As COD and CRD cases are often difficult to distinguish clinically, we have combined genetic data from the autosomal dominant forms of COD and CRD. In AD COD/CRD 21% of the cases were solved (Figure 3D) and in XL COD/CRD 74% (Figure 3E).

Figure 3 Frequency and genetic causes of cone disorders Estimates of the current frequency and genetic causes in cone disorders (CD). We only assessed studies in which cohort sizes were clearly indicated. A) ACHM. Mutations in five genes explain 93% of the cases, with the largest contribution by CNGB3, followed by CNGA3. For the included studies see supplemental table 1. B) Autosomal recessive and isolated COD. Eight genes each are mutated in a small proportion of the cases and together form the basis of disease in 25% of the cases. For the included studies see supplemental table 2; DNA samples in the study of Littink et al. (2010) have been scanned using homozygosity mapping (no Sanger sequencing) and therefore have been excluded from numerical analysis, C) Autosomal recessive and isolated CRD. Seventeen genes are implicated in AR/I CRD, together responsible for 23% of the cases, of which only ABCA4 is mutated in a significant proportion. For the included studies see supplemental table 3. D) Autosomal dominant COD and CRD. Ten genes are mutated in these conditions, mutations in which explain not more than 20% of the cases. For the included studies see supplemental table 4, and E) X-linked COD and CRD. For the included studies see supplemental table 5. Mutations in the ABCA4 gene are not only implicated in Stargardt disease, but also the most prevalent cause of AR CRD. However its involvement varies widely between studies. In the Caucasian population this percentage varies between 26-65%;34,40 in a recent Chinese study only 1/47 cases carried ABCA4 variants. This also suggests a much lower prevalence of Stargardt disease (STGD1) in this population as previously suggested.41,42 In CRD patients with two patho-

genic ABCA4 variants, the observed visual prognosis is significantly worse than in patients with mutations in other genes, with a mean age of legal blindness more than 20 years earlier. 34 One study suggested that CNGB3 (3/47) and PDE6C (2/47) are involved in CRD (Huang et al 2013), while previous studies only identified CNGB3 and PDE6C mutations in COD. Reasons for this discrepancy could include different genetic backgrounds, or insufficient phenotyping.

2.2.4. Color vision impairment

Normal human color vision is mediated by three types of cone photoreceptors that are maximally sensitive to light at 565 nm (the red cones, long [L] wavelength sensitive), at 535 nm (the green cones, middle [M] wavelength sensitive), and at 440 nm (the blue cones, short [S] wavelength sensitive). The rods are maximally sensitive to light at 500 nm, mediate vision in dim light, and contribute little to color sense. Humans thereby generally have trichromatic color perception. The most frequent form of color vision impairment is dichromacy, which is observed in ~6% of males and 0.4% of females. Dichromats have lost one class of pigment, either the L, M or S pigments, and are named protanopes, deuteranopes, and tritanopes, respectively, the latter of which are very rare. The more frequent protanopes and deuteranopes carry deletions of the red (OPN1LW) or green (OPN1MW) pigment genes, nonsense mutations in these genes, or missense mutations that influence their spectral tuning.43,44 Persons with X-linked blue cone monochromatism (XL-BCM) have the same clinical presentation as those with ACHM with photophobia, nystagmus, severe color vision disturbances, and a low visual acuity of ~0.10 in early childhood (table 1).45 To distinguish BCM from ACHM it is necessary to use color vision tests that specifically select for the blue color axis such as Berson plates.46 Color ERGs may also distinguish between responses from different cone types and may detect signals from intact blue cones on a background of absent signals from red and green cones.47 XL-BCM is very rare with a prevalence of 1:100,000 individuals.24 The red and green cone opsins are absent which in almost 40% of BCM can be explained by deletions in the locus control region, the region regulating transcription of the red and green opsin genes.45,48 Other BCM patients carry a p.Cys203Arg variant in a single green or red-green hybrid gene that remained after non-homologous recombination.48 Together with a cystein at amino acid position 126, this cystein forms a conserved disulfide bond that is important for protein stability. Bornholm eye disease is an XL disease that has been reported in a large Danish family originating from the Danish island of Bornholm, characterized by a low visual acuity, a reduced photopic ERG, moderate to high myopia, and deuteranopia. Linkage analysis mapped the defect to a chromosomal region encompassing the red and green opsin gene array, suggesting overlap with congenital color blindness (deuteranopia). Recently, families with Bornholm eye disease, that display dichromacy and myopia, were shown to carry sequence variants in the first OPN1LW gene copy, which presumably lowers the amount of functional protein.49-51 Oligocone trichromacy (OT) is a rare condition first described in 1973 by van Lith. 52 It is characterized by reduced visual acuity, mild photophobia, nearly normal color vision, a normal fundus appearance, and reduced cone responses with normal rod responses on multifocal ERG (table 1). It has been proposed that these patients have a reduced number of central cones or a reduced number in total (oligocone). The disease has an AR inheritance, and genetically there is considerable overlap with ACHM, as mutations in CNGA3, CNGB3 and GNAT2 have been associated with this form of CD. 53,54

2.2.5. Stargardt disease

Autosomal recessive Stargardt disease (STGD1), figures 2G and 2H, table 1) is the most prevalent inherited AR juvenile retinal dystrophy, with an estimated frequency of ~1:10,000. 55 Fundus flavimaculatus is a largely overlapping phenotype with a later onset, slower progression, and

37 Causes and consequences of inherited cone disorders

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more widespread distribution of flecks. 56-58 Hereafter, fundus flavimaculatus will not be mentioned separately. STGD1 can arise in the first decade, but can also have an onset much later in life (up to 70 years of age). The outcome is generally poor central vision developing only a few years after the age of onset, but the prognosis may be better due to foveal sparing in some cases. 59-61 STGD1 patients show a dark choroid in ~80% of the cases upon fluorescein angiography due to the lipofuscin accumulation, which prevents the background fluorescence of the choroid.62,63 A combination of a mild and a severe, or two moderately severe mutations in ABCA4 give rise to STGD1,64,65 whereas the combination of two severe mutations in ABCA4 are known to cause panretinal, severe CRD which, at the endstage, may be difficult to discriminate from typical retinitis pigmentosa (RP).66 The estimated carrier frequency of ABCA4 variants in the normal population can be as high as 1:20.65 As described in more detail in paragraph 3.2, a deficiency of ABCA4 results in accumulation of lipofuscin in the RPE which can be seen in and around the macula as yellowish white flecks. In animals as well as in patients, it was shown that a defective ABCA4 protein causes accumulation of N-retinylidene-PE in the RPE, which forms a potential toxic component inducing photoreceptor death.67,68 In persons with CRD and ABCA4 mutations there appears to be an intrinsic and severe defect in photoreceptors, which directly leads to photoreceptor loss and lipid accumulations in the RPE are mostly absent.

2.2.6. Other Maculopathies

Mutations in PRPH2 have been associated with AD central areolar choroidal dystrophy (CACD, figures 2K and 2L), “pseudo-Stargardt pattern dystrophy”69, pattern dystrophies like butterflyshaped pigment dystrophy (BSMD, figures 2M and 2N) and adult-onset foveomacular vitelliform dystrophy (Figures 2O and 2P), as well as AD retinitis pigmentosa.70 The PRPH2 protein product RDS/peripherin is a photoreceptor-specific glycoprotein crucial in photoreceptor outer segment discs development and maintenance (Figure 4, left lower panel).71-73 It has been hypothesized that abnormal RDS/peripherin protein results in an altered cone outer segment structure and potentially an altered rod outer segment structure.74 This may interfere with the photoreceptor outer segment-RPE interaction leading to accumulation of lipofuscin and byproducts in the RPE cells resulting in apoptotic cell death of RPE and photoreceptor cells. Pattern dystrophies due to PRPH2 mutations are presumed to arise by disruption of the photoreceptor disc membranes.75,76 Best vitelliform macular dystrophy (BVMD, figures 2I and 2J), a member of the group of ‘bestrophinopathies’, is caused by AD inherited mutations in the BEST1 (or VMD2) gene, which encodes the bestrophin-1 protein, a calcium-activated chloride channel located at the baso-lateral membrane of RPE cells, that also influences voltage-gated calcium channels within the RPE. Furthermore, BEST1 localized mostly to the ER close to the baso-lateral plasma membrane and is involved in the storage-dependent Ca-influx into RPE cells.77 The age at onset of central vision loss is highly variable ranging from the first to the sixth decade.78,79 The disease-course of BVMD begins with the asymptomatic carrier (‘pre-vitelliform’) stage, progressing to variable clumping of vitelliform material in the vitelliruptive or ‘scrambled-egg’ stage, culminating in variable degrees of chorioretinal atrophy and subretinal scarring in the cicatricial stage. Patients with AR bestrophinopathy have more extensive retinal abnormalities and carry a BEST1 mutation on both alleles.80 Hypothetically, causes of RPE dysfunction in BVMD are related to abnormal ionic transport leading to the accumulation of subretinal fluid and vitelliform material originating from photoreceptor outer segment-waste products and lipofuscin loaded pigmented cells.81 The overload of the RPE eventually leads to photoreceptor and RPE dysfunction. PRPH2 variants have also been associated with BVMD and like BEST1 mutations, are also associ-

ated with a high phenotypic heterogeneity and limited genotype-phenotype correlation.70,82 Genes with a minor contribution to AD macular dystrophies are C1QTNF5, EFEMP1, FSCN2, GUCA1B, HMCN1, IMPG1, RP1L1 and TIMP3. Three of these present a rare but particular phenotype; EFEMP1 mutations are a cause of AD Doyne honeycomb retinal degeneration (Malattia Leventinese)83 which presents with drusen deposits at the level of Bruch’s membrane in the macula around the edge of the optic nerve head.84 Occult macular degeneration is caused by heterozygous mutations in RP1L1 and is characterized by central cone dysfunction and vision loss with a normal appearing retina.85 Sorby’s fundus dystrophy develops due to mutations in TIMP3 and is recognized by loss of central vision from subretinal neovascularization, as well as atrophy of the choriocapillaris, RPE and retina.86 For seven other AD macular conditions, the underlying genetic causes are not yet known. Autosomal dominant macular dystrophy with cystoid macular edema (dominant cystoid mcular dystrophy, DCMD; a.k.a. cystoids macular dystrophy, CYMD, figure 2Q and 2R) causes visual acuity loss in the second decade of life with or without strabismus.87 As the disease progresses, the macula will develop a central zone of ‘beaten bronze’ atrophy. The DCMD locus was positioned to an approximately 20-cM region between markers D7S493 and D7S526 on chromosome 7p15-p21,88 but the gene has remained elusive. North Carolina macular dystrophy (NCMD) is an AD macular dystrophy with an early childhood onset and a stable disease course.89-91 The highly variable phenotype contains yellow drusen-like lesions with retinal pigmentary changes of variable severity.89,92 The corresponding locus, MCDR1, was identified on chromosome 6q16,93,94 but the gene and its presumed function are currently unknown. Benign concentric annular macular dystrophy (BCAMD) is described in a Dutch family and may be caused by a mutation in IMPG1.95 An early-onset AD macular dystrophy (MCDR3) resembling NCMD has been mapped to chromosome 5.96,97 The defect in a Greek family with macular dystrophy has been positioned on chromosome 19q (MCDR5).98

2.3. Clinical and genetic characteristics of syndromic cone disorders

In a minority of cases, cone-rod disorders are not limited to a degeneration of the retina, but show systemic involvement as well. Ocular signs and symptoms may precede or follow the onset of systemic features and it is therefore recommended to incorporate a comprehensive ophthalmologic examination into the regular clinical work-up of patients with additional nonocular features. CD can be part of a syndrome or complex disease like Danon disease, Alström syndrome, or Jalili syndrome. Danon is a rare genetic condition caused by mutations in the X-linked lysosomeassociated membrane protein gene (LAMP2), and consists of the triad muscle weakness, cardiomyopathy, and mental impairment. The mortality rate in males is high; the most frequent cause of death is a heart arrhythmia. Female carriers can show a milder phenotype, often restricted to cardiomyopathy.99 Ophthalmic involvement has been reported in several cases, varying from macular abnormalities to CRD.100,101 In one family, with a missense mutation in LAMP2, affected males presented with all features of CRD. The onset in this family was relatively late (middleage) and visual acuity declined to legal blindness within two decades thereafter.101 Another example is the very rare AR Alström syndrome. It is characterized by CRD, hearing loss, obesity, diabetes, short stature, cardiomyopathy, and a progressively failure of lungs, liver and kidney. The disease manifests in early childhood and leads to a reduced life expectancy. Age of onset, severity of symptoms and prognosis may vary depending on genetic background. Alström syndrome is caused by mutations in the ALMS1 gene, which codes for a ciliary protein present in basal bodies, centrosomes and cytosol of the cells. Patients with Alström disease present with a low visual acuity, nystagmus and photophobia on occasion, atrophic lesions of

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the retina, and an abnormal cone-rod ERG. Phenotypically this ciliopathy shows similarities to Bardet-Biedl syndrome.102 Jalili syndrome (cone dystrophy with amelogenesis imperfecta) is caused by autosomal recessive mutations in CNNM4, encoding a metal transporter protein. Patients present with a demineralization of both primary and secondary dentition with or without COD. A large Arab family with Jalili syndrome showed low visual acuity since early childhood, photophobia and nystagmus in some cases, absent color vision, and a bull’s eye maculopathy or atrophic macular degeneration. Cone responses were more reduced than rod responses,103 consistent with a retinal diagnosis of CORD. A novel syndrome of North Carolina-like macular dystrophy with progressive sensorineural hearing loss (MCDR4) was linked to chromosome 14q.104

2.4. Differential diagnosis

CDs form a genetically complex disease spectrum with a large phenotypic variability. Different mutations can result in similar phenotypes, whereas many genetic factors may contribute to the phenotypic variability of one causative mutation. Traditionally, Leber congenital amaurosis (LCA) has been considered as the congenital form of retinal dystrophy and it is typically associated with a very low vision at birth accompanied by roving nystagmus, amaurotic pupils, and a high hypermetropic refractive error. Visual acuity ranges from 0.10 Snellen visual acuity to no light perception. Eye poking (oculodigital reflex) to provoke visual stimuli can be observed in young children. Most often no fundus abnormalities are present, but in later stages round subretinal pigment clumps or bone-spicule-like pigment changes can develop. ERG responses are undetectable for both rods and cones before the age of one year. With an inconclusive ERG result, it is sometimes necessary to repeat the ERG after the age of 1 year to obtain the correct diagnosis. The prevalence of LCA varies from 1:30,000 to 1: 81,000.105,106 In most cases the inheritance modus is AR, but cases of AD inheritance have been described. Systemic disorders that are associated with LCA or a LCA-like ocular phenotype are Alström syndrome,107 Batten disease,108,109 Joubert syndrome,110 peroxisomal diseases ,111 and Senior Løken syndrome.112

3. Cone disorder mechanisms 3.1. Phototransduction cascade

The processes involved in the conversion of light into an electric neural signal is called phototransduction. The phototransduction cascade takes place in the outer segments of the rod and cone (Figure 4, upper right panel) photoreceptors. Defects in various components of the phototransduction cascade can lead to CD: the red and green opsins, the cone transducin α subunit, the cone phosphodiesterase α’ and γ subunits, the cone-specific cGMP-gated α and β subunits, the retinal guanylyl cyclase-1, and the voltage-gated potassium channel subunit. Deletions of, or mutations in, the red and green opsin genes may cause color blindness (see 2.2.4.). A gene conversion event in which exon 3 of the red opsin gene, carrying the p.Trp177Arg variant, was transferred into the red opsin gene, was shown to underlie X-linked COD.43 Mutations in the GNAT2-encoded α-subunit of cone transducing leads to ACHM.113-115 The identified mutations are null alleles, which are predicted to prevent either the formation of the trimeric transducin protein complex or its interaction with cone opsin and phosphodiesterase-γ.115,116 Gene defects in PDE6C and PDE6H, encoding the α’ and γ subunits of the cone cGMP phosphodiesterase, are a cause of ACHM and COD.117,118 Mutations in the PDE6C gene are null alleles leading to absence of the α’ subunit, or missense mutations causing a loss or reduction of the catalytic activity resulting in reduced hydrolysis of cGMP.119,120 The absence of transducin or phosphodiesterase activity can be considered functionally analogous since in both cases the phototransduction cascade is interrupted and the photoreceptor remains in the dark state.119

Mutations in the CNGA3 and CNGB3 genes encoding the α- and β-subunits of the cone cGMPgated channel can cause ACHM and COD.26,113,114,121 A defect or lack of the cGMP-gated channel impairs the resting state dark current and the establishment of a proper cone photoreceptor membrane potential (Chang et al 2009), causing apoptosis by perpetual influx of calcium ions.34,122 The majority of CNGB3 mutations are truncating, resulting in the absence or altered protein levels of the β-subunit. 32 Compared to the β-subunit of the pore, which mostly comprises truncating mutations in the CNGA3 gene, the cone α-subunit is more sensitive to changes. Amino acid substitutions in the highly conserved CNGA3 protein have an even more severe phenotypic impact. The reason may lie in the fact that CNGA3 can form a functional channel in the absence of CNGB3, while CNGB3 cannot establish the ion currents without the presence of CNGA3.123,124 CNGA3 missense mutations can lead to an interfering conformational change of the active channel due to an altered secondary structure.124 The impaired channel will cause cellular mistrafficking125,126 and altered targeting to the plasma membrane due to impaired surface expression.127,128 Remarkably, unlike COD and CRD, autosomal dominant inheritance has not been observed for ACHM, despite the fact that many cone-specific proteins act in multimeric complexes in which dominant-negative disease mechanisms would be plausible. The KCNV2 gene encodes a voltage-gated potassium channel subunit in cone and rod photoreceptors and mutations cause COD with supranormal rod responses on ERG. The subunit encoded by KCNV2 is unable to form a functional potassium channel but needs heteromerization with other channel components, thereby altering the potassium current in the cell and affecting its excitability potential.129,130 It is unclear how mutations in this gene cause repolarization disturbances in rods.131,132 Specific heterozygous GUCY2D missense mutations are a cause of AD CRD, whereas homozygous or compound heterozygous inactivating mutations in this gene can cause AR-LCA. GUCY2D encodes the membrane-bound retinal guanylyl cyclase-1 protein (RetGC-1), which is expressed in both types of photoreceptors but predominantly in the cone outer segment.133-136 In mammalian photoreceptor cells RetGC-1 and RetGC-2 together synthesize cyclic 3’, 5’-guanosine monophosphate (cGMP) from guanosine triphosphate. RetGC-1 restores the Ca2+-sensitive cGMP levels after light activation of the phototransduction cascade, together with its associated activator proteins GUCA1A and GUCA1B.137 Cyclase activity is regulated by Ca2+, which binds to the GC-associated proteins, GCAP1 and GCAP2 encoded by GUCA1A and GUCA1B, respectively. The majority of dominant missense mutations causing AD COD and AD CRD are found in the Ca2+-binding EF hands of the proteins. Similar to the dominant GUCY2D mutations, these mutations generally alter the cyclase sensitivity to inhibit the Ca2+ levels after a light flash.

3.2. Retinoid cycles

To maintain the phototransduction cascade, recycling of the chromophore all-trans-retinal to 11-cis-retinal is required. This process is referred to as the retinoid or visual cycle. The rod and cone visual cycles take place in their respective outer segments, and the RPE cells (Figure 4, left upper panel). Moreover, part of the visual cycle of the cones takes place in the cone OS and the Müller cells (Figure 4, left middle panel). Although several genes encoding proteins of the retinoid cycles have been found to be defective in inherited retinal dystrophies, only the ABCA4 and SEMA4A genes are implicated in CDs (Table 2). Upon photo-excitation all-trans-retinal dissociates from opsin and is subsequently transferred to the cytoplasmic space by ABCA4. In rods, ABCA4 is located at the rim of the outer segments discs and essential in the transport of all-trans-retinal from the inner to the outer leaflet of the disks through an ABCA4-mediated flippase of N-retinylidene-phosphatidylethanolamine

41 Causes and consequences of inherited cone disorders

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(PE), the Schiff base conjugate of retinal and PE. In the cytoplasm, all-trans-retinal separates from PE and is converted to all-trans-retinol.68,138-140 In cones, N-retinylidene-PE is translocated from the exocytoplasmic leaflet (lumen/extracellular) to the cytoplasmic leaflet, and upon dissociation from PE, all-trans-retinal is reduced to all-trans-retinol by retinol dehydrogenase 8 (RDH8) (Figure 4, left upper panel).141,142 A defective ABCA4 protein leads to the accumulation of A2E in RPE cells which basically consists of all-trans-retinal conjugated to N-retinylethanolamine (N-retinylidene-N-retinylethanolamine; N-retinylidene-PE) and another all-trans-retinal molecule cells,143,144 which is toxic to these cells in higher concentrations.145 In cone and rod photoreceptors, all-trans-retinal is converted to all-trans-retinol (i.e. vitamin A), and absorbed by the RPE cell. In these cells, vitamin A is converted to all-cis-retinal and recycled to be introduced back into the phototransduction cascade.146 Under light conditions cones have a higher demand for vitamin A compared to rods, which can be provided through an alternate visual cycle between the cone photoreceptor cells and the M端ller cells. In the fovea, M端ller cells exist in a 1:1 ratio to cones. In cone-dominated retinas (from ground squirrel and chicken) it was found that all-trans-retinol was isomerized to 11-cis-retinol directly, without the intermediate molecule all-trans-retinyl.147 Recently, Kaylor and colleagues showed that alltrans-retinol is isomerized by dihydroceramide desaturase-1 to 11-cis-retinol, 9-cis-retinol and 13-cis-retinol, of which 11-cis-retinol is subsequently bound to cellular retinal-binding protein (CRALBP). The interphotoreceptor retinoid-binding protein (IRBP) transports 11-cis-retinol to the cone OS. 9-cis-retinol diffuses directly to the interphotoreceptor matrix and into cone OS, and together with 11-cis-retinol is oxized by an unknown retinol dehydrogenase to 9/11-cis-retinal which subsequently is combined with apo-opsin to form a new chromophore.148 Although more knowledge on the cone visual cycle has been gained, compared to the rod cycle challenges remain regarding the differences between the cycles. More studies could possibly address these challenges, potentially revealing novel candidate genes for CD. Semaphorin 4A (SEMA4A) plays a role in endosomal sorting in the RPE (Figure 4, left upper panel). As a response to oxidative stress, SEMA4A acts as a switch converting endosomal sorting in the lysosome to exosomal release, thereby preventing light-induced photoreceptor apoptosis. In the absence of oxidative stress, SEMA4A sorts retinoid-binding proteins with retinoids, a RAB11-facilitated process by which 11-cis-retinal is regenerated and transported back to photoreceptors.149

3.3. Photoreceptor development and structure

During development, photoreceptor specification is initiated by transcription factors OTX2 and RB by influencing the multipotent retinal neuroepithelial cells to exit the cell cycle. It is hypothesized that CRX is the competence factor in photoreceptor precursor cells after mitosis.150 Cells that only express CRX have the fate to become cone precursors, while NRL-expressing cells will develop into rod photoreceptor cells with subsequent expression of NR2E3.150 The transcription factor RAX2 (QRX) is thought to synergistically increase the transactivating function of CRX and NRL and also to interact with CRX.151 Other, as yet unknown transcription factors are necessary to complete the development to mature photoreceptors. Dominant gain-of-function mutations in CRX were found to be a cause of AD CRD,152 by impairing CRX-mediated transcriptional regulation of photoreceptor genes.153 Recessive lossof-function, and dominant mutations in NR2E3 cause enhanced S-cone syndrome, AR RP and rod related syndromes.154-157 The molecular mechanism underlying phenotypes due to NR2E3 mutations involves the absence of the NR2E3 repressor function on cone-specific gene promoters, either by the absence of NR2E3 protein because of nonsense mutations or aberrant

splicing, absence of DNA binding because of mutations in the DNA-binding domain, or impaired co-repressor binding because of mutations in the ligand-binding domain.158 The rod photoreceptor outer segments represent a unique and complex example of specialized sensory cilia by their dense stacks of rhodopsin-laden discs. Disc renewal takes place at the proximal side and it has been estimated that 10% of the rod photoreceptor discs at the distal tips of the OS in humans are removed every day by undergoing phagocytosis by the overlying RPE cells.159 The process of disc morphogenesis is critical for the viability of the photoreceptor cells as shown by several vertebrate models, where blockage of disc morphogenesis resulted in cell death and retinal degeneration.160,161 A relevant difference between cone and rod photoreceptors is the continuity of the disc membranes in cones and the presence of separate discs in rods. The transmembrane protein peripherin/RDS forms homotetramers, or heterotetramers with ROM1. Some heterozygous variants in the gene encoding RDS, PRPH2, are associated with AD cone disorders.162-165 Other heterozygous variants in PRPH2, in combination with heterozygous variants in ROM1, are associated with digenic RP.166-168 These molecules are proposed to play a critical role in disk membrane morphogenesis and to keep the rod discs in a flattened configuration by connecting to similar molecules on the other side of the disc through disulphide bridges (Figure 4, left lower panel). Possibly, RDS has a similar function in cones to flatten the invaginations. PROM1 interacts with protocadherin 21 (PCDH21) and actin filaments, which are also crucial for disk membrane morphogenesis. For ADAM9 it has been suggested that it may be involved in adhesion at the junction of the outer segments (OS) and the RPE. Alternatively, the transmembrane ADAM9 protein may be involved in the remodeling of the extracellular matrix between the RPE and the OS or in the shedding of factors essential for the maintenance of the extracellular matrix.169 FSCN2 and ELOVL4, two genes involved in AD macular degeneration,170,171 are expressed in different parts of the photoreceptor cells and are also thought to have a role in maintaining the structure and/or physiological function of photoreceptor cells.

3.4. Transport processes

The narrow ‘channel’ or ‘conveyer belt’ connecting the inner segment with the outer segment of the photoreceptor cells is called the connecting cilium (CC; see Figure 4, right lower panel). In the process of vision, transport of proteins from the inner segments critically occurs through docking at the basal body (or adjacent membrane) and subsequent transport via the CC towards the outer segment.172 Intraflagellar transport (IFT) complexes tightly regulate the anterograde flow of proteins necessary for the assembly, maintenance and renewal of outer segment discs,173 as well as the retrograde transport of proteins. Several proteins involved in CD have been identified at the CC. These proteins include AIPL1, C8orf37, RAB28, RPGR, RPGRIP1, TULP1, and UNC119. Mutations in RPGR causing COD are localized in ORF15, a highly repetitive region coding for a glutamic acid and glycine-rich C-terminal domain, while mutations causing RP are found throughout the protein. It has been hypothesized that the phenotype is determined by the type of opsin which is mis-localized, depending on the type of mutation.174,175 Alternatively, the genetic background may determine the disease outcome to be cone-dominated or rod-dominated.176 The transition zone protein RPGR has a wide expression, but mutations predominantly affect photoreceptor function, with few cases of hearing loss, sino-respiratory infections and sperm dysfunction.177-180 RPGR binds to RAB8a,181 and via crystal modeling it was proposed that RPGR has an interplay with PDEδ and Arl2/3 to regulate ciliary targeting of farnesylated cargo.182

43 Causes and consequences of inherited cone disorders

1.3

RPGRIP1 is linked to cilium integrity with RPGR via Nek4 serine/threonine kinase183 and localizes exclusively to the photoreceptor CC. Absence of RPGRIP1 in mice resulted in a strongly decreased formation of photoreceptor outer segments and a rapid degeneration of these neurons, leading to vision loss.184 AIPL1 functions as a chaperone of rod PDE6 and interacts with its α-, β-, and γ-subunits 185 and is thereby indirectly also involved in the phototransduction cascade. The cone PDE6 α’ but not the γ subunit carries a carboxy-terminal prenylation motif (as do the rod α and β PDE6 subunits), and it has been hypothesized that AIPL1 also acts as a chaperone for cone PDE6.186 It recently has been shown that AIPL1 is essential for cone PDE6 stability.187 AIPL1 is necessary for proper assembly of the rod PDE6-complex185 and also functions as a chaperone of other farnesylated proteins. The absence of AIPL1 results in disorganized rod and cone outer segments, although presence of viable rod cells may prevent a rapid degeneration.186 Interestingly, null mutations in RAB28 were recently found to be associated with AR CRD.188 RAB28 belongs to a superfamily (>60 members in humans) of small GTPases that fulfill a myriad of intracellular transport functions. Strikingly, RAB28 is uniquely farnesylated at its carboxy terminus, whereas almost all other RABs contain two geranylgeranyl groups for membrane attachment and very few RABs can be farnesylated or geranylgeranylated. The expression of RAB28, as is the case for most RABs, is not restricted to the retina. It is not known why mutations in RAB28 only result in a retinal phenotype. UNC119, also known as Retina Gene 4 protein (RG4), has been shown to be crucial for transducin transport in the absence of light.189 In one mouse model UNC119 can dissociate transducin subunits from each other and the membrane, and subsequently inhibits rhodopsin-dependent activation of transducing.190 Furthermore, UNC119 has the capacity of binding myristoylated proteins, e.g. NPHP3, which is regulated by the small GTPase ARL3.191 Mutations in the gene coding for RIMS1 are a cause of AD CRD, which is characterized by a progressive loss of photoreceptors along with retinal degeneration.192 RIMS1 was found to be essential to short- and long-term synaptic plasticity by affecting the readily releasable pool of vesicles193,194 and RIMS1 proteins were found to be required for normal Ca2+-triggering of exocytosis.195

3.5. Synaptic transduction

A subset of genes associated with CD encode proteins that are localized in the synaptic region, i.e. CACNA1F, CACNA2D4, RIMS1, and UNC119. The transmission of L-glutamate at the photoreceptor synapses to the horizontal and bipolar cells is a calcium-dependent event. Transmitter release is increased with depolarization, as voltage-dependent calcium channels in a depolarized state have an increased capability of channel opening.196 CACNA1F and CACNA2D4 are involved in a continuous calcium-dependent transmitter release and when disturbed cause CD. The reduction of functional calcium-channel densities in synaptic terminals may lead to inefficient photoreceptor-signal transmission and may account for the electronegative ERG.197 The RIMS1 protein plays a role in basic synaptic vesicle release as well as long- and short-term pre-synaptic plasticity.193,194,198 The RIMS1 p.Arg655His missense mutation identified in CRD abolishes the hyperpolarization of current activation, likely resulting in impaired synaptic transmission of ribbon synapses of the visual system.199 UNC119 may also have an important function in photoreceptor neurotransmission as it also is localized at the ribbon synapses formed between photoreceptors and the horizontal and bipolar cells of the retina.200

3.6. Miscellaneous

Several other CD-associated proteins cannot be assigned to one of the five major functionalities of the cone photoreceptor, RPE cells, and Müller cells, and are listed below. Sphingolipids consist of a sphingoid base linked to a variety of polar groups and fatty acid chains.201 CERKL is an endoplasmic reticulum and golgi apparatus membrane associated protein, which seems to be involved in the sphingolipid metabolism due to the shared homology with CERK.202 However its exact role is currently unknown. Knockdown mice showed reduced levels of several sphingolipids, such as glucosylceramide or sphingomyelin. Enzymes in the synthesis/degradation pathway of these sphingolipids are associated with sphingolipidoses, which among other features also can show retinal degeneration.203 Furthermore ceramides are involved in oxidative stress and apoptosis, and it has been shown that CERKL protects from induced oxidative stress in vitro.202 CERKL has been shown to localize in mouse cone photoreceptors and ganglion cells,204-206 and in vitro assays suggest that CERKL interacts with GUCA1A and GUCA1B.207 Morpholino oligonucleotide injected zebrafish showed abnormal eye development with lamination defects, photoreceptor outer segment developmental abnormalities, reduced eye size and increased apoptosis of retinal cells, suggesting that CERKL is relevant for survival and protection of retinal tissue.208 In humans, PITPNM3 is shown to be expressed in the brain, spleen and ovary, although the analysis of expressed sequence tags predicted a wider expression pattern.209,210 The zebrafish homologue of PITPNM3 is predominantly expressed in the inner segments of cone photoreceptor cells.211 In the rat expression however is seen throughout the retina in all cell layers including the inner segment and outer plexiform layers containing photoreceptor terminals with predominant expression in Müller cells.212 Members of the PITP-family play a role in multiple processes such as phospholipase C-mediated inositol signaling, ATP-dependent Ca2+-activated secretion, lipid metabolism, trafficking from Golgi-membranes and exocytosis.213 The importance of a PIT domain was observed in rdgB mutant flies with light-dependent retinal degeneration rescued by transfer of the PIT-domain.214 The signaling pathway for mammalian PITPNM3 is not completely understood.209 Extracellular matrix components or plasma membrane components proteins encoded by BEST1, C1QTNF5, EFEMP1, HMCN1 and TIMP3, are expressed in the RPE. A heterozygous p.Ser163Arg variant in C1QTNF5, encoding the short-chain collagen CTRP5, causes late-onset macular degeneration. It was proposed that mutant CTRP5, which is secreted by the RPE, is unable to fulfill its adhesive property between the RPE and Bruch’s membrane.215 Although the functions of fibulin-3 (EFEMP1) and fibulin-6 (HMCN1) are unknown, interaction of fibulin-3 with TIMP3, an inhibitor of matrix metalloproteinases, has been demonstrated. TIMP3 mutations are a cause of Sorsby maculopathy, a drusen associated dystrophy.216 It has been hypothesized that misfolding and accumulation of mutant fibulin-3 underlies drusen formation although the protein itself is not a major component of the drusen.217 Another component of the rod and cone photoreceptor extracellular matrix is IMPG1 (a.k.a. SPACR). Although its exact function is unknown, IMPG1 could be important in RPE-photoreceptor attachment.218 Alternatively, mutations in IMPG1 lead to impaired metabolism of the photoreceptors and/or RPE resulting in an accumulation of vitelliform deposits in the retina.219 RP1L1 was shown to closely interact with RP1 via its two DCX domains, which functions in the assembly and stabilization of axonemal microtubules.220 Whereas autosomal recessive and dominant mutations in RP1 lead to rod photoreceptor dysfunction, autosomal recessive variants in RP1L1 lead to late-onset RP; heterozygous variants are associated with AD occult macular dystrophy.85,221,222

45 Causes and consequences of inherited cone disorders

1.3

Figure 4 Schematic representation of the roles of the non-syndromic cone disorder-associated proteins in the human retina Schematic representation of five major functionalities of human cone photoreceptor cells (PC), M端ller cells and the RPE. The locations and functions of proteins involved in non-syndromic CDs are depicted in different colors. Proteins which are relevant for the different cellular processes and not mutated in CDs are given in black lettering. Some CD-associated proteins are not shown in this figure either because of their wide-spread localization in the retina (EFEMP1,

FSCN2, HMCN1), or because they localize to structures not shown (TIMP3). Left upper panel) The retinoid cycle taking place in cone photoreceptor cells and the RPE. Upon photoactivation (indicated by a lightning symbol), 11-cis-retinal is converted into all-trans-retinal and dissociates from activated cone opsins, OPN1LW and OPN1MW (activated opsins indicated by an asterix). All-trans-retinal is then recycled to 11-cis-retinal via several enzymatic steps in the RPE. Transport of all-trans-retinal is mediated by ABCA4. Left middle panel) Retinoid cycle in cones and Müller cells. Upon photoactivation, isomerization of 11-cis to all-trans-retinal in a cone opsin takes place. After dissociation, all-trans-retinal is reduced to all-trans-retinol by RDH8 and then released from the cone outer segment into the interphotoreceptor matrix where it is bound by IRBP and taken up by the Müller cells. All-trans-retinol is isomerized by dihydroceramide desaturase-1 (DES1) to 11-cis-retinol, 9-cis-retinol and 13-cis-retinol, of which 11-cis-retinol is subsequently bound to cellular retinal-binding protein (CRALBP). The interphotoreceptor retinoid-binding protein (IRBP) transports 11-cis-retinol to the cone outer segments. 9-cis-retinol diffuses directly to the interphotoreceptor matrix and into cone outer segments, and together with 11-cis-retinol is oxized by an unknown retinol dehydrogenase to 9/11-cis-retinal which subsequently is combined with apo-opsin to form a new chromophore. Left lower panel) Developmental and structural proteins. Three transcription factors mutated in non-syndromic CDs are CRX, NR2E3 and RAX2. Other proteins important in the morphogenesis and structure of the cone photoreceptor cell are EYS, IMPG1, PRPH2, PROM1, and PCDH21. Right upper panel) The phototransduction cascade in cone photoreceptor cells. Upon activation, amplification of the signal is mediated by the α-subunit of transducin and the α’- and γ-subunits of cone phosphodiesterase, encoded by PDE6C and PDE6H. This results in closure of the cGMP-gated channel composed of CNGA3 and CNGB3, a hyperpolarization of the cell and a reduction in glutamate release at the synaptic region. The KCNV2 gene encodes a voltage-gated potassium channel subunit together with other channel components and regulated the potassium current in the cell and affects its excitability potential. PDE6 and transducin subunits are marked as alpha (α), alpha’ (α’), beta (β), and gamma (γ). Right lower panel) CD-associated proteins involved in transport processes in the cone photoreceptor cell. AIPL1 is a chaperone of the putative farnesylated protein PDE6C. Farnesylated proteins are marked with a red zig-zag icon. C8orf37, RAB28, RIMS1, and TULP1, based on immunolocalisation studies act predominantly in transport processes toward and at the base of the connecting cilium, whereas RPGR and RPGRIP1 act throughout the connecting cilium. UNC119 is responsible for ciliary delivery of myristoylated proteins as well as the dissociation of transducin in separate subunits in mice. ATP, adenosine-triphosphate; GTP, guanosine-5’-triphosphate; GDP, guanosine diphosphate; GMP, guanosine monophosphate; cGMP, cyclic GMP; IRBP, interphotoreceptor retinol binding protein, the product of RBP3; MII, metarhodopsin II; P, phosphorylation; RAL, retinal; RE, retinyl esters; REH, retinyl ester hydrolase; ROL, retinol.

4. Mammalian models for cone disorders 4.1. Mouse models for cone disorders

To learn more about the etiology, progression, underlying disease mechanisms, and to explore treatment of CDs, rodent animal models have been used. Rodent models have significantly improved our knowledge of retinal diseases as the disease process can be followed entirely, and invasive studies can be done. The lifespan and development of these animals permit a degenerative process to be followed in a matter of weeks to months also enabling thereby the effects of treatment to be monitored. The largest disadvantage of the mouse as a model for CD is the lack of the cone-dominated region or macula in mice, as well as the fact that humans have genes encoding three different types of cones (long, middle and short wavelengths), whereas the mouse only contains cones sensitive for middle and short wavelengths.215 Therefore, retinal phenotypes

47 Causes and consequences of inherited cone disorders

1.3

may differ significantly between mice and humans. In some cases, mice do not mimic the human phenotype, as is the case for the Adam9,216 Cerkl,196 Gcap1 and Gcap2217; 218 mutant mice, or have not been described in detail for genes like CDHR1, GUCY2D and RIMS1. Secondly, the eye of the mouse differs in size and anatomy from humans hindering gene delivery approaches. Finally, the relatively short life span of a mouse can limit the relevance of the model. The ability to genetically modify mouse models has increased throughout the past decades. Besides genetic modification, a number of spontaneous and chemically induced models have been identified. In Table 2, 42 genes are listed that are associated with non-syndromic CDs. Natural occurring or generated mouse models are available for 31/42 (74%) CD-associated genes, including 33 knockouts, 14 knock-ins, two knockdowns, eight reporter models, three chemically induced, two targeted, two trapped, one floxed and 10 spontaneous mouse models (Table 2). For 10 of the CD associated genes, no mouse model is available. This is also true for the EYS gene, which represents the only CD-associated gene that is absent in the mouse.16; 219 To date mouse model studies have contributed to our understanding of disease, in addition gene therapy has been successfully applied in 10 of these models (Table 2). Compared to rod phototransduction studies in mice, studies in cones have been limited to ERG studies, mainly due to the low percentage of cones (3%) and fragility of the cone outer segments. To further study the mechanism, function and etiology of cones, mouse models were designed to increase the number of cones in the mouse retina.220 The Nrl knockout forces the cell fate of rod photoreceptor cells into cone-like photoreceptors.212-223 In a second model, EGFP was expressed in mouse cones to increase detection and identification of cones,224 and in the third model, rod phototransduction was blocked by a knockout of the rod transducin Îą-subunit (Gnat1 -/-).225

4.2. Other animal models of cone disorders

Compared to small animal models like mouse or rat, larger animal models have a few advantages. Anatomically, the retinas of dogs and cats are more similar to those of humans, as they have a cone-rich area, which is similar to the macula. Chicken have a cone-rich retina in which cones outnumber the rods at 6:1, while in humans the cone-rod ratio is 1:20. Chicken cones have a higher density in the rod-free region analogous to the human fovea.234,235 Chicken also have retinal glia (MĂźller) cells, a fully sequenced genome and relatively low maintenance costs. The size of the eyes makes the chick especially suitable for experimental manipulation.236 Next to the cone-rich retina of chickens, pigs are also equipped with a cone-dominated retina. Although no naturally occurring cone disease related defects have been discovered in pigs, Mussolino et al (2011) showed that the porcine retina is suitable for developing novel AAV-mediated therapies using AAV2/5 or AAV2/8 to transduce both RPE and photoreceptor cells without safety issues.237 Xenopus laevis is an ideal animal model to study the role of the interphotoreceptor matrix in RPE-photoreceptor interactions,238 due the fact that 30% of its photoreceptors are cones, and because they have relatively large photoreceptors, which is helpful in cellular, transgenic and imaging studies.239 The Xenopus genome is available240,241 and strategies using morpholino oligonucleotide antisense knock-down offer a powerful reverse genetics approach.242 Currently there are only few large animal models which are suitable for investigating inherited CDs. Canine models with gene mutations in ADAM9, BEST1, CNGB3, RPGR and RPGRIP1, are currently used to develop therapy models. In three dog models the retinal degeneration was successfully treated30,243,244 (Table 2). A great potential in the discovery of novel canine models for CDs resides in the several hundred dog breeds affected by progressive retinal atrophy with a currently unidentified gene defect (RWJ Collin (Nijmegen), K. Stieger (Giessen) and H. Lohi, (Helsinki), personal communications). Other large animal models for CD are sheep, which carry a spontaneous nonsense mutation in CNGA3 to investigate ACHM,245 and a feline model with a single nucleotide deletion in CRX.246

Cones and rods

RPE

RPE, lens and ciliary Trapped, KI, KI, epithelium reporter**

Cones and rods

ONL and INL

Ubiquitously

Cones and rods

Cones

Cones and rods

RPE

Endoplasmatic reticulum

Cones and rods

AD-CRD, AR-LCA

AD-MD, AR-Bestrinopathy

AD-MD

AR-CRD, AR-RP

XL-CRD, XL-CSNB

AR-COD

AR-CRD, AR-RP

AR-ACHM, AR-COD

AR-ACHM, AR-COD, AR-CRD

AD-CRD, AR- AD- de novo LCA, AD-RP

AD-MD

AR-CRD, AR-RP

AIPL1

BEST1

C1QTNF5

C8orf37

CACNA1F

CACNA2D4

CDHR1

CERKL

CNGA3

CNGB3

CRX

EFEMP1

ELOVL4

EYS

286-291

283-285

280; 281

277

122; 274

204; 268

271; 272

270

266-268

263-265

259; 260

253-255

224

68; 247

Pig

Cat

Dog

Sheep

Rat

Dog

292

246

278; 279

245

269

261

251; 252

Dog

Mouse

Photoreceptor morphogenesis and maintenance

Catalyzing very-long-chain fatty acid synthesis

Unknown

Transcription factor

Subunit of cGMP-regulated Mouse, cation channel dog

Subunit of cGMP-regulated Mouse cation channel

Sphingolipid metabolism

282

30; 276

275; 276

262

256-258

248-250

Gene Ref. therapy gene model therapy

Involved in the assembly of OS discs

Calcium channel

Transport across the CC

Unknown

Calcium channel

PDE chaperone

Outer segment-RPE junction protein

All-trans-retinal flippase

Ref. Other Ref. mouse model animal model animal model Potential function

No mouse ortholog -

KO, KO, KI, KI, reporter,targeted

KI, KO**, KI, KO

KO, chemically induced

KO, spontaneous

KD, KD

KI, KO

Spontaneous

KO, spontaneous

KI, KO

RPE

AR-CRD

ADAM9

Mouse model KO

Expression site

AR-COD, AR-CRD, AR- Cones and rods RP, AR-STGD1

Gene

ABCA4

Associated phenotypes

Table 2 Non-syndromic cone disorder genes, their associated human phenotypes, animal models, and gene therapy studies A thorough literature search resulted in estimates of the current frequency and genetic causes in CD. Corrections were made for patient cohorts reported repetitively.

1.3

Causes and consequences of inherited cone disorders

Cones

Cones and rods

RPE

Interphotoreceptor space

Cones and rods

Neural retina

Cones

Retina (rat)

Ubiquitously

Cones and rods

Ubiquitous

AR-ACHM

AD-COD, AD-CRD

AD-MD, AD-RP

AD-CRD, AR-CRD, AR-LCA

AD-MD

AR-CDSRR

AR-ESC, AR-RP

XL-COD

AR-ACHM, AR-COD

AD-CRD

AD-STGD, AR-CRD

AD-CRD, AD-MD, AD-RP,

AR-CRD

FSCN2

GNAT2

GUCA1A

GUCA1B

GUCY2D

HMCN1

IMPG1

KCNV2

NR2E3

OPN1LW

OPN1MW

PDE6C

PDE6H

PITPNM3

PROM1

PRPH2

RAB28

293; 294

225; 298

297-299

KI, spontaneous**, chemically induced

Reporter, KI, KO, reporter

Spontaneous

KO, spontaneous

316; 317

312-315

311

310

308; 309

Reporter, reporter, 301-303 KO

KO, KO

KI, KO, KO

Rhesus macaque

Pig, zebrafish

307

304; 305

Mouse

Transport towards the CC -

Membrane morphogenesis Mouse

Photoreceptor membrane morphogenesis

Membrane turnover of photoreceptor cells (transport)

Phosphodiesterase subunit γ

Phosphodiesterase subunit α’

Green light sensitive opsin

Red-light sensitive opsin

Transcription factor

Ion channel subunit

Unknown

Generation cGMP

Ca2+-activated interactor of GUCY2D

Subunit of cone transducin Mouse

311; 312

306

300

296

Gene Ref. therapy gene model therapy

Actin structure assembly of CC plasma membrane; photoreceptor disc formation

Ref. Other Ref. mouse model animal model animal model Potential function

Spontaneous, spon- 268; 295 taneous

Spontaneous, KO, KO

Cones and rods

AD-MD, AD-RP

Gene

Mouse model

Expression site

Associated phenotypes

Table 2 (continue)

Cones and rods

Ubiquitous

AD-MD

XL-CRD, XL-RP

AR-CRD, AR-LCA

AD-CRD, AD-RP

Cones and rods

Reporter**, KO, KO**, KO, KO, KI

KO, trapped

KO, chemically induced**

Reporter, targeted, spontaneous

332

328-331

326; 327

175

174; 321; 322

220

Dog

Dog, dog

324

323

Mouse, dog

Dog

Transport towards the CC -

Extracellular matrix remodelling

Interactor of CRBP1 and endosomal sorting

Transport across the CC, intracellular trafficking

Transport across the CC

Stabilization of microtubules

Neurotransmittor release and synaptic plasticity

Transcription factor

237; 318

236

Gene Ref. therapy gene model therapy

*, mode of inheritance is unknown; **, Mouse Genome Informatics direct data submission; ACHM, achromatopsia; AD, autosomal dominant; AR, AD-CRD autosomal recessive; Neural retina KO 333 protocadherin - cone dystrophy Transport across the CCrod response; CD, cone dystrophy; CDHR1, encodes 21; CDRSS, with supranormal CRD, conerod dystrophy; CC, connecting cilium; CSNB, congenital stationary night blindness; ESC, enhanced S-cone syndrome; KD, knockdown; KI, knock-in; KO, knockout; LCA, Leber congenital amaurosis; MD, macula dystrophy; RAX2, retina and anterior neural fold homeobox 2, encodes QRX; RP, retinitis pigmentosa; RPE, retinal pigment epithelium; OS, outer segment; XL, X-linked.

AR-COD, AR-CRD, AR-RP

AD-Sorsby fundus dystrophy

RPE

Presynaptic ribbons KO, KI**, KO, floxed 194; 320 in retina and brain

AD-CRD

ONL and INL

*CRD

Mouse model

Expression site

Ref. Other Ref. mouse model animal model animal model Potential function

Associated phenotypes

Causes and consequences of inherited cone disorders

1.3

5. Therapeutics for cone disorders

Currently, to (partially) restore gene function in CD, several strategies are being applied with the use of animal models. A specific vector is always necessary to deliver the target gene into the cell, as naked DNA is not able to enter the cell efficiently. The success of retinal gene augmentation is dependent on the vector and delivery method. Vector choice depends on the size of the cDNA of the relevant gene, the type of targeted cells, stability of expression and immunogenicity. Although the eye is a complex organ, the advantages of the eye are its immune privileged nature, its small size, accessibility, and functional assessment. Adeno-associated viruses (AAVs) have been successfully used as vectors for gene therapy. AAVs enable the incorporation of a healthy copy of the gene that is defective in the patient. An advantage of this technology is its low toxicity due to the absence of the original viral coding sequences as shown in its application in muscle, brain, liver, lung, RPE and the neural retina. A restriction of this technology is its limited cargo size, only enabling the transport of cDNAs of genes up to 4.7 kb, and therefore potentially are thereby able to accommodate 36 of the 42 CD-associated genes (see Table 3). Putative immune responses to the viral capsid may also limit recurrent treatments. In Cnga3 -/- mice, AAV5-mediated gene replacement therapy restored cone function and thereby visual processing. The non-functional cones were capable of producing photoreceptor responses and transferring the generated signals to the bipolar cells. Treated mice showed normalized levels of cGMP in the cones as well as a delayed cone cell death, and the inflammatory response typically seen in retinal degeneration was reduced. 31,334 Treatment was successful when the subretinal injections were performed in 1- and/or 3-month-old knockout mice, and the therapeutic effect was still present after 8 months. Improved cone function using the AAV5 vector was confirmed in another Cnga3 study.276 A similar strategy was used to treat a deficiency of CNGB3-associated ACHM. In two canine models it was shown that long-term restoration of cone function and diurnal vision could be achieved by subretinal delivery of AAV5 vectors expressing human CNGB3 under the control of a 2.1 kb human red cone opsin promoter.30 The same authors were also able to circumvent the unexplained reduction in therapeutic effect after 54 weeks in dogs by administration of ciliary neurotrophic factor (CNTF). Administration of this neuroprotective and axogenic factor for retinal ganglion cells however led to immature photoreceptors with shorter outer segments and reduced gene expression. This effect was reversible with photoreceptors returning to expected developmental maturity and function thereafter. This approach to photoreceptor replacement enabled successful gene therapy in dogs up to 42 months. 335 As the treatment with CNTF in the dog model showed measurable phenotypic improvements, the first clinical trials in humans with CNGB3 mutations using intraocular implants releasing CNTF are currently ongoing (http://clinicaltrials.gov/ct2/show/NCT01648452?term=achromatopsia&rank=1). Long-term improvement of retinal function was also found in a study using AAV8-mediated therapy. In both M- and S-cones, the Î˛-subunit of the CNG-channel could be detected after subretinal AAV8 vector injection. In addition, increased levels of the Îą-subunit of the CNGchannel were found, reduced cone death, improved density and a positive effect of the outer segment structure of the cone and subcellular compartmentalization of the cone opsins.33

The current status of treatment of CNGA3 and -B3-associated channelopathies in animal models have shown solid and encouraging results and hold great promises for future treatment of patients with similar defects. 336 A human Phase 1 trial for patients with CNGA3 mutations is being developed (B. Wissinger, personal communication). The human situation however may be more complex and limitations may occur due to the age-dependent results for CNGB3, which argues to treat only younger patients. Possibly, the same or a similar treatment with CNTF could create treatment opportunities for older patients too. In GNAT2 knockout mice, AAV-mediated therapy has also proven to be successful.296 However, for those genes in which AAV-mediated therapy is not feasible due to their size, other strategies need to be developed. The ABCA4 open reading frame (ORF) measures 6.8 kb and therefore an alternative approach using the nanoparticle (a.k.a. non-virus) mediated therapy has been applied. Compacted DNA nanoparticles were constructed with the human ABCA4 cDNA and the IRBP or mouse opsin promotor. Nanoparticles have a high transfection efficiency in post-mitotic cells, are biodegradable, and cause minimal toxicity even after repeated doses to the eye, lung and brain.248 and refs therein This non-viral approach has been performed subretinally on Abca4-/- mice. ABCA4 transgene expression was identified up to 8 months after injection resulting in significant structural and functional rescue of Stargardt disease, with improved dark adaptation recovery and reduced lipofuscin granules in the RPE.248 These results suggest that nanoparticle-mediated gene delivery has the potential to target many diseases caused by mutations in large genes. Alternatively, lentiviral vectors can be used337 which can accommodate cDNAs up to ~10 kb. This strategy is currently also used for the development of ABCA4 gene therapy. Equine infectious anemia virus (EIAV)-derived lentiviral vectors expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters were subretinally injected in Abca4-/- mice. High transduction efficiency of rod and cone photoreceptors and a significant reduction of accumulation of A2E were observed in these mice suggesting the potential of lentiviral gene therapy in treating ABCA4-associated maculopathies.249 Similar results in rabbit and macaque were shown in the study by Binley et al. (2013). In this study â&#x20AC;&#x2DC;StarGenâ&#x20AC;&#x2122; was used, a lentivirus vector containing a normal copy of the human ABCA4 gene, which is currently in a Phase I/IIa stage with individuals affected with STGD1 (http://clinicaltrials.gov/ct2/show/NCT01367444?term=abca4 &rank=6). 338 One of the challenges for gene therapy is the limited time window to achieve a successful treatment outcome. Some CDs exhibit a fast progression of disease and therefore the window of opportunity of treatment is smaller compared to slow or non-progressive phenotypes. Hence, treatment opportunities may expand beyond our initial expectations now that SD-OCT and adaptive optics may determine the viability of the cone cell bodies, which have shown the capability of being reactivated in mice.339 In persons with very early onset CD, gene augmentation therapy however should ideally start as early as possible.

53 Causes and consequences of inherited cone disorders

1.3

Table 3 Non-syndromic cone disorder genes, their human open reading frame sizes, the mouse model names, synonyms, and the retinal phenotype description Gene

ORF (bp)

Model name**

Synonym**

Retinal phenotype

ABCA4

2,273

6,819*

tm1Ght, tm1Kpal

Abcr-, Abca4-

Delayed rod dark adap

ADAM9

819

2,457

tm1Bbl

mdc9-

No retinal phenotype

AIPL1

384

1,152

tm1Mad, tm1Tili, tm1Visu

Aipl1 , Aipl1

BEST1

605

1,815

tm1.1Amar, tm1Lmar

Best1W93C,Vmd2-

Altered eye electrophy

C1QTNF5

243

3,914

Gt(OST522586)Lex, tm1.1Geno, tm1.1Itl, tm1.1(KOMP)Vlcg, rd6, rdx

C1qtnf5Ser163Arg, C1qtnf5S163R, C1qtnf5tm1.1Igl, CTRP5S163R, Mfrp174delG

Increased retina vascul zygous for the same kn

C8orf37

208

624

CACNA1F

1,977

5,931*

tm1.1Sdie, tm1Ntbh, nob2

Cav1.4 , nob2, Cacna1f Cacna1fG305X

CACNA2D4

1,137

3,411

lob

Severe loss of retinal si

CDHR1

859

2,577

tm1(cre)Kbal, tm1Nat

Pcdh21/Cre, prCAD-

Progressive degenerati

CERKL

558

1,674

tm1.1Geno

No photoreceptor phe potentials

CNGA3

676

2,028

tm1Biel, cpfl5

CNG3-

Progressive loss of con

CNGB3

809

2,427

tm1Dgen

Cone degeneration and

CRX

299

897

tm1Clc, tvrm65

Crx-

A lack of photorecepto pineal-specific genes, an

EFEMP1

494

1,482

tm1Eap, tm1Lex, tm1Lmar, tm2Lmar

Efemp1R345W, Efemp1ki

Knock-out mice display epithelium

ELOVL4

315

945

tm1Kzh, tm1Rayy, tm1Sie, tm1Wked, tm2Kzh

Elovl4del, E_mut-, Elovl4270x

Knock-out mice die be abnormalities

EYS

3,144

9,432*

FSCN2

517

1,551

ahl8, tm1Sykk, tm2Sykk

Fscn2

GNAT2

354

1,062

cpfl3, c.518A>G

Progressive degenerati

GUCA1A

202

606

tm1.1Hunt, tm1Itl, tm1Jnc

COD3, GCAP-, GCAP1-GCAP2-, GCAPs-

Photoreceptor degene

GUCA1B

201

603

tm1Amd, tm1Jnc

GCAP-, GCAP1-GCAP2-, GCAPs-

Abnormal rod electrop

GUCY2D

1,103

3,309

tm1Mom, tm1Sdm

GCD-ITL, Gucy2d-

Not analyzed for retina

HMCN1

5,636

16,908*

IMPG1

798

2,394

KCNV2

545

1,635

NR2E3

410

1,230

tm1Dgen, rd7

Rosettes and a reduced

OPN1LW

364

1,092

OPN1MW

364

1,092

tm1(OPN1LW)Nat

Opn1mw , R

A knock-in allele encod female mice with a ~45

PDE6C

858

2,574

cpfl1

Abnormal cone photor

PDE6H

252

PITPNM3

974

2,922

PROM1

865

2,595

tm1.1(DTA)Toko, tm1Pec, tm1(cre/ ERT2)Gilb, tm1Rafi

Prom1lacZ,DTA, Prom1C-L

Abnormal retina morp

Complete retinal degen tion and impaired ERG

nob2

deltaEx14-17

Impaired eye electroph paired retinal synapse m

R109H

, Fscn2 neo(-), Fscn2 neo p

Retinal generation with with age

Retinal phenotype description**,+ Delayed rod dark adaptation. Model for juvenile macular degeneration No retinal phenotype Complete retinal degeneration and lack of ERG responses. Hypomorphic mutants display less severe retinal degeneration and impaired ERG responses

Altered eye electrophysiology

C1qtnf5S163R, C1qtnS163R , Mfrp174delG

Increased retina vasculature leakage, retinal degeneration, and features of late-onset macular degeneration Mice homozygous for the same knock-in generated by a different group are normal -

2, Cacna1f

deltaEx14-17

Impaired eye electrophysiology, abnormal retinal neuronal layer, bipolar cell, and horizontal cell morphology, and impaired retinal synapse morphology Severe loss of retinal signaling associated with abnormal photoreceptor ribbon synapses and cone-rod dysfunction Progressive degeneration of retinal photoreceptor cells and a slight reduction in light responses

rCAD-

No photoreceptor phenotype, retinal apoptosis and decreased amplitudes and increased implicit time of oscillatory potentials Progressive loss of cone photoreceptor cells Cone degeneration and decreased photopic response A lack of photoreceptor outer segments and rod and cone activity, reduced expression of several photoreceptor- and pineal-specific genes, and altered circadian behavior

emp1ki

Knock-out mice display a normal phenotype. Homozygous R345W mice develop deposits below the retinal pigment epithelium

t-, Elovl4270x

Knock-out mice die before or around birth. Mice heterozygous for a null allele breed poorly and display mild retinal abnormalities -

n2 neo(-), Fscn2 neo p

Retinal generation with structural abnormalities of the outer segment and depressed rod and cone ERGs that worsen with age Progressive degeneration of photoreceptors and normal ERG responses

, GCAP1-GCAP2-,

Photoreceptor degeneration and loss of cone and rod function

1-GCAP2-, GCAPs-

Abnormal rod electrophysiology

y2d-

Not analyzed for retinal phenotype

rom1C-L

Rosettes and a reduced number of nuclei in the retinal outer nuclear layer A knock-in allele encoding a derivative of the human red cone pigment results in hemizygous male and homozygous female mice with a ~45-nm red shift in retinal sensitivity Abnormal cone photoreceptor function Abnormal retina morphology, vasculature, and electrophysiology

55 Causes and consequences of inherited cone disorders

1.3

Table 3 (continue) Gene

ORF (bp)

Model name**

Synonym**

Retinal phenotype d

PRPH2

346

1,038

tm1Nmc, rd2, Nmf193

Prph2delta307, rds-307, Prph2Rds, Rd-2, rds, rds-, Rds, RdsRd2

Slow retinal degenerat increased numbers of M

RAB28

221

663

RAX2/QRX

184

552

RIMS1

1,692

5,076*

tm1Sud , tm3.1Sud, tm2Sud, tm3Sud

RP1L1

2,401

7,203*

tm1Jnz

RPGR

1,152

3,456

tm1Tili, tm1Wbrg, rd9

RPGR-, Rpgrdeltaexon4

Ectopic location of con photoreceptors by 2-6

RPGRIP1

1,286

3,858

tm1Tili, nmf247

RPGRIP-

Photoreceptor cell dys

SEMA4A

761

2,283

tm1Kik, Gt(OST393408)Lex

Sema4aGt22Lex

Severe retinal degenera receptors

TIMP3

212

636

tm1.1(KOMP)Vlcg, tm1Hest, tm1(KOMP)Vlcg, tm1Osya, tm1Rkho, tm1Web

timp-3-, Timp-3-null, Timp3S156C

Knock-out mice die pr branching, reduced alve for Sorsby fundus dystr

TULP1

542

1,626

tm1Pjn

Tulp1-

Retinal degeneration

UNC119

240

720

tm1Gina

RIM1alpha-, RIM1alphabeta-, RIM1 S413A-KI, fRIM1, RIM1alphabetafloxed

No retinal phenotype

Knock-out mice exhibi duced ERG amplitudes

Retinal degeneration c progresses rapidly afte

*ORF sizes in italics are larger than 4.7 kb and therefore not suitable targets for adeno-associated virus-therapy. **Data collected from MGI Jackson lab, +phenotype among different models can vary; aa, amino acids; bp, basepair; ERG, electroretinogram; ORF, open reading frame.

6. Future perspectives 6.1. Identification of CD-associated genes

WES, combined with homozygosity mapping or linkage analysis, has proven successful in the discovery of novel retinal disease genes. 340-342 In a recent Saudi Arabian study of predominantly AR inherited retinal dystrophies, ~80% of DNA samples analyzed using homozygosity mapping and/or WES were solved by the identification of mutations in either known or novel genes. 343 In six of 86 families with causal variants, mutations in different new genes were found. This suggests that most of the undiscovered CD genes (Figure 3) are mutated in very low percentages of affected individuals. In the next few years, WES and whole genome sequencing (WGS) will facilitate the identification of novel gene defects in individuals for which WES could not identify the pathologic defect. WGS can robustly identify heterozygous deletions, duplications, inversions, and intronic variants. 344 However, proving causality of rare variants in candidate CD genes will be challenging, as in most cases only few family members are available for segregation analysis. This may be partially solved by testing large patient cohorts through collaborative efforts. In addition, the causality of novel variants can be tested through cellular and animal model studies.

Retinal phenotype description**,+

s-307, Prph2 , Rd-2, RdsRd2 Rds

M1alphabeta-, RIM1 M1, RIM1alphabetafloxed

eltaexon4

-null, Timp3S156C

Slow retinal degeneration with thinning and loss of the outer nuclear layer, loss of photoreceptor outer segments, and increased numbers of M端ller cells No retinal phenotype Knock-out mice exhibit retinal photoreceptor abnormalities, including scattered outer segment disorganization, reduced ERG amplitudes, and progressive retinal rod cell degeneration Ectopic location of cone opsins, reduced levels of rhodopsin in rod cells, and partial degeneration of both cone and rod photoreceptors by 2-6 months of age Photoreceptor cell dysmorphology. By 3 months of age mutant animals show near complete loss of photoreceptor cells Severe retinal degeneration with reduced retinal vessels, depigmentation and dysfunction of both rod and cone photoreceptors Knock-out mice die prematurely with lethargy, ruffled hair, and a hunched posture, displaying impaired bronchiole branching, reduced alveologenesis and abnormal mammary gland involution. S156C knock-ins provide a mouse model for Sorsby fundus dystrophy Retinal degeneration Retinal degeneration characterized by thinning of the outer nuclear layer of the retinal that is visible at 6 months and progresses rapidly after 17 months to end-stage by 26 months

6.2. Molecular diagnostics

Up to a few years ago it was not possible to, stabilize or improve impaired vision in persons with CD. With the emerging options for treatment of CD, early molecular diagnostics has become highly relevant. In addition, early molecular diagnosis may be important in cases in which genetic defects are associated with extra-ocular features, some of which may develop later in life and be amenable to pre-symptomatic management, e.g. in nephronophthisis, in which the kidney is involved.

6.3. Transcriptomics

Introns are riddled with rare variants that theoretically can have an impact on RNA splicing if they are situated in an intronic splice enhancer or inhibitor site, or if they increase the splicing potential of a cryptic splice site, leading to the transcription of an aberrant mRNA product. The latter was recently shown for ABCA4, as a few deep intronic variants led to aberrant splicing in persons with STGD1.345 Farkas et al (2013) identified 79,915 novel alternative splice events and more than one hundred novel genes expressed in human retinal tissue, which will aid the difficult task of discriminating benign from causal variants. 15-36% of the novel splicing events resulted in mRNAs with an open reading frame and are likely be transcribed into protein coding transcripts. 346 This demonstrates that the retinal transcriptome is more compli-

57 Causes and consequences of inherited cone disorders

1.3

cated than previously thought, and the knowledge of naturally occurring splice events may aid the interpretation of RNA analysis of known retinal disease genes in patients. Importantly, only approximately 35% of the genes involved in retinal dystrophies are expressed at high enough levels in lymphoblastoid cells to scrutinize the transcriptome from an individualâ&#x20AC;&#x2122;s blood sample (K. Neveling, unpublished data). Alternatively, photoreceptor precursor cells can be differentiated from patientâ&#x20AC;&#x2122;s keratinocytes through generation of induced pluripotent stem cells (iPSC). 347 iPSCs can be generated from adult somatic cells by expressing a set of transcription factors, and subsequently can be differentiated into retinal cells.348 and refs therein. RNA analysis of such cells is likely to more closely mimic the situation in the diseased tissue, enabling the detection of splice defects that might not be detectable in lymphoblastoid cells. The generation of a homogeneous population of fully differentiated cells remains a challenge, in particular regarding the neural retina.

6.4. Alternative cone rescue strategies

A great promise in treating early onset degenerative diseases, like CD, are cell replacement therapies using retinal progenitor cells derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). iPSCs can be generated from adult somatic cells by expressing a set of transcription factors, and subsequently can be differentiated into retinal cells. 349 These cells could be very valuable in cell-based therapies. Proof of concept using mouse retinal progenitor cells harvested in a specific developmental time window (postnatal day 4-8) was obtained recently.349 It is critical to transplant a sufficient number of cells that can populate a significant fraction of the diseased retina. The transplanted cells differentiated into adult photoreceptor cells, were positioned at the correct location, and formed connections with bipolar cells, 349-352 demonstrating the potential of this approach to rescue retinal function in future clinical trials.

7. Conclusions

The early aim of CD research has been the identification of the genetic bases of CD. Advances in this field compounded by technologies such as WES and WGS technologies have created a new era for molecular diagnostics in CD. In the next few years, the remaining genetic causes of CD will most likely be elucidated representing an enormous achievement. The prevalence of novel genetic defects however will be very low, and in particular regarding rare missense variants, functional modeling of their effects in animal models or cellular systems will be crucial to understand their mode of action. The ultimate aim remains the development of therapies that rescue cone photoreceptors. For the 31/42 CD-associated genes in which mammalian models are available, 14 have successfully been used for gene augmentation studies and a few human trials are underway. Nevertheless, large naturally occurring animal models, In particular canine models with progressive retinal atrophy, need to be identified for CDs as they can more closely mimic the human diseases and are a more relevant model with respect to the development of gene therapy. The rescue of cone photoreceptors not only is important to treat cone dysfunctions, but also will be crucial to maintain vision in rod-dominated diseases.

Acknowledgements

We would like to thank Louise F. Porter for critically reading this manuscript. This study was financially supported by the Foundation Fighting Blindness USA (grants BR-GE-051004890RAD, C-GE-0811-0545-RAD01) to A.I.d.H., and F.P.M.C., the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, the Gelderse Blinden Stichting, the Landelijke Stichting voor Blinden en Slechtzienden, the Stichting Blinden-Penning, the Stichting Macula Degeneratie fonds, the Rotterdamse Stichting Blindenbelangen (to F.P.M.C. and C.C.W.K.). None of the authors have any financial interest or conflicting interest to disclose.

Supplemental tables 1-5 A thorough investigation of all public available literature resulted in estimates of the current frequencies and genetic causes of CD. Corrections were made for patient cohorts reported repetitively. Supplemental Table 1 Occurrences and frequency of mutations in achromatopsia Study

CNGA3

CNGB3

GNAT2

353

12/36

0/36

3/63

55/63

0/63

354

4/16

8/16

2/16

123

9/22

355

4/9

5/9

356

3/16

10/16

357

358

9/19

5/19

121

126

359

1/5

360

113

3/13

114

53/162

361

1/14

362

9/11

363

163/256

115

5/77

116

364

365

PDE6C

119

6/26

118

3/10

120

7/77

117 Overall mutation frequency

PDE6H

1/217 165/407 (35.5%)

273/449 (58.7%)

10/195 (2.2%)

16/113 (3.3%)

1/217 (0.2%)

59 Causes and consequences of inherited cone disorders

1.3

Supplemental Table 2 Occurrences and frequency of mutations in autosomal recessive cone dystrophy PDE6H

Study

ABCA4

8/90

366

367

4/18

197

CACNA2D4

CNGA3

CNGB3 3/90

KCNV2 PDE6C

PDE6H

1/34

114

3/48

368

3/60

131

10/11

369

370

132

371

4/154

118

1/104 1/159

372* 373 Overall mutation frequency

TULP1

4/90

1 13/109 (3.9%)

1/34 (0.3%)

3/48 (0.9%)

10/154 (3.0%)

44/277 (13.1%)

1/104 (0.3%)

*Roosing et al. (2013) only analyzed one variant of TULP1 in this cohort.

1/217 1/217 (0.2%)

1/1 (0.3%)

1/159 (0.3%)

Supplemental Table 3 Occurrences and frequency of mutations in autosomal recessive cone rod dystrophy Study 374â&#x20AC; 41 66 375 40 376 377 378 379 380 381 382 383 384 385 386 387 388 366 389 367 390 391 392 34 358 393 169 340 394 395 396 397 398 399 400 401 402 403 404 188 405 372* Overall mutation frequency

ABCA4 33/123 1/47 1 13/55 13/20 4/8 11/37 5/8 2 1 16/30 18/54 1 1 2 1 1 2/14 3 1 16/46 3 1 1/5 17/65 16/34 26/86

ADAM9

C8orf37

CDHR1

CERKL 2/123

CNGB3 3/47

4 4 1 2 1 7/21 1 3

176/542 (15.7%)

4/4 (0.4)

4/4 (0.4%)

11/25 (1.0%)

3/47 (0.3%)

61 Causes and consequences of inherited cone disorders

1.3

Supplemental Table 3 (continue) CNGB3 3/47

3/47 (0.3%)

Study 374â&#x20AC; 41 66 375 40 376 377 378 379 380 381 382 383 384 385 386 387 388 366 389 367 390 391 392 34 358 393 169 340 394 395 396 397 398 399 400 401 402 403 404 188 405 372* Overall mutation frequency

CRB1

CRX

2/29

1/29

EYS 1/123

FSCN2

GUCY2D

KCNV2 1

29/186 1/47 1

2/29 (0.2%)

1/29 (0.1%)

29/186 (2.6%)

1/47 (0.1%)

1/1 (0.1%)

CRB1

2/29

CRX

EYS 1/123

FSCN2

GUCY2D

KCNV2 1

1/29 29/186 1/47 1

2/29 (0.2%)

1/29 (0.1%)

29/186 (2.6%)

1/47 (0.1%)

1/1 (0.1%)

63 Causes and consequences of inherited cone disorders

1.3

Supplemental Table 3 (continue) KCNV2 1

1/1 (0.1%)

Study 374† 41 66 375 40 376 377 378 379 380 381 382 383 384 385 386 387 388 366 389 367 390 391 392 34 358 393 169 340 394 395 396 397 398 399 400 401 402 403 404 188 405 372* Overall mutation frequency

PDE6C

PROM1 1/123

RAB28

RPE65

2/47

RPGRIP1

TULP1

1/47

1/29

0/29

1 2/617 2 2/47 (0.2%)

1/1 (0.1%)

2/617 (0.2%)

1/29 (0.1%)

3/76( 0.3%)

1/91 1/91 (0.1%)

† Littink et al. (2010) was excluded in the statistics. In this study the samples had been scanned with homozygosity mapping only without the addition of Sanger sequencing, *Roosing et al. (2013) only analyzed one variant of TULP1 in this cohort.

Supplemental Table 4 Occurrences and frequency of mutations in autosomal dominant cone and cone-rod dystrophy Study 406 162 407 408 400 409 410 411 412 413 414 415 152 416 417 418 419 420 421 422 423 424 425 426 133 427 134 428 429 135 430 431 432 433 434 136 435 436 437 438 439 209 440 441 442 163 164 165 443 444 200 Overall mutation frequency

AIPL1 1/15

CRX

GUCA1A

GUCY2D

1/7 3 1 1/29 1 0/88 4/67 1 1 3/30 4 2/30 1 1

3/22

3/7

PITPNM3

1.3

1 3 1 1/119 1 1 1 1 4/24 4 1 1 1 4 3/40 2 2 3/38 1 1 11/27 1 2/22 1 1 1 2 5/163

1/15 (0.2%)

24/260 (4.3%)

17/173 (3.0%)

43/155 (7.7%)

7/165 (1.2%)

65 Causes and consequences of inherited cone disorders

Supplemental Table 4 (continue) PITPNM3

2 5/163

7/165 (1.2%)

Study 406 162 407 408 400 409 410 411 412 413 414 415 152 416 417 418 419 420 421 422 423 424 425 426 133 427 134 428 429 135 430 431 432 433 434 136 435 436 437 438 439 209 440 441 442 163 164 165 443 444 200 Overall mutation frequency

PROM1

PRPH2

RIMS1

SEMA4A

UNC119

2/22

5 3 4 1 3 1 4 8/8 (1.4%)

10/30 (1.8%)

1/1 (0.2%)

4/4 (0.7%)

1/20 1/20 (0.2%)

Supplemental Table 5 Occurrences and frequency of mutations in X-linked cone and cone-rod dystrophy Study

CACNA1F

445

OPN1LW/OPN1MW

RPGR

1.3

446

447

448

2/6

449

7/8

358 Overall mutation frequency

2/10 1/1 (2.78%)

3/3 (8.34%)

19/32 (52.8%)

67 Causes and consequences of inherited cone disorders

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406. Sohocki, M.M., Perrault, I., Leroy, B.P., Payne, A.M., Dharmaraj, S., Bhattacharya, S.S., Kaplan, J., Maumenee, I.H., Koenekoop, R., Meire, F.M., et al. (2000). Prevalence of AIPL1 mutations in inherited retinal degenerative disease. Mol. Genet. Metab. 70, 142-150. 407. Huang, L., Xiao, X., Li, S., Jia, X., Wang, P., Guo, X., and Zhang, Q. (2012). CRX variants in cone-rod dystrophy and mutation overview. Biochem. Biophys. Res. Commun. 426, 498-503. 408. Itabashi, T., Wada, Y., Sato, H., Kawamura, M., Shiono, T., and Tamai, M. (2004). Novel 615delC mutation in the CRX gene in a Japanese family with cone-rod dystrophy. Am. J. Ophthalmol. 138, 876-877. 409. Kitiratschky, V.B., Nagy, D., Zabel, T., Zrenner, E., Wissinger, B., Kohl, S., and Jagle, H. (2008). Cone and cone-rod dystrophy segregating in the same pedigree due to the same novel CRX gene mutation. Br. J. Ophthalmol. 92, 1086-1091. 410. Rivolta, C., Peck, N.E., Fulton, A.B., Fishman, G.A., Berson, E.L., and Dryja, T.P. (2001). Novel frameshift mutations in CRX associated with Leber congenital amaurosis. Hum. Mutat. 18, 550-551. 411. Sohocki, M.M., Daiger, S.P., Bowne, S.J., Rodriquez, J.A., Northrup, H., Heckenlively, J.R., Birch, D.G., Mintz-Hittner, H., Ruiz, R.S., Lewis, R.A., et al. (2001). Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Hum. Mutat. 17, 42-51. 412. Sankila, E.M., Joensuu, T.H., Hamalainen, R.H., Raitanen, N., Valle, O., Ignatius, J., and Cormand, B. (2000). A CRX mutation in a Finnish family with dominant cone-rod retinal dystrophy. Hum. Mutat. 16, 94. 413. Jacobson, S.G., Cideciyan, A.V., Huang, Y., Hanna, D.B., Freund, C.L., Affatigato, L.M., Carr, R.E., Zack, D.J., Stone, E.M., and McInnes, R.R. (1998). Retinal degenerations with truncation mutations in the cone-rod homeobox (CRX) gene. Invest. Ophthalmol. Vis. Sci. 39, 2417-2426. 414. Sohocki, M.M., Sullivan, L.S., Mintz-Hittner, H.A., Birch, D., Heckenlively, J.R., Freund, C.L., McInnes, R.R., and Daiger, S.P. (1998). A range of clinical phenotypes associated with mutations in CRX, a photoreceptor transcription-factor gene. Am. J. Hum. Genet. 63, 13071315. 415. Swain, P.K., Chen, S., Wang, Q.L., Affatigato, L.M., Coats, C.L., Brady, K.D., Fishman, G.A., Jacobson, S.G., Swaroop, A., Stone, E., et al. (1997). Mutations in the cone-rod homeobox gene are associated with the cone-rod dystrophy photoreceptor degeneration. Neuron 19, 1329-1336. 416. Lines, M.A., Hebert, M., McTaggart, K.E., Flynn, S.J., Tennant, M.T., and MacDonald, I.M. (2002). Electrophysiologic and phenotypic features of an autosomal cone-rod dystrophy caused by a novel CRX mutation. Ophthalmology 109, 1862-1870. 417. Paunescu, K., Preising, M.N., Janke, B., Wissinger, B., and Lorenz, B. (2007). Genotypephenotype correlation in a German family with a novel complex CRX mutation extending the open reading frame. Ophthalmology 114, 1348-1357 e1341. 418. Payne, A.M., Downes, S.M., Bessant, D.A., Taylor, R., Holder, G.E., Warren, M.J., Bird, A.C., and Bhattacharya, S.S. (1998). A mutation in guanylate cyclase activator 1A (GUCA1A) in an autosomal dominant cone dystrophy pedigree mapping to a new locus on chromosome 6p21.1. Hum. Mol. Genet. 7, 273-277. 419. Downes, S.M., Holder, G.E., Fitzke, F.W., Payne, A.M., Warren, M.J., Bhattacharya, S.S., and Bird, A.C. (2001). Autosomal dominant cone and cone-rod dystrophy with mutations in the guanylate cyclase activator 1A gene-encoding guanylate cyclase activating protein-1. Arch. Ophthalmol. 119, 96-105. 420. Wilkie, S.E., Li, Y., Deery, E.C., Newbold, R.J., Garibaldi, D., Bateman, J.B., Zhang, H., Lin, W., Zack, D.J., Bhattacharya, S.S., et al. (2001). Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy. Am. J. Hum. Genet. 69, 471-480. 421. Nishiguchi, K.M., Sokal, I., Yang, L., Roychowdhury, N., Palczewski, K., Berson, E.L., Dryja,

93 References introduction

T.P., and Baehr, W. (2004). A novel mutation (I143NT) in guanylate cyclase-activating protein 1 (GCAP1) associated with autosomal dominant cone degeneration. Invest. Ophthalmol. Vis. Sci. 45, 3863-3870. 422. Jiang, L., Katz, B.J., Yang, Z., Zhao, Y., Faulkner, N., Hu, J., Baird, J., Baehr, W., Creel, D.J., and Zhang, K. (2005). Autosomal dominant cone dystrophy caused by a novel mutation in the GCAP1 gene (GUCA1A). Mol. Vis. 11, 143-151. 423. Michaelides, M., Wilkie, S.E., Jenkins, S., Holder, G.E., Hunt, D.M., Moore, A.T., and Webster, A.R. (2005). Mutation in the gene GUCA1A, encoding guanylate cyclase-activating protein 1, causes cone, cone-rod, and macular dystrophy. Ophthalmology 112, 1442-1447. 424. Sokal, I., Dupps, W.J., Grassi, M.A., Brown, J., Jr., Affatigato, L.M., Roychowdhury, N., Yang, L., Filipek, S., Palczewski, K., Stone, E.M., et al. (2005). A novel GCAP1 missense mutation (L151F) in a large family with autosomal dominant cone-rod dystrophy (adCORD). Invest. Ophthalmol. Vis. Sci. 46, 1124-1132. 425. Jiang, L., Wheaton, D., Bereta, G., Zhang, K., Palczewski, K., Birch, D.G., and Baehr, W. (2008). A novel GCAP1(N104K) mutation in EF-hand 3 (EF3) linked to autosomal dominant cone dystrophy. Vision Res. 48, 2425-2432. 426. Kitiratschky, V.B., Behnen, P., Kellner, U., Heckenlively, J.R., Zrenner, E., Jagle, H., Kohl, S., Wissinger, B., and Koch, K.W. (2009). Mutations in the GUCA1A gene involved in hereditary cone dystrophies impair calcium-mediated regulation of guanylate cyclase. Hum. Mutat. 30, E782-796. 427. Van Ghelue, M., Eriksen, H.L., Ponjavic, V., Fagerheim, T., Andreasson, S., Forsman-Semb, K., Sandgren, O., Holmgren, G., and Tranebjaerg, L. (2000). Autosomal dominant cone-rod dystrophy due to a missense mutation (R838C) in the guanylate cyclase 2D gene (GUCY2D) with preserved rod function in one branch of the family. Ophthalmic Genet. 21, 197-209. 428. Weigell-Weber, M., Fokstuen, S., Torok, B., Niemeyer, G., Schinzel, A., and Hergersberg, M. (2000). Codons 837 and 838 in the retinal guanylate cyclase gene on chromosome 17p: hot spots for mutations in autosomal dominant cone-rod dystrophy? Arch. Ophthalmol. 118, 300. 429. Downes, S.M., Payne, A.M., Kelsell, R.E., Fitzke, F.W., Holder, G.E., Hunt, D.M., Moore, A.T., and Bird, A.C. (2001). Autosomal dominant cone-rod dystrophy with mutations in the guanylate cyclase 2D gene encoding retinal guanylate cyclase-1. Arch. Ophthalmol. 119, 16671673. 430. Udar, N., Yelchits, S., Chalukya, M., Yellore, V., Nusinowitz, S., Silva-Garcia, R., Vrabec, T., Hussles Maumenee, I., Donoso, L., and Small, K.W. (2003). Identification of GUCY2D gene mutations in CORD5 families and evidence of incomplete penetrance. Hum. Mutat. 21, 170-171. 431. Ito, S., Nakamura, M., Ohnishi, Y., and Miyake, Y. (2004). Autosomal dominant cone-rod dystrophy with R838H and R838C mutations in the GUCY2D gene in Japanese patients. Jpn. J. Ophthalmol. 48, 228-235. 432. Ito, S., Nakamura, M., Nuno, Y., Ohnishi, Y., Nishida, T., and Miyake, Y. (2004). Novel complex GUCY2D mutation in Japanese family with cone-rod dystrophy. Invest. Ophthalmol. Vis. Sci. 45, 1480-1485. 433. Yoshida, S., Yamaji, Y., Yoshida, A., Kuwahara, R., Yamamoto, K., Kubata, T., and Ishibashi, T. (2006). Novel triple missense mutations of GUCY2D gene in Japanese family with cone-rod dystrophy: possible use of genotyping microarray. Mol. Vis. 12, 1558-1564. 434. Smith, M., Whittock, N., Searle, A., Croft, M., Brewer, C., and Cole, M. (2007). Phenotype of autosomal dominant cone-rod dystrophy due to the R838C mutation of the GUCY2D gene encoding retinal guanylate cyclase-1. Eye 21, 1220-1225. 435. Small, K.W., Silva-Garcia, R., Udar, N., Nguyen, E.V., and Heckenlively, J.R. (2008). New mutation, P575L, in the GUCY2D gene in a family with autosomal dominant progressive cone degeneration. Arch. Ophthalmol. 126, 397-403. 436. Garcia-Hoyos, M., Auz-Alexandre, C.L., Almoguera, B., Cantalapiedra, D., Riveiro-

Alvarez, R., Lopez-Martinez, M.A., Gimenez, A., Blanco-Kelly, F., Avila-Fernandez, A., TrujilloTiebas, M.J., et al. (2011). Mutation analysis at codon 838 of the Guanylate Cyclase 2D gene in Spanish families with autosomal dominant cone, cone-rod, and macular dystrophies. Mol. Vis. 17, 1103-1109. 437. Xiao, X., Guo, X., Jia, X., Li, S., Wang, P., and Zhang, Q. (2011). A recurrent mutation in GUCY2D associated with autosomal dominant cone dystrophy in a Chinese family. Mol. Vis. 17, 3271-3278. 438. Xu, F., Dong, F., Li, H., Li, X., Jiang, R., and Sui, R. (2013). Phenotypic characterization of a Chinese family with autosomal dominant cone-rod dystrophy related to GUCY2D. Doc. Ophthalmol. 126, 233-240. 439. Zhao, X., Ren, Y., Zhang, X., Chen, C., Dong, B., and Li, Y. (2013). A novel GUCY2D mutation in a Chinese family with dominant cone dystrophy. Mol. Vis. 19, 1039-1046. 440. Kohn, L., Kohl, S., Bowne, S.J., Sullivan, L.S., Kellner, U., Daiger, S.P., Sandgren, O., and Golovleva, I. (2010). PITPNM3 is an uncommon cause of cone and cone-rod dystrophies. Ophthalmic Genet. 31, 139-140. 441. Michaelides, M., Gaillard, M.C., Escher, P., Tiab, L., Bedell, M., Borruat, F.X., Barthelmes, D., Carmona, R., Zhang, K., White, E., et al. (2010). The PROM1 mutation p.R373C causes an autosomal dominant bullâ&#x20AC;&#x2122;s eye maculopathy associated with rod, rod-cone, and macular dystrophy. Invest. Ophthalmol. Vis. Sci. 51, 4771-4780. 442. Yang, Z., Chen, Y., Lillo, C., Chien, J., Yu, Z., Michaelides, M., Klein, M., Howes, K.A., Li, Y., Kaminoh, Y., et al. (2008). Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice. J. Clin. Invest. 118, 2908-2916. 443. Johnson, S., Halford, S., Morris, A.G., Patel, R.J., Wilkie, S.E., Hardcastle, A.J., Moore, A.T., Zhang, K., and Hunt, D.M. (2003). Genomic organisation and alternative splicing of human RIM1, a gene implicated in autosomal dominant cone-rod dystrophy (CORD7). Genomics 81, 304-314. 444. Abid, A., Ismail, M., Mehdi, S.Q., and Khaliq, S. (2006). Identification of novel mutations in the SEMA4A gene associated with retinal degenerative diseases. J. Med. Genet. 43, 378-381. 445. Jalkanen, R., Mantyjarvi, M., Tobias, R., Isosomppi, J., Sankila, E.M., Alitalo, T., and BechHansen, N.T. (2006). X linked cone-rod dystrophy, CORDX3, is caused by a mutation in the CACNA1F gene. J. Med. Genet. 43, 699-704. 446. Demirci, F.Y., Rigatti, B.W., Wen, G., Radak, A.L., Mah, T.S., Baic, C.L., Traboulsi, E.I., Alitalo, T., Ramser, J., and Gorin, M.B. (2002). X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15. Am. J. Hum. Genet. 70, 1049-1053. 447. Yang, Z., Peachey, N.S., Moshfeghi, D.M., Thirumalaichary, S., Chorich, L., Shugart, Y.Y., Fan, K., and Zhang, K. (2002). Mutations in the RPGR gene cause X-linked cone dystrophy. Hum Mol Genet 11, 605-611. 448. Ebenezer, N.D., Michaelides, M., Jenkins, S.A., Audo, I., Webster, A.R., Cheetham, M.E., Stockman, A., Maher, E.R., Ainsworth, J.R., Yates, J.R., et al. (2005). Identification of novel RPGR ORF15 mutations in X-linked progressive cone-rod dystrophy (XLCORD) families. Invest. Ophthalmol. Vis. Sci. 46, 1891-1898. 449. Ruddle, J.B., Ebenezer, N.D., Kearns, L.S., Mulhall, L.E., Mackey, D.A., and Hardcastle, A.J. (2009). RPGR ORF15 genotype and clinical variability of retinal degeneration in an Australian population. Br. J. Ophthalmol. 93, 1151-1154.

95 References introduction

1.4 Chapter 1.4 Outline and aim of this thesis

The leading causes of hereditary blindness worldwide are retinal disorders, primarily affecting the photoreceptors leading to severe visual impairment. Early-onset cone-dominated phenotypes such as achromatopsia (ACHM), cone dystrophy (COD) and cone-rod dystrophy (CRD) are grouped as cone dysfunctions (CD). In order to develop effective therapeutic approaches the genetic etiology of CD needs to be elucidated. Throughout the years, dozens of genes have been found to cause CD, but still significant percentages of CD cases are unexplained. The aim of this thesis was to elucidate and characterize genetic aberrations underlying autosomal recessive cone dystrophies. The identification of the genetic defects was facilitated by using homozygosity mapping and next generation sequencing. Chapter 1 provides an introduction into the fascinating process of vision and the technologies homozygosity mapping and next generation sequencing, which were used to unravel genetic defects in CD. Furthermore, it contains a comprehensive description of all clinical phenotypes, the cellular processes in which CD-associated proteins are involved, the animal models and the current status of gene therapy for CDs. Chapter 2 describes the prevalence and novel discovered association of mutations in CNGA3 and CNGB3 with progressive COD. Chapter 3 summarizes the novel genotype-phenotype correlations that we have established. Maternal uniparental isodisomy allowed the discovery of TULP1 mutations in a COD and a CRD case, and in an case of oligocone trichromacy CEP290 mutations were identified, which are associated to a wide spectrum of phenotypes. Chapter 4 presents three novel genes to be involved in CD. Mutations in PDE6C were discovered using homozygosity mapping, whereas mutations in RAB28 and MFSD8 were discovered using genetic linkage studies and WES. Chapter 5 provides the general discussion of this thesis. It covers the novel disease mechanisms underlying PDE6C, RAB28 and MFSD8-associated CDs and future considerations when elucidating novel CD-associated genes. Furthermore, a review is given of all prenylated retinal proteins that are mutated in persons with inherited retinal diseases (IRD). Moreover, other IRD-associated proteins involved in the process of prenylation are described and discussed with respect to putative overlap in mechanisms of disease.

99 Outline and aim of this thesis

1.4

Alberta A.H.J. Thiadens, Susanne Roosing, Rob W.J. Collin, Norka van Moll-Ramirez, Janneke J.C. van Lith-Verhoeven, Mary J. van Schooneveld, Anneke I. den Hollander, L. Ingeborgh van den Born, Carel B. Hoyng, Frans P.M. Cremers, Caroline C.W. Klaver

Ophthalmology 2010, 117, 825-830 100

Chapter 2 Comprehensive analysis of the achromatopsia genes CNGA3 and CNGB3 in progressive cone dystrophy

101

Abstract

Objective: To investigate whether the major achromatopsia genes (CNGA3 and CNGB3) play a role in the etiology of progressive cone dystrophy (COD). Design: Prospective multicenter study. Participants: Probands (N=60) with autosomal recessive (AR) COD from various ophthalmogenetic clinics in the Netherlands. Methods: All available ophthalmologic data from the AR COD probands were registered from medical charts and updated by an additional ophthalmologic examination. Mutations in the CNGA3 and CNGB3 genes were analyzed by direct sequencing. Main outcome measure: CNGA3 and CNGB3 mutations and clinical course in AR COD probands. Results: In three AR COD probands (3/60; 5%) we found two mutations in the CNGB3 gene. Two of these probands had compound heterozygous mutations (p.R296Yfs*9 / p.R274Vfs*12 and p.R296Yfs*9 / c.991-3T>G). The third proband revealed homozygous missense mutations (p.R403Q) with two additional variants in the CNGA3 gene (p.E228K and p.V266M). These probands did not have a congenital nystagmus, but had a progressive deterioration of visual acuity, color vision, and photopic electroretinogram, with onset in the second decade. In six other unrelated probands, we found six different heterozygous amino acid changes in the CNGA3 (N=4) and CNGB3 (N=2) gene. Conclusions: The CNGB3 gene accounts for a small fraction of the later onset progressive form of cone photoreceptor disorders, and CNGA3 may have an additive causative effect. Our data indicate that these genes are involved in a broader spectrum of cone dysfunction, and it remains intriguing why initial cone function can be spared despite similar gene defects.

102

Cone dystrophy (COD) is a progressive cone disorder with an estimated prevalence of 1:30,000-1:40,000. Patients have normal cone function initially, but present with visual loss and color vision disturbances in the first or second decade.1-3 Macular abnormalities can be present, and the optic nerve may show a variable degree of temporal pallor. On electroretinogram (ERG), cone responses progressively deteriorate and rod responses are initially normal, but can diminish slightly over time. Although the course of disease may vary, the visual acuity generally worsens to legal blindness before middle age. For COD, several genes have been identified for the autosomal dominant4-6 and X-linked forms,7; 8 but little is known about the genetic causes of the most prevalent autosomal recessive (arCOD) form. Three genes have been implicated in this form: the ABCA4, CACNA2D4 and the KCNV2 genes.9-12 They play a role in only a fraction of patients, thus, most AR COD patients have an unknown genetic cause. In contrast to the progressive nature of COD, achromatopsia is a congenital cone disorder. The estimated prevalence is 1:30,000, and the inheritance is autosomal recessive.13; 14 The clinical course is characterized by low visual acuity, nystagmus, photophobia, severe color vision defects, and no recordable or only residual cone function on ERG. The genetic basis of achromatopsia has been elucidated to a large extent during the last decade. Three genes have been implicated: CNGA3, CNGB3, and GNAT2.15-18 The contribution of the CNGB3 gene in achromatopsia varies per population, but it explains the vast majority of achromatopsia cases (50-80%) in the Netherlands and Germany.19; 20 The CNGA3 and CNGB3 genes are cone-specific, and code for the Îą and Î˛ subunits of the cyclic nucleotide gated channel type 3 (CNG3), respectively. They are located on the membrane of cone outer segments, and play an important role during the phototransduction cascade. In the dark, high levels of cGMP open the channel, facilitate free flow of ions, and prohibit formation of an action potential. In the presence of light, cGMP levels decrease, resulting in closure of the channel, hyperpolarization of the cell, and initiation of phototransduction.21 We hypothesize that genes with such a vital role for cone function may also be involved in other disorders in which cones are primarily affected. On this subject, only one study reported mutations in CNGB3 in three relatives with AR COD;22 and one study described a heterozygous CNGA3 mutation in one patient with COD.16 In our study, we expanded the possible role of CNGA3 and CNGB3 in cone photoreceptor disorder by investigating a large series of AR COD patients for mutations in these genes.

Patients and Methods

Study population Patients (N=60; 65% males, range 10-79 years, mean age 39 years, SD 16 years) who had been diagnosed with autosomal recessive COD were ascertained from various ophthalmogenetic centers in the Netherlands. Inclusion criteria for the study were: progressively deteriorating visual acuity, color vision disturbances in three axes, decreasing cone responses over time with normal or only slightly reduced rod responses on ERG, and no presence of nystagmus. The study was approved by the Medical Ethics Committee of Erasmus Medical Center and adhered to the tenets of the Declaration of Helsinki. All participants provided signed, informed consent for participation in the study, retrieval of medical records, and use of blood and DNA for research. Clinical examination All patients received a questionnaire which addressed the medical history and family pedigree. Medical charts were retrieved from their ophthalmologists, and all available data on Snellen visual acuity, color vision (American Optical Hardy-Rand-Rittler Test, Ishihara Test for Color Blindness, or Farnsworth Panel D15 Test), slitlamp and fundus examination, and

103 CNGA3 and CNGB3 gene in progressive cone dystrophy

ERGs were collected. Color vision defects were classified as mild, moderate or severe in the three color axes (red, green and blue). Patients with incomplete or unavailable data from the last five years were invited for an additional ophthalmologic examination, ERG, and fundus photography. ERGs incorporated the recommendations of the International Society for Clinical Electrophysiology of Vision.23

Family A

Family B I

p.R274Vfs*12/+

p.R296Yfs*9/+

p.R296Yfs*9/p.R274Vfs*12

p.R296Yfs*9/c.991-3T>G

III

III p.R296Yfs*9/+

p.R274Vfs*12/+ p.R274Vfs*12/+

Family C I

II CNGA3 CNGB3

p.E228K/+ +/+

p.E228K/p.V266M p.E228K/p.V266M +/+ p.R403Q/p.R403Q

III

CNGA3 CNGB3

p.E228K/+ p.R403Q/+

Figure 1 Pedigrees of three families with autosomal recessive progressive cone dystrophy showing segregation of CNGA3 and CNGB3 gene mutations Open square and circle, unaffected males and females; black square and circle, affected males and females; dashed symbols denote deceased individuals. Molecular Genetic Analysis Blood samples were obtained from probands, affected relatives, and parents. DNA was isolated from peripheral blood lymphocytes by standard procedures. The coding region and intron/ exon boundaries of the CNGA3 and CNGB3 genes were amplified by PCR using the conditions and primer sequences provided in Supplemental Table 1. Direct sequencing was performed on sequence analyzer 3730 (Applied Biosystems, Inc. [ABI], Foster City, CA) with sequence analy-

104

sis software (Vector NTI Advance, version 10; Invitrogen Corp., Carlsbad, CA). Segregation analysis was performed when mutations were detected.

Results

Mutation analysis Table 1 shows the mutations that were detected. We found two variants in the CNGB3 gene in 3/60 (5%) patients, and confirmed their independent segregation in the families (Figure 1). In two patients, the mutations affected exonic splicing and caused a frameshift; and in one patient the mutation caused an aminoacid change mutation, p.R403Q. This variant could not be detected in 100 ethnically matched control subjects. One patient had two additional variants in the CNGA3 gene. Segregation analysis in this family (Figure 1) showed that the unaffected sister of the proband had the two CNGA3 variants without a CNGB3 variant, while the unaffected children were heterozygous for the CNGB3 variant and one CNGA3 variant. This suggests that the pathogenicity was predominantly caused by the CNGB3 variants. One missense variant, p.V266M, was novel. Neither CNGA3 variant could be detected in 100 ethnically matched control subjects. In two other unrelated probands, we found two different heterozygous mutations in the CNGB3 gene: c.1148delC (p.T383Ifs*13) and c.1208G>A (p.R403Q). In four other unrelated probands we detected four different CNGA3 variants: c.1856C>T (p.A619V); c.284C>T (p.P95L); c.1618G>A (p.V540I) and c.1694C>T (p.T565M). In these patients, we did not find a second variant on the other allele in the coding region of the CNGA3, CNGB3 and GNAT2 gene, despite complete sequencing (Table 1). Clinical findings Clinical findings are summarized in Table 2. The three probands with two variants had a progressive deterioration of visual acuity, color vision, and photopic ERG since their teens. Two patients had passed their driving exam at age 18 years, with a best corrected visual acuity (BCVA) of at least 0.50 decimals Snellen. In all three patients, the BCVA deteriorated rapidly in the second decade. They did not have a congenital nystagmus. The macular appearance varied in each patient. Two patients showed degeneration of the macula over time; one had progressive pigmentary changes since age 10, one developed a bullâ&#x20AC;&#x2122;s eye maculopathy in the second decade and one patient still had a normal macular appearance at the age of 45 years. The optic discs did not show any abnormalities in the three probands; no temporal pallor was apparent (Figure 2).

Figure 2 Fundus appearance of probands with progressive cone dystrophy and mutations in the CNGA3 and CNGB3 genes. A) Fundus photograph of the left eye of proband A-II-1, performed at age 45 years. No macular abnormalities are apparent. B) Fundus photograph of the left eye of proband B-II-1, performed at age 57 years, showing pigmentary changes in the macular region. C) Fundus photograph of the left eye of proband C-II-3, performed at age 47 years, showing a bullâ&#x20AC;&#x2122;s eye maculopathy with atrophy of retinal pigment epithelium.

105 CNGA3 and CNGB3 gene in progressive cone dystrophy

2/6

7/8

19/32 (52.8%)

2/10

p.R403Q

p.R274Vfs*12 8

c.1694C>T

c.1618G>A

c.284C>T

c.1856C>T

c.682G>A

p.T565M

p.V540I

p.P95L

p.A619V

p.E228K

Protein change

Exon

c.796G>A

DNA variant

CNGA3 allele 2

p.V266M

Protein change

106

A-II-1

B-II-1

C-II-3

0.70

0.50

First exam

No.

0.20

Last exam

First exam

Age

Patient

Last exam

Best Corrected Visual Acuity: better eye

0.70

0.50

0.40

First exam

0.16

0.20

Last exam

Best Corrected Visual Acuity: worse eye

Mild

Severe

First exam

All axes All axes

Medium

All axes

First exam

All axes

Last exam

Involved color axis

Severe

Last exam

Color vision test

Reduced

First exam

bullâ&#x20AC;&#x2122;s eye Reduced maculopathy

Pigmentary changes

No foveal reflex Pigmentary changes

Normal

Last exam Normal

First exam

Macular appearance

Normal

First exam

Absent

Normal

Last exam

Scotopic Electroretinogram

Severely Normal reduced

Absent

Last exam

Photopic Electroretinogram

Table 2 Clinical findings at first and last exam of three probands with autosomal recessive progressive cone dystrophy and mutations in the CNGA3 and CNGB3 genes

p.R403Q

c.1208G>A

c.819_826del8

c.1208G>A

p.T383Ifs*13

splice defect

c.1148delC

p.R403Q

p.R296Yfs*9

c.991-3T>G

c.1208G>A

c.886_896del11insT

DNA Exon variant

CNGA3 allele 1

C-II-3

p.R296Yfs*9

DNA variant

Protein change

B-II-1

c.886_896del11insT

Exon

CNGB3 allele 2

A-II-1

DNA variant

Protein change

Exon

Patient

CNGB3 allele 1

Table 1 Mutations in the CNGA3 and CNGB3 genes in three probands with autosomal recessive progressive cone dystrophy and in the six unrelated probands with a heterozygous variant in these genes

RPGR

Discussion

It has been well established that mutations in the CNGA3 and CNGB3 genes are a common cause of achromatopsia. We now report that mutations in these genes can also cause a progressive cone disorder. In contrast to achromatopsia, the AR COD patients with these mutations had no clinical signs of cone dysfunction at birth, but developed progressive loss of cone function during the first two decades of life. The truncating mutations in CNGB3 were described earlier in patients with achromatopsia, as was the combination of the splice defect in exon 9 and the frameshift mutation p.R296Yfs*9.19 The combination of p.R296Yfs*9 and p.R274Vfs*12 has not been reported earlier. Our observed mutations in the CNGB3 gene were all located in the transmembrane helices (Figure 3). This is likely to lead to defective pore formation and may ultimately lead to apoptosis by the continuous influx of Ca2+ ions.24 The question remains why the severe mutations in these patients did not disturb initial cone function, but caused deterioration only after the first decade. There are no functional studies of absent β-subunits of the CNG3 channel, but mice lacking the α-subunits due to CNGA3 deficiency have been described.25 These mice never had recordable cone responses on ERG. However, histology showed presence of cones at birth and progressive cone cell loss only after the second postnatal week. Residual cones could still be identified after 22 months.25 Apparently, there is functional redundancy of an unidentified cGMP-dependent protein, particularly early in development, which is also capable of ion transport and initiation of an action potential. Another explanation could be that the CNGA3 protein can compensate initially for some function of CNGB3.

Pore

Figure 3 Localization of the mutations with respect to the topological model of the CNGB3 and CNGA3 polypeptides (http://smart.embl-heidelberg.de,October 2008). CaM = Ca2+ calmodulin domains; COOH = carboxylic acids; cGMP = cyclic guanosine monophosphate. The third patient had a homozygous missense mutation in CNGB3 (p.R403Q) in combination with a compound heterozygous variant in CNGA3 (p.E228K; p.V266M). In a previous report, Nishiguchi et al. found a homozygous p.R403Q mutation without CNGA3 mutations, in a 31-year-old patient with few macular drusen, a slightly decreased visual acuity, normal color vision, and normal cone responses on full-field ERG.26 p.R403Q has also been described in a patient with COD in a compound heterozygous genotype with p.T383Ifs*13.22 Although this combination is more pathogenic, the phenotype was comparable to our patient. p.R403Q is located in a well conserved domain of the cone β-subunit in the middle of the pore domain 107 CNGA3 and CNGB3 gene in progressive cone dystrophy

of the CNG-channel (Figure 3). This mutation replaces the positively charged residue arginine by a neutral glutamine. The substitution may directly affect ion transfer through the channel pore, 27 or may change the electrostatic environment of the pore region and influence Ca2+ binding.27 Both will result in dysfunction of the channel. Evidence for channelopathy was provided by an in vitro study which investigated the effect of p.R403Q in recombinant cone CNGchannels.27 p.R403Q appeared to increase sensitivity for cGMP. This will sustain the opening of the channel, cause increased Ca2+ entry, and eventually cause cell dysfunction and apoptosis.24 Remarkably, cGMP affinity of CNGB3 containing the p.R403Q variant was more comparable to the wild-type protein than cGMP affinity of CNGB3 containing p.F525N, a variant associated with achromatopsia.27 Whether the heterozygous CNGA3 mutations have an additional pathogenic effect in the third patient is unclear. p.E228K is located in a fully conserved site in the transmembrane region of the cone α-subunit (Figure 3), and was found homozygously without CNGB3 mutations in a proband with achromatopsia.28 This observation indicates that the p.E228K variant can be pathogenic, and suggests that this variant could have an additive effect to the AR COD phenotype in our proband. The eldest daughter of this proband, who is a heterozygous carrier of this CNGA3 variant and of a CNGB3 variant, is still unaffected at the age of nine years. The next decade will show whether she will develop any cone dysfunction, and provide further proof of a true digenic disease model. The newly identified p.V266M variant appears to be well tolerated and is unlikely to affect protein function. What may be the consequences of mutations in both CNGB3 and CNGA3? The CNG-channels in the cone are heterotetramers with two α- and two β-subunits. In a mouse model inter-subunit interaction between CNGA3 and CNGB3 was identified with immunolabeling. Matveev et al. concluded that this interaction was necessary to obtain a heterotetrameric complex in the cone CNG-channels.29 Our hypothesis is that a missense mutation in the CNGA3 gene in the presence of two missense mutations in CNGB3 further deteriorates an already compromised CNG3 channel. The single CNGA3 and CNGB3 variants that we found in six probands all reside in evolutionary conserved areas. 30 If a second mutation is present, it could be located in an area of the CNGA3, CNGB3 or GNAT2 gene that was not analyzed (deep intronic, promotor), or it may have been a large deletion that was missed due to the nature of our PCR-based analysis. However, a second mutation may not be present. In this scenario, the heterozygous variant does not contribute to the COD phenotype. In conclusion, the CNGB3 gene accounts for only a small fraction of the later onset progressive form of cone photoreceptor disorders, and CNGA3 may have an additive causative effect. It is likely that the majority of AR COD is due to other genetic causes. The finding that cone function can be maintained in the early years of life despite mutations coding for essential proteins of the cell suggests that protective mechanisms are present. Identification of these pathways may help develop strategies to prolong the life of the cone photoreceptor cell.

Acknowledgements

Supported by Prof. Dr. Henkes Stichting, Nijmeegse Oogonderzoek Stichting, Stichting Wetenschappelijk Onderzoek Oogziekenhuis (SWOO), The Rotterdam Eye Hospital, Macula Degeneratie Fonds (MD Fonds), Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (ANVVB), Dr. F.P. Fischer Stichting, Gelderse Blinden Stichting, Landelijke Stichting voor Blinden en Slechtzienden (LSBS), Stichting Blindenhulp, Stichting Blinden-penning, Stichting Nederlands Oogheelkundig Onderzoek (SNOO), Stichting Ondersteuning Oogheelkunde ’s-Gravenhage (OOG), Stichting ter Verbetering van het Lot der Blinden.

108

Supplemental table 1 Primers used for sequencing of CNGB3 and CNGA3 Sequence (5’3’) Gene & exon

Forward

Reverse

Product size (basepairs)

ggcacagtcataaatacagaggg

ggagactatactaggatttgg

393

CNGB3 Exon 1 Exon 2

agtacatcataaacagtcaattt

gcattttcatcacctgacatg

425

Exon 3

ttggcctgaaggtgtctacc

gctcaaatatccactctcccc

400 561

Exon 4

tgccccaccatctgtagc

ccaaaagcagaccctgagaa

Exon 5

cggtgtttggttaagaaattc

ccctgtgactcaaagtcacag

309

Exon 6

acagtccagaggcagaatgg

caattatccatgcagatagcc

481

Exon 7

gagattggaaggaaccaacc

ttgagaggcagaaacttcagg

298

Exon 8

gccttaaaaagccccatgc

ccatctttctgccctcatca

397

Exon 9

ctataactacagggtagcaat

tcatatccctgccaaattcc

388

Exon 10

cagtcaagacattgccatcag

gcatttaccagccattgaatgg

430

Exon 11

cccaagaatagtggtctttcg

gatcaacagtgcttttccatt

385

Exon 12

cagggcattagaaggaagta

ctgtaaggtagcagagactag

340

Exon 13

aggtatggaggtccaataga

cctagagaaattatgtgga

405

Exon 14

cacacccaagtctatctgagc

actctgagagcacgttattgc

468

Exon 15

ggaggcaaacagtactcacg

ggtttgattgtgctgagagc

472

Exon 16

aatcacctggaccctcacc

ctctgagatagggagaaccg

357

Exon 17

cttgatcacagtgagatatg

acgcccactctaattccatt

410

Exon 18

tcttggtggtgatcttagcc

ccttgagaaacgaaaggcaa

543

CNGA3 Exon 2

cttgatgagctgggtttgc

cccccacagtctagatcagc

283

Exon 3

tctcactcctggctgtgtcc

ccccatctagcactttttcc

279 335

Exon 4

agggaaagactggggtttgg

caaacaggatggagcaaagc

Exon 5

gtaatcccctggtgaaatgg

gggagcaggagcactaagg

228

Exon 6

gccctaggctctctaaaacc

gggagaggtggagctctgg

274

Exon 7

ttacatgatccagcgtcttcc

taatgtcccatccaccatgc

294

Exon 8.1

gcatactgtgtagccgtgagg

ggttttgggacagactcctg

655

Exon 8.2

gttcaggattgggaacttgg

ccgtgaggcattcatattcg

331

Exon 8.3

gtgggtgttctgatttttgc

cccaatatctcccttcttgc

390

Exon 8.4

gacacgctgaagaaggttcg

gctgcttcatcttcatctgg

559

Exon 8.5

ctggacaccctgcagacc

ttcaaccctgaccaagttcc

271

Primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_ www.cgi, April 2007) or using Exon Primer from the Genome Browser (http://genome.ucsc.edu, April 2007). Exon 1 of the CNGA3 gene is non-coding.

109 CNGA3 and CNGB3 gene in progressive cone dystrophy

References 1. Michaelides, M., Hunt, D.M., and Moore, A.T. (2004). The cone dysfunction syndromes. Br J Ophthalmol 88, 291-297. 2. Michaelides, M., Hardcastle, A.J., Hunt, D.M., and Moore, A.T. (2006). Progressive cone and cone-rod dystrophies: phenotypes and underlying molecular genetic basis. Surv Ophthalmol 51, 232-258. 3. Simunovic, M.P., and Moore, A.T. (1998). The cone dystrophies. Eye 12 ( Pt 3b), 553-565. 4. Jiang, L., Katz, B.J., Yang, Z., Zhao, Y., Faulkner, N., Hu, J., Baird, J., Baehr, W., Creel, D.J., and Zhang, K. (2005). Autosomal dominant cone dystrophy caused by a novel mutation in the GCAP1 gene (GUCA1A). Mol Vis 11, 143-151. 5. Nishiguchi, K.M., Sokal, I., Yang, L., Roychowdhury, N., Palczewski, K., Berson, E.L., Dryja, T.P., and Baehr, W. (2004). A novel mutation (I143NT) in guanylate cyclase-activating protein 1 (GCAP1) associated with autosomal dominant cone degeneration. Invest Ophthalmol Vis Sci 45, 3863-3870. 6. Kitiratschky, V.B., Wilke, R., Renner, A.B., Kellner, U., Vadala, M., Birch, D.G., Wissinger, B., Zrenner, E., and Kohl, S. (2008). Mutation analysis identifies GUCY2D as the major gene responsible for autosomal dominant progressive cone degeneration. Invest Ophthalmol Vis Sci 49, 50155023. 7. Yang, Z.L., Peachey, N.S., Moshfeghi, D.M., Thirumalaichary, S., Chorich, L., Shugart, Y.Y., Fan, K., and Zhang, K. (2002). Mutations in the RPGR gene cause X-linked cone dystrophy. Hum Mol Genet 11, 605-611. 8. Demirci, F.Y., Rigatti, B.W., Wen, G., Radak, A.L., Mah, T.S., Baic, C.L., Traboulsi, E.I., Alitalo, T., Ramser, J., and Gorin, M.B. (2002). X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15. Am J Hum Genet 70, 1049-1053. 9. Wycisk, K.A., Zeitz, C., Feil, S., Wittmer, M., Forster, U., Neidhardt, J., Wissinger, B., Zrenner, E., Wilke, R., Kohl, S., and Berger, W. (2006). Mutation in the auxiliary calcium-channel subunit CACNA2D4 causes autosomal recessive cone dystrophy. Am J Hum Genet 79, 973-977. 10. Kitiratschky, V.B., Grau, T., Bernd, A., Zrenner, E., Jagle, H., Renner, A.B., Kellner, U., Rudolph, G., Jacobson, S.G., Cideciyan, A.V., Schaich, S., Kohl, S., and Wissinger, B. (2008). ABCA4 gene analysis in patients with autosomal recessive cone and cone rod dystrophies. Eur J Hum Genet 16, 812-819. 11. Wissinger, B., Dangel, S., Jagle, H., Hansen, L., Baumann, B., Rudolph, G., Wolf, C., Bonin, M., Koeppen, K., Ladewig, T., Kohl, S., Zrenner, E., and Rosenberg, T. (2008). Cone dystrophy with supernormal rod response is strictly associated with mutations in KCNV2. Invest Ophthalmol Vis Sci 49, 751-757. 12. Ben Salah, S., Kamei, S., Senechal, A., Lopez, S., Bazalgette, C., Bazalgette, C., Eliaou, C.M., Zanlonghi, X., and Hamel, C.P. (2008). Novel KCNV2 mutations in cone dystrophy with supernormal rod electroretinogram. Am J Ophthalmol 145, 1099-1106. 13. Sundin, O.H., Yang, J.M., Li, Y., Zhu, D., Hurd, J.N., Mitchell, T.N., Silva, E.D., and Maumenee, I.H. (2000). Genetic basis of total colourblindness among the Pingelapese islanders. Nat Genet 25, 289-293. 14. Pokorny, J., Smith, V.C., Pinckers, A.J., and Cozijnsen, M. (1982). Classification of complete and incomplete autosomal recessive achromatopsia. Graefes Arch Clin Exp Ophthalmol 219, 121-130. 15. Kohl, S., Baumann, B., Broghammer, M., Jagle, H., Sieving, P., Kellner, U., Spegal, R., Anastasi, M., Zrenner, E., Sharpe, L.T., and Wissinger, B. (2000). Mutations in the CNGB3 gene encoding the beta-subunit of the cone photoreceptor cGMP-gated channel are responsible for achromatopsia (ACHM3) linked to chromosome 8q21. Hum Mol Genet 9, 2107-2116. 16. Wissinger, B., Gamer, D., Jagle, H., Giorda, R., Marx, T., Mayer, S., Tippmann, S., Broghammer,

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M., Jurklies, B., Rosenberg, T., Jacobson, S.G., Sener, E.C., Tatlipinar, S., Hoyng, C.B., Castellan, C., Bitoun, P., Andreasson, S., Rudolph, G., Kellner, U., Lorenz, B., Wolff, G., Verellen-Dumoulin, C., Schwartz, M., Cremers, F.P., Apfelstedt-Sylla, E., Zrenner, E., Salati, R., Sharpe, L.T., and Kohl, S. (2001). CNGA3 mutations in hereditary cone photoreceptor disorders. Am J Hum Genet 69, 722-737. 17. Kohl, S., Baumann, B., Rosenberg, T., Kellner, U., Lorenz, B., Vadala, M., Jacobson, S.G., and Wissinger, B. (2002). Mutations in the cone photoreceptor G-protein alpha-subunit gene GNAT2 in patients with achromatopsia. Am J Hum Genet 71, 422-425. 18. Sidjanin, D.J., Lowe, J.K., McElwee, J.L., Milne, B.S., Phippen, T.M., Sargan, D.R., Aguirre, G.D., Acland, G.M., and Ostrander, E.A. (2002). Canine CNGB3 mutations establish cone degeneration as orthologous to the human achromatopsia locus ACHM3. Hum Mol Genet 11, 1823-1833. 19. Kohl, S., Varsanyi, B., Antunes, G.A., Baumann, B., Hoyng, C.B., Jagle, H., Rosenberg, T., Kellner, U., Lorenz, B., Salati, R., Jurklies, B., Farkas, A., Andreasson, S., Weleber, R.G., Jacobson, S.G., Rudolph, G., Castellan, C., Dollfus, H., Legius, E., Anastasi, M., Bitoun, P., Lev, D., Sieving, P.A., Munier, F.L., Zrenner, E., Sharpe, L.T., Cremers, F.P.M., and Wissinger, B. (2005). CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia. Eur J Hum Genet 13, 302-308. 20. Thiadens, A.A., Slingerland, N.W., Roosing, S., van Schooneveld, M.J., van Lith-Verhoeven, J.J., van Moll-Ramirez, N., van den Born, L.I., Hoyng, C.B., Cremers, F.P., and Klaver, C.C. (2009). Genetic etiology and clinical consequences of complete and incomplete achromatopsia. Ophthalmology 116, 1984-1989 e1981. 21. Kaupp, U.B., and Seifert, R. (2002). Cyclic nucleotide-gated ion channels. Physiol Rev 82, 769824. 22. Michaelides, M., Aligianis, I.A., Ainsworth, J.R., Good, P., Mollon, J.D., Maher, E.R., Moore, A.T., and Hunt, D.M. (2004). Progressive cone dystrophy associated with mutation in CNGB3. Invest Ophthalmol Vis Sci 45, 1975-1982. 23. Marmor, M.F., Fulton, A.B., Holder, G.E., Miyake, Y., Brigell, M., and Bach, M. (2009). ISCEV Standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol 118, 69-77. 24. Biel, M., Seeliger, M., Pfeifer, A., Kohler, K., Gerstner, A., Ludwig, A., Jaissle, G., Fauser, S., Zrenner, E., and Hofmann, F. (1999). Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3. Proc Natl Acad Sci U S A 96, 7553-7557. 25. Michalakis, S., Geiger, H., Haverkamp, S., Hofmann, F., Gerstner, A., and Biel, M. (2005). Impaired opsin targeting and cone photoreceptor migration in the retina of mice lacking the cyclic nucleotide-gated channel CNGA3. Invest Ophthalmol Vis Sci 46, 1516-1524. 26. Nishiguchi, K.M., Sandberg, M.A., Gorji, N., Berson, E.L., and Dryja, T.P. (2005). Cone cGMPgated channel mutations and clinical findings in patients with achromatopsia, macular degeneration, and other hereditary cone diseases. Hum Mutat 25, 248-258. 27. Bright, S.R., Brown, T.E., and Varnum, M.D. (2005). Disease-associated mutations in CNGB3 produce gain of function alterations in cone cyclic nucleotide-gated channels. Mol Vis 11, 1141-1150. 28. Reuter, P., Koeppen, K., Ladewig, T., Kohl, S., Baumann, B., Wissinger, B., and Achromatopsia Clinical Study, G. (2008). Mutations in CNGA3 impair trafficking or function of cone cyclic nucleotide-gated channels, resulting in achromatopsia. Hum Mutat 29, 1228-1236. 29. Matveev, A.V., Quiambao, A.B., Browning Fitzgerald, J., and Ding, X.Q. (2008). Native cone photoreceptor cyclic nucleotide-gated channel is a heterotetrameric complex comprising both CNGA3 and CNGB3: a study using the cone-dominant retina of Nrl-/- mice. J Neurochem 106, 2042-2055. 30. Muraki-Oda, S., Toyoda, F., Okada, A., Tanabe, S., Yamade, S., Ueyama, H., Matsuura, H., and Ohji, M. (2007). Functional analysis of rod monochromacy-associated missense mutations in the CNGA3 subunit of the cone photoreceptor cGMP-gated channel. Biochem Biophys Res Commun 362, 88-93.

111 CNGA3 and CNGB3 gene in progressive cone dystrophy

112

Chapter 3 Novel genotype-phenotype correlations for cone dysfunctions

113

Susanne Roosing, L. Ingeborgh van den Born, Carel B. Hoyng, Alberta A.H.J. Thiadens, Elfride de Baere, Rob W.J. Collin, Robert K. Koenekoop, Bart P. Leroy, Norka van Moll-Ramirez, Hanka Venselaar, Frans C.C. Riemslag, Frans P.M. Cremers, Caroline C.W. Klaver, Anneke I. den Hollander

Ophthalmology 2013;120:1239-1246 114

Chapter 3.1 Maternal uniparental isodisomy of chromosome 6 reveals a TULP1 mutation as a novel cause of cone dysfunction

115

3.1

Abstract

Purpose: The majority of the genetic causes of autosomal recessive (AR) cone dystrophy (COD) and cone-rod dystrophy (CRD) are currently unknown. We employed a high-resolution homozygosity mapping approach in a cohort of patients with COD or CRD to identify new genes for autosomal recessive (AR) cone disorders. Design: Case series Participants: A cohort of 159 AR COD and 91 CRD patients Methods: The genomes of 83 AR COD and 73 CRD patients were analyzed for homozygous regions using single nucleotide polymorphism (SNP) microarrays. One patient showed homozygosity of SNPs across chromosome 6 and segregation analysis was performed using microsatellite markers. Direct sequencing of all retinal disease genes on chromosome 6 revealed a novel pathogenic TULP1 mutation in this patient. A cohort of 159 COD and 91 CRD individuals was screened for this particular mutation using the restriction enzyme HhaI. The medical history of patients carrying the TULP1 mutation was reviewed and additional ophthalmic examinations were performed, including electroretinography (ERG), perimetry, optical coherence tomography (OCT), fundus autofluorescence (FAF), and fundus photography. Main Outcome Measures: TULP1 mutations, age at diagnosis, visual acuity, fundus appearance, color vision defects, visual field, ERG, FAF and OCT findings. Results: In one patient homozygosity mapping and subsequent segregation analysis revealed maternal uniparental disomy (UPD) of chromosome 6. A novel homozygous missense mutation (p.Arg420Ser) was identified in TULP1, while no mutations were detected in other retinal disease genes on chromosome 6. The mutation affects a highly conserved amino acid residue in the Tubby domain, and is predicted to be pathogenic. The same homozygous mutation was also identified in an additional, unrelated CRD patient. Both patients carrying the p.Arg420Ser mutation presented with a bullâ&#x20AC;&#x2122;s eye maculopathy. The first patient had progressive loss of visual acuity with a relatively preserved ERG, while the second patient developed loss of visual acuity, peripheral degeneration and severely reduced ERG responses in a cone-rod pattern. Conclusions: Maternal UPD of chromosome 6 unmasked a mutation in the TULP1 gene as a novel cause of cone dysfunction. This expands the disease spectrum of TULP1 mutations from Leber congenital amaurosis and early-onset retinitis pigmentosa to cone-dominated disease.

116

Introduction

Cone dystrophy ( COD) and cone-rod dystrophy (CRD) are progressive retinal cone disorders with an estimated prevalence of 1:30.000-1:40.000 worldwide.1-3 Individuals with COD have normal cone function at birth, but develop progressive loss of cones and central vision in the first or second decade of life.4 Most persons develop severe visual acuity loss before the age of 40.4 Clinical features of cone dystrophy are poor visual acuity, disturbances in color vision, a fundus appearance varying from normal, a bull’s eye maculopathy or total atrophy of the macular region, with a variable degree of temporal pallor of the optic nerve. Visual fields display a central scotoma and full-field electroretinography (ERG) records a progressive deterioration of cone derived amplitude responses. Both diseases are characterized by loss of cone photoreceptors and progressive visual decline, but CRD can be distinguished from COD by early involvement of rod photoreceptors. Symptoms of CRD resemble those of COD, although patients with CRD may also experience nyctalopia owing to rod dysfunction. 5 Multiple genes have been shown to be involved in both disorders. The most prevalent mode of inheritance is autosomal recessive; Seven genes have been implicated in AR COD (ABCA4, CACNA2D4, CNGA3, CNGB3, GNAT2, KCNV2, PDE6C); and six in AR CRD (ABCA4, ADAM9, CERKL, EYS, PROM1 and RPGRIP1). In both disorders mutations in these genes explain disease in only a minority of all patients, and most genetic causes are currently still unknown.6; 7 The goal of this study was to identify novel causes of AR COD and CRD using homozygosity mapping. Homozygosity mapping has proven itself to be an effective tool in elucidating genetic defects in recessive traits in consanguineous as well as non-consanguineous families.8-10 This study demonstrates that homozygosity mapping can reveal uniparental disomy, facilitating the identification of a novel gene for cone disorders.

Methods

Subjects and clinical evaluation Patients (COD N= 159; CRD N= 91) were ascertained from various ophthalmic centers in the Netherlands, Belgium, the United Kingdom, and Canada. Individuals with COD were included when they showed a progressive decline of visual acuity, color vision disturbances, and reduced cone amplitude responses on ERG, with normal rod responses for ~5 years. Inclusion criteria for CRD were a progressive decline of visual acuity, color vision disturbances, and on ERG a reduction of both cone and rod responses, with cones equally or more severely reduced.5 After identification of the genetic defect, the medical history of the respective patients was reviewed and additional ophthalmic examinations were performed. This included best-corrected visual acuity (BCVA; Snellen chart), slit-lamp biomicroscopy, ophthalmoscopy, color vision testing (Hardy–Rand–Rittler color vision test and Lanthony Panel D-15 tests), and Goldmann kinetic perimetry (targets V-4e, and I-4e to I-1e). Electroretinography (ERG) was performed according to the extended protocol for the full-field ERG (ERG, Rod- and Cone b-wave series) of the International Society for Clinical Electrophysiology of Vision (ISCEV).11 In addition we also did S-cone-specific ERG testing in one patient.12; 13 After one-minute adaptation to an amber background to deactivate the L- and M-cones, blue light-flashes were delivered once every two seconds to stimulate the S-cones. This process was repeated, but instead patients followed a one-minute adaptation to a blue background to deactivate the S-cones and amber light-flashes were delivered to stimulate the L- and M-cones. Spectral-domain optical coherence tomography (SD-OCT; Heidelberg Spectralis HRA+OCT, Heidelberg Engineering, Germany) was used to obtain cross-sectional images of the macular region through dilated pupils, one horizontally and one vertically (30° wide, 51 frames per line). In addition we performed a volume scan (20x15°, 37 lines, 36 frames per line). Fundus autofluorescence (FAF) imaging (Heidelberg Spectralis HRA+OCT, Heidelberg Engineering, Germany) of the macula was performed with 488 nm wavelength using a 30° lens: a mean image out of 16 single images was calculated. Fundus photographs

117 A novel TULP1 mutation as a cause of cone dysfunction

3.1

centered on the macular area as well as on the four peripheral quadrants was performed using standard procedures. This study was approved by the Institutional Review Board, and adhered to the tenets of the declaration of Helsinki. All subjects provided written informed consent prior to participation in the study. Molecular Genetic Analysis Blood samples were obtained of all probands, and (if possible) their parents, affected and unaffected siblings. DNA was isolated from peripheral blood lymphocytes by standard procedures. After exclusion of mutations in the known genes CNGA3, CNGB3, KCNV2, and PDE6C, genome-wide homozygosity mapping was performed in 83 individuals affected by COD using the Affymetrix GeneChip Genome-Wide Human Mapping 5.0 (Affymetrix, Santa Clara, CA, USA). Homozygosity mapping in another 73 individuals with CRD was performed using the Affymetrix GeneChip Human Mapping 250K NspI array (Affymetrix, Santa Clara, CA,USA, N=32) and Affymetrix GeneChip Genome-Wide Human Mapping 6.0 (Affymetrix, Santa Clara, CA,USA, N=41).7 Genotypes were called with Genotype Console software (Affymetrix), and homozygous regions were calculated with Partek Genomic Suite software (Partek Inc., St. Louis, MO, USA). For one Dutch patient and all his available family members, microsatellite analysis was performed for selected markers on chromosome 6. Microsatellites D6S291, D6S963, D6S1552, D6S1568, D6S1582, D6S1611, D6S1618, D6S1645 and were amplified with primers containing a M13 forward and reverse tail. A second PCR was performed with a fluorescently labelled M13-forward primer and an unlabelled M13-reverse primer. Polymerase chain reaction (PCR) products were mixed with a fluorescent size marker (Applied Biosystems), and samples were analyzed on a 3100 or 3730 DNA Analyzer (Applied Biosystems). Fragment lengths were analyzed with GeneMapper software (Applied Biosystems). Haplotypes were constructed based upon the size of the alleles of the microsatellites. Sequence analysis of all exons and intron-exon boundaries was performed for ELOVL4, EYS, IMPG1, LCA5, RDS and TULP1 with the Big Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA). Primers were designed with Primer3 software, and primer sequences are listed in Supplemental Table 1. Screening of patients and controls for the observed TULP1 mutation p.Arg420Ser was performed by amplification of exons 12 and 13 (499 basepairs (bp)) followed by HhaI digestion of the amplified products. Wildtype alleles were digested into 355 bp and 145 bp fragments, whereas mutant alleles remained undigested. The pathogenicity of the mutation was determined in silico using the Grantham- and PhyloP-score (phylogenetic P-value), SIFT, and Polyphen-2 (v2.1.0r367). The 3D-structure of TULP1 (residues 290-542) was determined experimentally and can be found in PDB-file 2FIM. YASARA was used to visualize this structure and to study the effect of the mutation.14

Results

Molecular genetic analysis Homozygous SNP calls across the entire chromosome 6 were observed in one single Dutch patient (patient 1; Supplemental Table 2), suggesting uniparental disomy (UPD), a phenomenon involving the inheritance of two homologous chromosomes from one parent.15 Considering the high chance of identifying the causative mutation on chromosome 6, all relevant retinal disease genes located on this chromosome were analyzed. Screening of ELOVL4, EYS, IMPG1, LCA5, and RDS did not reveal a pathogenic mutation. Analysis of TULP1 identified a novel homozygous missense mutation that substitutes the amino acid arginine by a serine (c.1258C>A; p.Arg420Ser). To determine the origin of the patientâ&#x20AC;&#x2122;s UPD, haplotype analysis using eight microsatellite markers surrounding TULP1 was performed (Figure 1). This demonstrated that patient 1 inherited two

118

copies of the mutated allele from his mother. Segregation analysis in five unaffected siblings and the unaffected mother confirmed the presence of the mutation on the maternal allele. The patientâ&#x20AC;&#x2122;s mother was 45 years old when he was born, had a normal pregnancy with a normal gestational period, although the patient was born with a very low birth weight. A cohort of 159 unrelated individuals with COD and 91 with CRD (including individuals who were previously genotyped with SNP microarrays) was screened for this particular mutation, which lead to the identification of another Dutch individual (patient 2) carrying p.Arg420Ser in a homozygous state. TULP1 was located in the largest homozygous region (43.2 Mb) of patient 2 (Supplemental Table 3). Patients 1 and 2 shared a common 8.2-Mb homozygous haplotype surrounding the TULP1 mutation, suggesting that they are related through a common ancestor. Segregation analysis confirmed that both parents of patient 2, who are first cousins, carry the p.Arg420Ser mutation in heterozygous state (Figure 1B). The mutation was not detected in 159 healthy ethnically matched control individuals nor in our in-house exome (n=672) database and online exome (Exome Variant Server, http://evs.gs.washington.edu/EVS/, 1 October 2012) and mutation databases (HGMD, www.hgmd.cf.ac.uk/ac/index.php 1 October 2012; 1000genomes project, www.1000genomes.org, 1 October 2012).

3.1

Figure 1 Pedigrees with two Dutch patients with cone dysfunction due to a novel mutation (c.1258C>A; p.Arg420Ser) in the TULP1 gene A) Microsatellite analysis in patient 1 and his family members demonstrated maternal UPD of chromosome 6. The patient inherited two copies of the maternal chromosome carrying the TULP1 mutation p.Arg420Ser. Microsatellite marker positions are based on the UCSC human genome reference sequence, version hg19. B) Segregation analysis of the same TULP1 mutation in family members of patient 2. Both parents, who are first cousins, carry the p.Arg420Ser mutation in heterozygous state. In silico analysis of the p.Arg420Ser mutation in TULP1 The p.Arg420Ser mutation identified in TULP1 affects a residue which is located in the C-terminal Tubby-domain (Figure 2A). The arginine residue at position 420 is highly conserved among mammalian and fish TULP1 orthologs, but is not conserved in chicken and frog nor in the homologous proteins TULP2, TULP3 and TUB. To evaluate the pathogenicity of the mutation, in silico analysis

119 A novel TULP1 mutation as a cause of cone dysfunction

using a variety of prediction programs was performed. Arginine shows a moderate physicochemical dissimilarity with serine (Grantham score: 110 [0-215]). SIFT predicts that no other residue than the wildtype arginine is tolerated at this position, and Polyphen-2 predicts that the substitution is possibly damaging, with a score of 0.925 (sensitivity: 0.80; specificity: 0.94). At nucleotide level, position 1258 is moderately high conserved with a phyloP score of 2.20. The 3D-structure of TULP1 residues 290-542 consists of a beta-barrel with the last C-terminal residues forming an alpha-helix centered at the core of this barrel. Arginine 420 is located in one of the beta-sheets that surrounds the central helix (Figure 2B). Its side chain is located on the protein surface where it can make a hydrogen bond to residue Asn411 and a saltbridge to residue Glu409. These interactions stabilize the beta-sheet domain, which might be required for the correct positioning of the central helix and could be involved in interactions with other proteins. Due to the differences in charge and size between Arginine and Serine, mutation p.Arg420Ser will lead to a change at the protein surface which will affect the stability of the domain (Figure 2C).

Figure 2 Evolutionary conservation and effect on the TULP1 structure of the TULP1 mutation p.Arg420Ser A) Alignment of a part of the C-terminal Tubby domain of TULP1 orthologs and TULP1 family members. The arginine residue at position 420 is highly conserved among mammalian and fish TULP1 orthologs, but is not conserved in chicken and frog nor in the homologous proteins TULP2, TULP3 and TUB. Identical amino acids are indicated in black boxes, conserved residues in gray boxes. B) Visualization of the beta-barrel in the TULP1 structure using the PDB-file 2FIM. Red represents the arginine residue at position 420 and the magenta dots indicate unknown parts of the structure. C) Visualization of the p.Arg420Ser mutation in the TULP1 structure. The wildtype amino acid is shown in green, whereas the mutant amino acid is depicted in red. The structure will be destabilized due to the change of the positively charged arginine to the neutral serine. D) Visualization of the p.Arg420Pro mutation in the TULP1 structure. Destabilization of the structure will occur by the change of the positive arginine to the neutrally charged proline and by the introduction of a more rigid residue.

120

Table 1 Clinical evaluation of cone disorders with TULP1 mutation Patient 1 (52 yrs)

Patient 2 (36 yrs)

Origin

The Netherlands

Age at diagnosis

Visual acuity (refraction)

At onset: RE: 80/200 LE: 120/200

At onset: RE: 50/200 LE: 32/200

Current age: RE: CF LE: 10/200

Current age: RE: 10/200 LE : 10/200

Mild myopia (SE-3D)

High myopia (SE -12.5D)

Fundus

-Macular bull’s eye -Peripheral mottling -Vessels slightly attenuated -Optic disc mild pallor

Macular bull’s eye -Mid-periphery subtle RPE changes and far-periphery bone-spicules -Vessels severely attenuated -Optic disc moderate pallor

Autofluore scence

Central hyperfluorescence, paracentral ring of nonfluorescence surrounded by hyperfluorescence

Hypofluorescent macula surrounded by hyperfluorescent ring

OCT

Thinned retina; decreased photoreceptor layer in fovea and paracentral loss of RPE

Thinned retina; photoreceptor layer absent in posterior pole and loss of RPE

Color vision

Errors in all color axes (Ishihara, Panel D15)

Errors in all color axes (Panel D15, HRR)

Visual field (Goldmann)

-Central scotoma -Periphery normal

-Large central scotoma -Peripheral field loss and constriction

ERG

Responses within normal limits

Scotopic and photopic responses significantly reduced

CF = counting fingers, D = diopters, ERG = electroretinogram, HRR = Hardy-Rand-Rittler color vision test, LE = left eye, OCT = optical coherence tomography, RE = right eye, RPE = retinal pigment epithelium, SE = spherical equivalent Clinical features of patients with TULP1 mutations Table 1 shows a summary of the clinical findings in both patients with the TULP1 mutations. Both were re-invited to the clinic for a complete ophthalmologic examination. Patient 1 had experienced a rapid visual decline from 80/200 RE and 120/200 LE at age 43 to counting fingers RE and 10/200 LE at age 52 with a myopic refraction (SE-3D). Fundoscopy detected a slight bull’s eye maculopathy, which is more apparent on the autofluorescence image, and psychophysical testing showed severe color vision defects without a specific axis of confusion. Goldmann perimetry revealed a central scotoma with an intact periphery. Dark adapted electrophysiology showed a mild delay of the onset of the b-wave of the isolated rod responses. The rod b-wave amplitudes and latencies were normal. Mixed dark adapted responses and light adapted responses, including 30-Hz flicker were in the low range of two standard deviations. OCT imaging revealed general thinning of the retina, and parafoveally, loss of RPE and photoreceptors. FAF showed a distinct hyperfluorescent ring (Figure 3A-D, and 3I).

121 A novel TULP1 mutation as a cause of cone dysfunction

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Figure 3 Clinical evaluation of cone dysfunctions caused by the TULP1 mutation A-D Clinical characteristics of patient 1. A) The fundus photograph showed a slight macular bull’s eye, peripheral mottling, slightly attenuated vessels and a mild pallor optic disc; B) Autofluorescence revealed a more apparent macular bull’s eye, central hyperfluorescence and a paracentral ring of nonfluorescence surrounded by hyperfluorescence; C) optical coherence tomography (OCT) displayed a thinned retina and a decreased inner and outer segmental region in the fovea and paracentral loss of RPE; D) Goldmann perimetry showed a central scotoma and a normal periphery; E-H Clinical characteristics of the patient 2: E) Fundus photographs revealed a macular bull’s eye, subtle mid-peripheral retinal pigment epithelium (RPE) changes, bone-spicules in the far periphery, severely attenuated vessels and a moderate pallor of the optic disc; F) Autofluorescence showed a hypofluorescent macula surrounded by a hyperfluorescent ring; G) OCT displayed a thinned retina and absence of the inner and outer segments. Also note the posterior staphyloma around the optic disc; H) Goldmann perimetry revealed a large scotoma and peripheral field loss; I) Electroretinogram (ERG) of patient 1 and 2. In patient 1 dark adapted electrophysiology showed a mild delay of the onset of the b-wave of the isolated rod responses. The rod b-wave amplitudes and latencies were normal. Mixed dark adapted responses and light adapted responses, including 30-Hz flicker were in the low range of two standard deviations. In patient 2 ERG responses showed significantly reduced cone and rod responses. Patient 2 also had progressive visual decline, as the visual acuity dropped from 50/200 RE and 32/200 LE at age 24 to 10/200 RE and 10/200 LE at age 36 with a myopic refraction (SE-12.5D). Psychophysical testing showed color vision without a specific axis. Goldmann perimetry revealed a large central scotoma, with constriction of peripheral isopters as well. ERG responses deteriorated over the years from mild cone dysfunction to a significantly reduced cone-rod

122

pattern. The fundus showed a macular bullâ&#x20AC;&#x2122;s eye with subtle RPE changes, bone spicules in the far periphery, severely attenuated vessels and a moderate pallor optic disc. The autofluorescencence image pointed to central hypofluorescence surrounded by a hyperfluorescent area. The OCT revealed thinned retinal layers with absent RPE and photoreceptor layer in the fovea (Figure 3E-I).

Discussion

In the present study, we describe two unrelated individuals with cone dysfunction due to a novel homozygous TULP1 mutation (p.Arg420Ser). Both patients presented with progressive loss of visual acuity, bullâ&#x20AC;&#x2122;s eye maculopathy, color vision deficiencies, central scotomas, abnormal imaging of the central retina, and progressive loss of cone function on ERG, indicating predominantly cone-mediated disease. This mutation occurred in 0.4% of our COD/CRD cohort, indicating that it is an infrequent cause of cone dysfunction. Previous studies have associated TULP1 mutations with predominantly rod-mediated diseases such as Leber congenital amaurosis (LCA) and early-onset retinitis pigmentosa (RP).16-18 Hanein et al. (2004) considered TULP1 mutations to cause type 2 LCA, which is a severe and progressive form of rod-cone dystrophy with nightblindness and peripheral involvement19. Several other studies described TULP1 mutations in RP and LCA with extinguished rod and cone ERG responses, who at early stages presented with nightblindness, a hallmark of initial loss of rod photoreceptors.10; 16; 20-22 All missense mutations that were identified in early-onset AR RP and LCA affect residues in the highly conserved C-terminal Tubby domain, which underlines the importance of this region for the physiology of photoreceptor cells. Interestingly, a different mutation (p.Arg420Pro, Figure 2D) affecting the same arginine residue of TULP1 was described in two siblings with early-onset RP.16 In that family, p.Arg420Pro was present in a compound heterozygous state with another missense mutation (p.Phe491Leu), which also affects a conserved residue in the C-terminal Tubby domain. The mutation p.Arg420Pro is predicted to also result in loss of interactions and affect the stability in a similar way to p.Arg420Ser. The wide spectrum of phenotypes attributed to TULP1 missense mutations suggests that the Tubby domain, and in particular the region spanning the Arg420 residue, may exert important functions specific to either rods or cones.23; 24 Indeed, previous studies have shown that TULP1 is expressed in both photoreceptor subtypes.23 Several cellular functions have been suggested for TULP1. TULP1 has been shown to enhance phagocytosis by the retinal pigment epithelium (RPE), an important process for the removal of apoptotic cells and cellular debris.25; 26 A reduced phagocytosis is shown for TULP1 mutations located towards the C-terminus, but p.Arg420Pro appeared to have no effect on RPE phagocytosis.25 TULP1 has also been suggested to be involved in vesicular trafficking of photoreceptor proteins, like rhodopsin, 27 both at the nerve terminal during synaptic transmission and at the inner segment during protein translocation at the other segment.28; 29 Tulp1 interacts with Actin and Dynamin-1, both known to be crucial in the scaffold of the cytoskeleton, vesicle docking/cycling at the synaptic terminals of photoreceptors, and in vesicular protein transport from the inner to the outer segment. The latter is supported by the abnormal distribution of two rhodopsin transport machinery proteins, Rab8 and Rab11, and the mislocalization of guanylate cyclase activation proteins GCAP1 and GCAP2 in Tulp1-/- mice. 30 Interestingly, synaptic malfunction after photoreceptor degradation was also observed in these mice, indicating that Tulp1 may also play a role in photoreceptor synapse development. 31 Both rod and cone opsins are mislocalized in Tulp1-/- mice. 32 Although the exact region involved in opsin transport remains to be elucidated, we speculate that the p.Arg420Ser mutation may have a more pronounced effect on cone opsin transport and a lesser effect on rhodopsin transport in rod photoreceptors, opposed to p.Arg420Pro and other missense mutations that cause early-onset RP or LCA. As the p.Arg420Ser and p.Arg420Pro mutations introduce different

123 A novel TULP1 mutation as a cause of cone dysfunction

3.1

side chains at the protein surface, they might have a different effect on protein interactions involved in either rod or cone opsin transport. Both patients carrying the p.Arg420Ser mutation had similarities in their clinical presentation, but we also noted several remarkable differences. Patient 2 had a younger age of onset (age 24) than patient 1 (age 43), was more severely affected, and his ERG showed significantly reduced cone and rod responses (Figure 3I), whereas patient 1 showed a mild delay, but normal amplitudes and latencies in the b-wave responses. Maybe the presence of high myopia may have deteriorated the clinical course in patient 2. Alternatively, patient 2 may carry additional deleterious alleles in other genes which contribute to the pathology, or patient 1 may carry protective alleles reducing photoreceptor degeneration. Interestingly, a recent screen for modifier loci identified a protective allele near Mtap1a, which reduces photoreceptor loss in Tulp1-/- mice. 33 Patient 1 had a significant hyperautofluorescent macular ring, which was even observed on the nasal side of the optic disc. A macular ring has been described in many RP patients, 34-36 but to our knowledge has not been described in patients with TULP1 mutations. The ring is considered to be the result of abnormal accumulation of the fluorophore lipofuscin in the RPE due to outer segment degeneration. The size and growth of this ring appears to correspond to the degree of abnormal retinal function and visual prognosis. 34 In this study, UPD facilitated the finding of the causal TULP1 mutation in patient 1. UPD is a phenomenon involving the inheritance of two homologous chromosomes from one parent.15 UPD allows two copies of a recessive mutation to be transmitted from a heterozygous carrier parent. 37 The majority of such cases appear to be associated with advanced maternal age. 38 This is in agreement with the observed UPD in this report, as the patientâ&#x20AC;&#x2122;s mother was 45 years old when he was born. UPD is a rare phenomenon, with an estimation of 1 in 5,000 or even less. 39 This is confirmed by the fact that of 156 COD/CRD, 186 RP and 93 LCA patients that were genotyped with SNP microarrays, the patient described in this study is the only case in which we detected homozygosity across an entire chromosome.8; 10 To date, seven cases of maternal UPD of chromosome 6 have been reported in literature.40-46 In several of these cases intrauterine growth retardation was observed. The mother of patient 1 had a normal pregnancy, but the patient was born with a very low birth weight, indeed suggesting intrauterine growth retardation. Chromosome 1 and 2 UPDâ&#x20AC;&#x2122;s were found in which mutations in MERTK, RPE65, USH2A were associated with retinal dystrophies.47-49 In conclusion, through homozygosity mapping of patients with cone disorders, a case with maternal UPD of chromosome 6 was identified. This unmasked a mutation in TULP1 as a novel cause of cone dysfunction in two unrelated patients. Our study expands the disease spectrum of TULP1 mutations from early-onset RP to cone-dominated disease. The observed phenotypic difference in patients carrying mutations affecting the same amino acid residue (p.Arg420Ser and p.Arg420Pro), may be a result of different structural alterations at the TULP1 protein surface, leading to defective opsin transport in either cones and/or rods. Further research is required to decipher the molecular mechanisms associated with the different diseases caused by TULP1 mutations, and will aid a better understanding of the function of TULP1 in cones and rods.

Acknowledgements

Supported by the Foundation Fighting Blindness USA (grants BR-GE-0510-04890RAD and C-GE-0811-0545-RAD01) (to A.I.d.H, F.P.M.C.), Prof. Dr. H.J. Flieringa Foundation SWOO, the Rotterdam Eye Hospital (to L.I.v.d.B, F.P.M.C., A.I.d.H.).

124

Supplemental Table 1 Chromosome 6 retinal disease gene primers Gene & exon

Forward

Reverse

Product size (bp)

1-2

gtgaagttgagccgagattag

taccctgcgtgggtttac

368

ctgcggttatttctggcggc

gcttccattcagcttcccac

408

gaagtgttgaaagtggaaacc

ccttctctccttagctccac

364

cggttcttgtttcagaagg

cctcaatcgctgtgtctc

376

6-7

gactataggcaggacaagagg

tccttacctagcactgtcttg

481

gaagcactttgcaaaccag

tctaggctcccaagtccag

238

9-10-11

ccagagcctcctaacttgg

cacagcaggacagagatgac

733

12-13

tgaattgctcagtcctaactc

agttggctatttcctaagctg

499

agccatctcagccatctc

cttgaatgaaggtcagcatc

296

gtgttgagtaactgagatggtg

gacacaggagcagttttcc

384

ttgaggagcaggagaagacg

tgatccgcagcatccgaaag

374

ttgggactcaaaggacagtg

atgccagaacagctaataagg

570

cacagtaacttctagcaatcg

cataccactgcacttcagtc

412

ccatgccttgtacattttgtg

tgacagagcgagactccatc

477

acaactgtgaaagtcctttgc

ataactgcgatatagctggag

520

aaatttgggcctgtgatagc

tcctttgcttctgttttccc

458

ttatgtcagcctgcacatgg

gtagttgtgttcagctaggc

286

agctaaaggcaggatactgg

atggaaagcagggaatgagg

315

gaagactcattctaggttagtc

cactgcaaagatagtgtcacc

472

cttaaaacaccattttgcagc

atgtgtcccaacactcagcc

498

acttctacagagattgctgg

agattcctggcagaactgc

376

gtggtccatcaccttgtcc

tagagacggggtttcaccg

486

agaattgagggaaaactgatgg

cataaaagagttcagtatatatacc

485

tctatgctcatttcttctttccttc

aaaataagtagaccgttcttgttcg

403

ttctccaggtaagaacccattc

ttaagtaaaagttagggttaaaaccag

311

ttggaataatgttaataggcttttc

tggctaagattaataagagcatttg

285

ggcttttgaacatggatatgac

agatttcctaggatgtagttggtg

559

ggaacttattttgtggcagatg

gactgttgagaatttgtttacgaag

427

ggtttcatcttagtagacagagaggc

cattgttaccatgaaacagttcg

368

tgcaccccacaactatcttc

aattgcccaaagaagcaatc

570

ttcagatgtcatcctaagtgg

cagacaagagacagaagtgc

293

ggatattttcattgttgctttgc

tgaatccaataagtgaacagtttg

655

gagatatcaaaatggccaggag

atcccaaggacactgagcac

273

caccacatactattagttcaag

attttaggaggccatcatcc

447

17-18

attctttagactaccactgattc

acataatgagcacatgtgtgc

551

TULP1

ELOVL4

EYS

125 A novel TULP1 mutation as a cause of cone dysfunction

3.1

Supplemental Table 1 (continue) Gene & exon

Forward

Reverse

Product size (bp)

agcagaacaaaattttgcaagg

catctggcagattatttcagg

425

agagaggtcttcatttcttgg

agctcttgttttatgaaagagc

395

aaacccaggaataaactctgc

ggaagaaatgactctgaaacc

322

gtcaaacaagtttgcacatcag

gaaagcaaaacatgggagtg

531

aactcattgtcaccccaagg

tcctgatgaaagcctaagtgc

486

aatggcaccacggataagag

gaggaatgccgagaaaagtg

572

gagctatccaatatgtctatgg

gcagaaaatatgcttttaccac

378

26A

ttatcccagtgccaaagtgg

agtaatgactgcctgtttagc

525

26B

actgttgctttatctgctacc

gaacatgttgcacatgtttgg

434

26C

tactgaggcttcaagcaacc

gtgggtcccatagttatgcc

549

26D

atccattgtcccttcacaaac

tacagacatggaggaagacg

439

26E

gatttttcagaagtcaccacc

ctgattacaatgaggctgttc

410

aaagaggcaggaaagagacg

aggaagagacatcctggtgg

443

acttcatgtctctccaaagtg

attgttagggatagcctttgc

273

tgcttctggctttgttttattg

tggaaaaacagactgacattgg

521

tgcataccctgaccagtttg

cgtaggaatgtgaagcaaaaac

335

gttaaaccttgatcagtattgg

acagctgtttcttgtttgtgc

475

tgcttcatgcactggtctgg

gtgttaccttttcaaatgaatgc

552

ctaatagcactcctaccaacc

tctgtagtcccaactacttgg

465

cttgaaaatgtccacacttgg

tttctggtgctttgttgagag

405

aactagccaacaatagcaacc

ctctcagaggacaatactgc

447

agtggaaagcacacagctac

ttgatcagtcaagtgctatcc

458

ttagttgctcaatgctgaagg

caattagagtgtccctgagg

410

cagccagttgcacatatacc

gtgaacttcgtggatgtagg

438

agcagagaattgagtggtatc

gaaagcaatccatatagctgg

410

tctctgcgcatttctgtattc

atgtgcatctgtttgtgttcc

438

aaattgacaagttagcatcagg

aagtactagtctatctgtgaag

432

gaactgcaggacagatgtac

cctaattctaagctccaatcc

323

ttgatgtactcacctacaagc

acgcatacacttgcagtgac

444

44A

cacaattgtgctcaagatctg

tacatttgagccaccttttgc

511

44B

tgaacctgtaggttttcaagg

tgaactggaggtttctcattc

422

44C

ttcttacagttgcctgtgtac

tttatgtggatcaatatcctcg

424

44D

ttctgttgtaataaatggcacc

acaatcagaaccttcagtgac

394

tgacagatgctcctccagaa

tcaattggtagccttgttgg

435

tagcagttgcaccacggtag

tgaatgaaagggaagggatg

430

actttgcttgggattgggta

cccttacgttgtggaaacca

329

ggtttccacaacgtaagggtta

acacagctcttagtggctgaca

346

tgcttacggaaaacactggag

catgatggttgtttccaaactg

543

aaaagatgagttcagaaggcac

cccctcagtacctaggaagaag

422

IMPG1

126

Product size (bp)

Gene & exon

Forward

Reverse

Product size (bp)

425

ttgattgcaaaacacttaagcc

tatgtcctatcggctcccac

335

395

ccattttcagctgttcccc

tccaggatttggcagagc

294

322

ccattcaaattctgatggcag

cattgaccttggaaa ggagc

620

531

gagcccatcaggaaagagg

acacaggaaggagtgagaaaag

533

486

ggactgttttattgtcccaacc

tctttggtggaaggttttgag

212

572

aggcagattcagttatccgag

aaggaaggatgctgtggatg

508

378

13-1

aatgatctacgcaaatgctatg

ctgggacagaaacatattcgc

459

525

13-2

tgcatcttcagatgacagcc

gcttggcggtttgtttctac

443

434

aattagcagcagctgagcac

tgtggcctaaagattgaaacg

375

549

ggtctcttgcctttgactgc

aaccatgggttgaaaggacc

381

439

ctcaaacggaagacacctgc

tgcactagaaaatgtcacccc

368

410

tggcaagcacatcctcttatc

tgtctggattttgatgaccc

288

443 273

LCA5

521

caatgggagctcgggtag

tccctgctttaagaaccacc

499

335

tcctaggagtggtctcatttcc

tctgttctcgcattactgagg

248

475

tgtggagaaaatagattgcacag

cctataaaacgtaaatcagcccac

465

552

4.1

acggacccacttgtgttagg

gttgcccgttctttctcttg

477

465

4.2

aggtcaagttagctgagctgc

tccaagcaaataccaaactgc

457

405

ttggtaaatctgtttcccagc

ttataccaacaaaccttttctaagtg

461

447

tgacaaagtgaggggttttg

tcaatgaggttcttttccttcc

269

458

ccaagctgagcaaaacatgc

ttaggtatatctcctaaaagccaaag

538

410

tctgtgttgcttagttcccc

tttctttctcaagggatgctg

256

438

9.1

aaatatggtggttttatgaaagttg

ttttgactaaatggatttgacctc

578

410

9.2

gaagccccaaaacatacagg

ttggcaaactatctatgtggtg

765

438 432

RDS

323

gtgggaagcaacccggac

agatcttcccagccagcg

276

444

tgatagggatgggggtgc

ctgtgtcccggtagtacttc

243

511

gctcgctggagaacaccct

tctgaccccaggactggaag

239

422

aagcccatctccagctgtct

cttaccctctacccccagctg

314

424

agattgcctctaaatctcctc

tgcactatttctcagtgttcg

290

394

435 430 329 346 543 422

127 A novel TULP1 mutation as a cause of cone dysfunction

3.1

Supplemental table 2 Homozygous regions identified in patient 1 Ranking

Chromosome

Start position

End position

Size (Mb)

# Homozygous SNPs

Ranking

Chromo

67,106,320

170,719,369

170.6

27168

46,212,732

57,489,108

11.3

779

56,930,577

67,061,694

10.1

762

105,661,099

107,446,812

1.8

413

78,334,621

79,781,601

1.4

258

113,437,540

114,518,218

1.1

220

109,214,236

110,409,180

1.2

217

56,453,118

58,215,268

1.8

211

112,050,154

113,057,813

1.0

210

Supplemental table 3 Homozygous regions identified in patient 2 Ranking

Chromosome

Start position

End position

Size (Mb)

# Homozygous SNPs

3,906,287

47,108,480

43.2

8165

212,339,909

239,532,850

27.2

5038

76,059,943

105,721,548

29.7

4427

85,585,248

115,108,714

29.5

4389

57,173,572

81,551,203

24.4

4346

12 20 9

62,239,467

87,333,478

25.1

4122

23,760,240

47,043,376

23.3

3229

64,757,325

85,923,635

21.2

3099

118,369,943

127,815,378

9.4

2016

135,645,476

148,658,165

13.0

1854

1459

6 5

128,991,071

138,407,083

9.4

117,702,425

122,959,129

5.3

891

22,173,106

25,198,128

3.0

657

11 12 10

48,367,867

52,695,154

4.3

563

1,841,598

3,813,951

2.0

449

25,382,609

28,102,584

2.7

367

78,696,092

81,235,379

2.5

308

33,620,575

38,489,062

4.9

297

53,707,089

54,918,720

1.2

287

10,417,491

12,008,371

1.6

285

150,071,531

151,464,242

1.4

282

52,879,681

53,963,968

1.1

264

58,845,146

60,201,267

1.4

263

57,270,662

63,224,267

6.0

255

58,984,219

60,612,388

1.6

214

47,841,423

50,234,746

2.4

207

80,592,770

82,065,456

1.5

206

25,222,938

26,215,931

1.0

203

128

3.1

129 A novel TULP1 mutation as a cause of cone dysfunction

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131 A novel TULP1 mutation as a cause of cone dysfunction

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40, 2795-2802. 33. Maddox, D.M., Ikeda, S., Ikeda, A., Zhang, W., Krebs, M.P., Nishina, P.M., and Naggert, J.K. (2012). An allele of microtubule-associated protein 1A (Mtap1a) reduces photoreceptor degeneration in Tulp1 and Tub Mutant Mice. Invest Ophthalmol Vis Sci 53, 1663-1669. 34. Chen, R.W., Greenberg, J.P., Lazow, M.A., Ramachandran, R., Lima, L.H., Hwang, J.C., Schubert, C., Braunstein, A., Allikmets, R., and Tsang, S.H. (2012). Autofluorescence imaging and spectral-domain optical coherence tomography in incomplete congenital stationary night blindness and comparison with retinitis pigmentosa. Am J Ophthalmol 153, 143-154. 35. Murakami, T., Akimoto, M., Ooto, S., Suzuki, T., Ikeda, H., Kawagoe, N., Takahashi, M., and Yoshimura, N. (2008). Association between abnormal autofluorescence and photoreceptor disorganization in retinitis pigmentosa. Am J Ophthalmol 145, 687-694. 36. Lima, L.H., Cella, W., Greenstein, V.C., Wang, N.K., Busuioc, M., Smith, R.T., Yannuzzi, L.A., and Tsang, S.H. (2009). Structural assessment of hyperautofluorescent ring in patients with retinitis pigmentosa. Retina 29, 1025-1031. 37. Fassihi, H., Wessagowit, V., Ashton, G.H., Moss, C., Ward, R., Denyer, J., Mellerio, J.E., and McGrath, J.A. (2005). Complete paternal uniparental isodisomy of chromosome 1 resulting in Herlitz junctional epidermolysis bullosa. Clin Exp Dermatol 30, 71-74. 38. Kotzot, D. (2004). Advanced parental age in maternal uniparental disomy (UPD): implications for the mechanism of formation. Eur J Hum Genet 12, 343-346. 39. Liehr, T. (2010). Cytogenetic contribution to uniparental disomy (UPD). Mol Cytogenet 3, 8. 40. van den Berg-Loonen, E.M., Savelkoul, P., van, H.H., van, E.P., Riesewijk, A., and Geraedts, J. (1996). Uniparental maternal disomy 6 in a renal transplant patient. Hum Immunol 45, 46-51. 41. Spiro, R.P., Christian, S.L., Ledbetter, D.H., New, M.I., Wilson, R.C., Roizen, N., and Rosenfield, R.L. (1999). Intrauterine growth retardation associated with maternal uniparental disomy for chromosome 6 unmasked by congenital adrenal hyperplasia. Pediatr Res 46, 510513. 42. Cockwell, A.E., Baker, S.J., Connarty, M., Moore, I.E., and Crolla, J.A. (2006). Mosaic trisomy 6 and maternal uniparental disomy 6 in a 23-week gestation fetus with atrioventricular septal defect. Am J Med Genet 140, 624-627. 43. Parker, E.A., Hovanes, K., Germak, J., Porter, F., and Merke, D.P. (2006). Maternal 21-hydroxylase deficiency and uniparental isodisomy of chromosome 6 and X results in a child with 21-hydroxylase deficiency and Klinefelter syndrome. Am J Med Genet 140, 2236-2240. 44. Salahshourifar, I., Halim, A.S., Sulaiman, W.A., and Zilfalil, B.A. (2010). Maternal uniparental heterodisomy of chromosome 6 in a boy with an isolated cleft lip and palate. Am J Med Genet 152A, 1818-1821. 45. Gumus, H., Ghesquiere, S., Per, H., Kondolot, M., Ichida, K., Poyrazoglu, G., Kumandas, S., Engelen, J., Dundar, M., and Caglayan, A.O. (2010). Maternal uniparental isodisomy is responsible for serious molybdenum cofactor deficiency. Dev Med Child Neurol 52, 868-872. 46. Sasaki, K., Okamoto, N., Kosaki, K., Yorifuji, T., Shimokawa, O., Mishima, H., Yoshiura, K.I., and Harada, N. (2011). Maternal uniparental isodisomy and heterodisomy on chromosome 6 encompassing a CUL7 gene mutation causing 3M syndrome. Clin Genet 80, 478-483. 47. Thompson, D.A., Gyurus, P., Fleischer, L.L., Bingham, E.L., McHenry, C.L., pfelstedt-Sylla, E., Zrenner, E., Lorenz, B., Richards, J.E., Jacobson, S.G., Sieving, P.A., and Gal, A. (2000). Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration. Invest Ophthalmol Vis Sci 41, 4293-4299. 48. Rivolta, C., Berson, E.L., and Dryja, T.P. (2002). Paternal uniparental heterodisomy with partial isodisomy of chromosome 1 in a patient with retinitis pigmentosa without hearing loss and a missense mutation in the Usher syndrome type II gene USH2A. Arch Ophthalmol 120, 1566-1571.

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49. Thompson, D.A., McHenry, C.L., Li, Y., Richards, J.E., Othman, M.I., Schwinger, E., Vollrath, D., Jacobson, S.G., and Gal, A. (2002). Retinal dystrophy due to paternal isodisomy for chromosome 1 or chromosome 2, with homoallelism for mutations in RPE65 or MERTK, respectively. Am J Hum Genet 70, 224-229.

3.1

133 A novel TULP1 mutation as a cause of cone dysfunction

Susanne Roosing,, Frans P.M. Cremers, Marijke N. Zonneveld-Vrieling, Herman Talsma, Francoise J.M. Klessens-Godfroy, Frans C.C Riemslag, Anneke I. den Hollander, L. Ingeborgh van den Born

Manuscript in preparation 134

Chapter 3.2 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

135

3.2

Abstract

Objectives: To identify the gene defect and to study the clinical characteristics and natural course of disease in a family diagnosed with oligocone trichromacy (OT). Methods: Extensive clinical and ophthalmologic assessment was performed for two siblings with OT. Subsequently, next generation sequencing (NGS) and Sanger sequence analysis of CEP290 was performed in the two siblings. Additionally, the identified CEP290 mutations were analyzed in persons with ACHM (n=23) and autosomal recessive or isolated cone dystrophy (n=145). Results: In the first decade of life, the siblings were diagnosed with OT based on low visual acuity, photoavesion, nystagmus, an absent cone response on electroretinography (ERG), but with normal color vision. Over time, the degenerative phenotype progressed from the periphery towards the macula. The siblings did not reveal any CEP290 associated non-ocular features. In both siblings NGS yielded a heterozygous nonsense mutation (c.451C>T; p.(Arg151*)) in CEP290. Subsequent Sanger sequencing revealed a second nonsense mutation (c.4723A>T; p.(Lys1575*)) in CEP290. mRNA analysis demonstrated nonsense-mediated altered splicing, confirming a suspected hypomorphic character of p.(Lys1575*), as exon 36 skipping was observed in a small fraction of CEP290 mRNA, which has previously also been demonstrated for the c.451C>T (p.Arg151*; p.Leu148_Glu165del) mutation. No additional cases carrying these variants were identified in the ACHM and CD cohorts. Conclusions: Compound heterozygous hypomorphic mutations in CEP290 may lead to OT, a novel phenotype belonging to the CEP290-associated spectrum of ciliopathies. Clinical relevance: These findings provide insight into the effect of CEP290 mutations on the clinical phenotype.

136

Introduction

Centrosomal protein 290 (CEP290, MIM#610142) encodes a centrosomal and ciliary protein of 290 kDa that is expressed in a variety of tissues. CEP290 has been shown to play an important role in ciliary trafficking and cilium assembly.1; 2 In the photoreceptor, CEP290 localizes to the connecting cilium, the transitional zone linking the inner and outer segments of rods and cones.1; 3-5 To date, more than one 100 CEP290 mutations have been identified leading to a spectrum of phenotypes ranging from isolated early-onset retinal dystrophy and Leber congential amaurosis (LCA, MIM#611755) to more severe syndromes like Senior-Loken, Bardet-Biedl, Joubert, and Meckel-Gruber syndromes.4; 6-9 Hypomorphic CEP290 mutations are generally associated with non-syndromic forms of LCA, and account for an estimated 15% of all LCA cases in the Caucasian population.4; 10; 11 Clinically most of these LCA patients, but not all, present with blindness from birth with nonrecordable electroretinograms.11-13 Littink et al. described a family with better preserved central vision; these patients were heterozygous carriers of the frequently occurring deep-intronic c.2991+1655A>G variant, which is a hypomorphic variant, as about 50% of the resulting mRNA contains a stopmutation in the inserted intronic sequence.4 On the other allele, the c. 451C>T (p.Arg151*) variant was identified.14 The latter was found to result in nonsense-associated altered splicing with an intact reading frame, possibly explaining the milder phenotype. In two patients of the family residual rod function was measured on eletroretinography (ERG). Coppieters and Perrault also suggested that CEP290-associated LCA is of the cone-rod type (LCA type I), despite the relatively well-preserved central macula.11; 15 In this study we describe a family with an unusual form of cone dysfunction evolving into a progressive dystrophy caused by hypomorphic CEP290 mutations. The two siblings were diagnosed as oligocone trichromates by Van Lith who described oligocone trichromacy (OT) as â&#x20AC;&#x2DC;general cone-dysfunction without achromatopsia (ACHM)â&#x20AC;&#x2122; in 1973.16 At that time, he described a boy of 13 years with nystagmus, photophobia, severely reduced visual acuity, normal fundi, normal rod responses and a remnant of cone function on ERG, suggesting a form of achromatopsia. Color vision however was unremarkable, leading Van Lith to postulate that all three types of cones must be present but reduced in number. Since then, eleven cases of OT with a similar clinical presentation have been described with severely reduced or non-recordable cone responses on ERG and normal or near normal trichromatic color vision. Concomitant signs may include nystagmus and photophobia. Mutations in CNGA3 (MIM#600053)17, CNGB3 (MIM#605080),18 GNAT2 (MIM#139340),19 and PDE6C (MIM#600827)18 have been detected in cases with OT, all of which encoding cone phototransduction pathway proteins,18 suggesting a pathomechanistic overlap with ACHM. In a number of these cases a single heterozygous missense variant has been identified, indicating that these cases may not be explained by mutations in these genes.18; 19 Unlike ACHM, no progressive deterioration of cones and visual function has been reported in the small number of described OT cases, so far. Here, we report 40 years follow-up of two siblings initially diagnosed with OT, but over time developed a progressive retinal dystrophy. The disease in these siblings was found to be caused by two hypomorphic variants in CEP290, and subsequently broadening the spectrum of CEP290associated disease.

Methods

The research procedures followed the tenets of the Declaration of Helsinki (1983) and were approved by the local ethics committee. Written informed consent was obtained from the subjects after explanation of the nature of the study. The father was Dutch and the mother originated from Belgium.

137 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

3.2

Clinical examination The first ophthalmic examinations of the subjects date from 1971, at ages 2 and 3. Since then they were followed regularly and underwent electrophysiology and color vision testing (Hardy Rand Rittler (HRR), Pseudoisochromatic Plates and Farnsworth Panel D-15 Test, saturated and desaturated version) at several occasions. Recent examination included best corrected visual acuity (Early Treatment Diabetic Retinopathy Study charts, Precision Vision Inc., La Salle, IL, USA), biomicroscopy, ophthalmoscopy, fundus photography, color vision testing (Panel D-15), and Goldmann kinetic perimetry. Extended ERG testing was performed according to the protocol of the International Society for Clinical Electrophysiology of Vision (ISCEV).20 Spectral domain optical coherence tomography (OCT) and fundus autofluorescence (FAF, Heidelberg Spectralis HRA+OCT, Heidelberg, Germany) were carried out as described previously,21 but failed partially due to photophobia and nystagmus. Both individuals were also seen by an internist to evaluate the presence of extra-ocular features. This included physical examination and extensive laboratory testing. Genetic testing To determine the genetic defect in the family with two affected siblings, blood samples for molecular genetic analysis were collected from all family members (Figure 3A). Genomic DNA was isolated from lymphocytes by standard procedures.22 DNA samples of both affected individuals was analyzed using GeneChip Genome-Wide Human single nucleotide polymorphism array 5.0 NspI (Affymetrix, Santa Clara, CA, USA), genotypes were called using Genotype Console (Affymetrix, Santa Clara, CA, USA) and homozygous regions were calculated with Partek Genomics Suite software (Partek, St. Louis, MO, USA). Using Sanger sequencing six genes known to be mutated in ACHM and cone dystrophy (CD, MIM#120970), (ABCA4 (MIM#601691), CNGA3, CNGB3, KCNV2 (MIM#607604), PDE6C and PDE6H (MIM#601190)), were excluded as the cause of their disease. Subsequently, DNA samples of the affected siblings, were analyzed by exome sequencing. The exomes were enriched according to the manufacturer’s protocol using Agilents’ SureSelect Human All Exon v.2 Kit (50Mb), which contains the exonic sequences of approximately 21,000 genes (Agilent Technologies, Inc., Santa Clara, CA, USA), and next generation sequencing was performed on a SOLiD4 sequencing platform (Life Technologies, Carlsbad, CA, USA). LifeScope software v2.1 (Life Technologies, Carlsbad, CA, USA), was used to map color space reads along the hg19 reference genome assembly. The DiBayes algorithm, with high-stringency calling, was used for single-nucleotide variant calling. The small Indel Tool was used to detect small insertions and deletions. Sequence analysis of the CEP290 gene The 54 exons and exon-intron boundaries of CEP290 (GenBank NM_025114) were screened in the DNA of the individuals for the presence of a second mutation using the primers previously described.23 Sequencing and segregation analysis was performed by Sanger sequencing. Subsequently, a subset of 23 ACHM and 145 CD individuals, mainly from the Netherlands, were specifically analyzed for the occurrence of the c.451C>T and c.4723T>A mutations. Analysis of hypomorphic character of the c.4723T>A variant The determine the effect of c.4723T>A on the CEP290 mRNA, RNA was isolated from EBVtransformed lymphocytes of the affected persons and three control individuals according to manufacturer’s protocol (Qiagen, Venlo, The Netherlands). The lymphocytes of the affected individuals were grown with or without cycloheximide to visualize the effect of the mutation and possible degradation of nonsense-containing mRNAs by nonsense mediated decay (NMD).24 Reverse transcription with iScript (Biorad, Veenendaal, The Netherlands) was performed on total RNA (1 µg) to obtain cDNA. RT-PCR experiments were performed using 2.5 µl cDNA with primers in exon 34 and 37 (35 cycles), with a subsequent nested PCR using 0.5 µl of the PCR-product and initial primers (15 cycles), followed by Sanger sequencing.

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Figure 1 Retinal imaging of two individuals with CEP290 variants A–B, Clinical characteristics of patient II-1 of family A. A) The fundus photograph showed a pink optic disc and mildly attenuated vessels. The retinal pigment epithelium (RPE) had a coarsegrained aspect in the posterior pole and mid-periphery, and a faintly recognizable foveola reflex with a subtle indication for bull’s eye maculopathy. B) Fundus autofluorescence (FAF) revealed a relatively normal appearance. D–F, C) Fundoscopy displayed a pink, myopic optic disc, mildly attenuated vessels and subtle RPE changes in the macula. A faintly recognizable foveola reflex

139 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

was noted, as well as perimacular RPE atrophy. Clinical characteristics of patient II-2 of family A. D) Autofluorescence of the right eye revealed a double hyperfluorescent ring and hypofluorescent spots along and inferior of the inferior vascular arcade. E) Optical coherence tomography (OCT) displayed no discernible photoreceptor complexes at the macula, but they were present at the peripheral part of the scan. F) Autofluorescence of the left eye revealed a double hyperfluorescent ring and hypofluorescent spots along and inferior of the inferior vascular arcade as well as the macula. Figure 3

Figure 2 Peripheral fundus imaging of individual II-2 A compilation of fundus photographs of the left eye of II-2 showed a pink and myopic optic disc. Mildly attenuated vessels and a bullâ&#x20AC;&#x2122;s eye maculopathy. A faintly recognizable foveola reflex was noted, as well as perimacular RPE atrophy, mild RPE changes in the superior quadrants. More pronounced RPE atrophy in inferior quadrants with bone-spicule pigmentations was documented.

Results

Clinical findings The patients were initially examined at the ages of 2 and 3 years, and presented with low visual acuity, severe photophobia and pronounced nystagmus. ERGs were performed under general anesthesia and revealed normal rod responses with absent cone responses. The fact that they could name colors correctly puzzled Van Lith, but they were too young to perform a color vision

140

test. A few years later, they were capable of executing the HRR, and Panel D-15 test correctly, leading to the diagnosis of OT. A remnant of cone function was measured on ERG of the youngest sib at the age of 13. Visual acuity was between 20/200 and 20/125 and remained stable over the years, while the youngest sibling developed myopia. Around the age of 27, color vision with the Panel D-15 test was still normal, but they developed more difficulties with pseudoisochromatic plates. ERG rod function was also reduced and perimetry revealed mild visual field defects. At the age of 39, the youngest patient presented with altitudinal visual field defects, threatening the macular area of the left eye. His sister had in the meantime developed tunnel vision. On funduscopy, both individuals had degenerative changes mainly in the inferior quadrants consisting of RPE atrophy with bone-spicule pigmentations. In the youngest patient, the degeneration had progressed towards the macula and small atrophic spots around the fovea were observed in the left eye imposing as a bullâ&#x20AC;&#x2122;s eye-like maculopathy (Figure 1C). It has to be taken into account that his myopia might also have played a role in the atrophic changes. In the past six years, a further deterioration of the visual fields and rod ERGs was observed, whereas visual acuity and color vision were still relatively well preserved. The data of the most recent examination are presented in Table 1, Figure 1 and 2. Due to photophobia and nystagmus, OCT and FAF failed partially. Interestingly, a double ring sign was observed on the FAF of the youngest patient, indicating the transitional zone between present and absent photoreceptors on OCT (Figure 1D). Examinations by an internist did not reveal any extra-ocular abnormalities that might be associated with the CEP290 variants. Genetic analysis Homozygosity mapping in the two affected siblings revealed two small overlapping homozygous regions of 1.1 Mb on chromosome 8 and a 1.0 Mb on chromosome 11 (Supplemental table 1). For the exomes of person II-1 and II-2, 49,732,464 and 55,772,367 reads were uniquely mapped to the gene-coding regions. The analysis uncovered 36,457 variants for individual I-11 and 37,944 variants for individual II-2. A total of 3,525 functionally relevant variants, occurring with a frequency of less than 1% in 1,302 individuals in an in-house exome database, were shared by both individuals (data not shown). As a control for initial sequencing analysis, genes known to be involved in ACHM were checked for pathogenic variants, but none were observed. Variants were included for further analysis when they met our criteria of a frequency <0.5% in genetic variant databases, as well as the in-house controls (n=2,604 alleles), and resulted in a nonsense, frame shift, (canonical) splice site or missense variant with a PhyloP >2.7. Under the hypothesis of autosomal recessive inheritance, we did not identify two heterozygous variants in one gene (>20% variation) in the shared variants among the siblings and also no homozygous (>80% variation) variants based were identified. Analysis of the two small overlapping homozygous regions (1.0 Mb and 0.9 Mb) in the NGS data showed a single variant in the largest region, which was excluded to be functionally relevant as it resided in a non-genic region. The second overlapping region for the siblings contained four variants present in both individuals, which were all discarded for functional relevance and high frequency. An expanded analysis of variants in known retinal disease genes for CD, cone-rod dystrophy (CRD, MIM#120977), LCA and retinitis pigmentosa (MIM#268000) yielded a single heterozygous nonsense variant (c.4723T>A; p.(Lys1575*)) in exon 36 of CEP290 in both affected persons, which was confirmed by Sanger sequencing. Further analysis of CEP290 in the exome data did not reveal a second hit, but as coverage of CEP290 was incomplete, all coding exons and intron-exon boundaries were analyzed by Sanger sequencing. This resulted in the identification of a second nonsense mutation (c.451C>T; p.(Arg151*)) in exon 7 in both siblings which segregated with the disease in the family (Figure 3A). Both mutations were not identified in the exome variant server (EVS, http://evs.gs.washington.edu/EVS/).

141 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

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Table 1 Clinical characteristics of two patients with variants in CEP290 Patient ID

II-1

II-2

Sex

Female

Male

Age at diagnosis

Age recent examination

Nystagmus

Present

Visual acuity RE

20/100+

20/80-

Visual acuity LE

20/1002+

20/160

Refraction

RE +1.25-2.25x19; LE: -0.25-3.00x170

RE: -14.25-1.75x5; LE: -10.25-2.25x155

Lens

Clear

Fundus

Pink optic discs, mildly attenuated aterioles, coarse-grained aspect RPE posterior pole and mid-periphery superior quadrants, faintly recognizable foveola reflex BE with subtle indication for bull’s-eye maculopathy, RPE atrophy in far periphery of superior quadrants, RPE atrophy in mid- and far periphery of inferior quadrants with scarce bonespicule pigmentations

Pink, myopic optic discs, mildly attenuated arterioles, subtle RPE alterations macula RE, bull’s-eye maculopathy LE, faintly recognizable foveola reflex BE, perimacular RPE atrophy, mild RPE changes superior quadrants, RPE atrophy inferior quadrants with bone-spicule pigmentations

Fundus autofluorescence

Relatively normal

Double hyperfluorescent ring, hypofluorescent spots along and inferior to the inferior vascular arcade BE, and in macula LE

OCT

Failed

No discernible photoreceptor complexes at the macula, but present at the peripheral part of the scan.

Color vision (Panel D-15)

Saturated: normal Desaturated: minor errors RE, multiple errors. LE, no specific axis

RE: de- and saturated: multiple errors mainly in tritan axis LE: failed

Visual field (Goldmann)

Radius < 100

Altitudinal defect BE, partially including the centre RE, central scotoma LE

ERG Dark adapted

Mildly reduced isolated rod responses with significantly reduced rod derived ‘mixed’ responses

Significantly reduced isolated rod and rod derived ‘mixed’ responses

ERG Light adapted

Non-recordable

Miscellaneous

Anorexia, depressions, followed by the diagnosis of schizofrenia at age 29

None

BE = both eyes, ERG = electroretinogram, LE = left eye, OCT = optical coherence tomography, RE = right eye, RPE = retinal pigment epithelium. Analysis of the hypomorphic character of c.4723A>T. Compared to the thus far described phenotypes associated to CEP290 mutations, the affected individuals of this family showed a relatively mild non-syndromic retinal phenotype. We hypothesized that the c.4723A>T variant potentially had a hypomorphic character as was already experimentally proven for this families’ other allele (c.415C>T)14, because the exon consists of 108 bp. If deleted, the resulting open reading frame will miss the coding information for 36 amino acids (aa), but will be in frame. Analysis of the cDNA for both siblings using primers surrounding the mutated exon 36, confirmed the hypothesis of the hypomorphic character (Figure 3B). Sequence

142

analysis of the normal sized product bands showed only a fraction of the mutant allele, while the smaller sized product showed and absence of exon 36 (Figure 3C). The mutant allele was recovered to the normal levels due to the cell treatment with cycloheximide, which inhibits nonsense mediated decay (Figure 3D). Figure 1 A I

Family A

M1/+

M2/+

II-1

500 bp 400 bp

II-2

C1 C2 -

C3 -

Normal transcript Aberrant transcript*

300 bp

CHX

200 bp

M1/M2

Non-specific bands

M1/M2 100 bp

M1: c.451C>T M2: c.4723A>T C

exon 35

exon 36

GUSB

WT Mutant

G CCA G A G A GG A G CA AAG AG A A A T T G T GT A G A A A C AT A

WT Mutant

G CCA G A G A GG A G CA AAG AG A A A T T G T GA A G A A A C AT T

exon 35

exon 37 *

WT G CCA G A G A GG A T T T AAT G A A A CAG A C A CC C A C T C CA

3.2

E exon 35

exon 36

Figure 3 mRNA analysis of individuals with OT carrying CEP290 c.4723A>T variant A) The pedigree with OT and the segregation of CEP290 variants c.415C>T (p.(Arg151*), M1) and c.4723A>T (p.(Lys1575*), M2); B) CEP290 cDNA analysis of the effect of the c.4723A>T variant on splicing of exon 36 in the patient cell lines treated with (+) or without (-) cycloheximide (CHX) to prevent degradation by nonsense-mediated decay (NMD) versus non-treated control cell lines. Agarose gel electrophoresis revealed a major 432 bp product representing the normal transcript in all tested samples. Hence, the difference (Δ) between treated and untreated samples in the affected individuals can be explained by the partial effect of NMD. An aberrant product (*) is seen in the affected persons at 324 bp, whereas this product is not found in controls. This product size corresponds to a transcript lacking exon 36. The lower products represent nonspecific product as observed by sequencing. The comparison for input was made by the analysis of the housekeeping gene GUSB; C) Sequence analysis of the abundant cDNA product in the CHX treated samples shows the exon transition from 35 to 36 with the recovered mutant allele at wild-type level. The right panel shows sequences of the aberrant product from exon 35 to 37, skipping exon 36; D) Sequence analysis of the untreated normal mRNA product shows a reduced presence of the mutant allele due to partial NMD, visible by a reduced peak size of the mutant T-allele (M2 Δ) compared to the wildtype A-allele.

143 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

144

p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*)

Mutation

c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T

c.4723A>T c.4723A>T c.4723A>T

c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T

p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT

p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT

p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT

Protein effect based on RNA study

c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T c.4723A>T

c.4393C>T c.4393C>T No second allele

c.451C>T c.1709C>G c.2991+1655A>G c.2991+1655A>G c.2991+1655A>G c.4696G>C c.4393C>T

Mutation

Second allele

p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*) p.(Lys1575*)

p.(Arg1465*) p.(Arg1465*)

p.(Arg151*) p.(Ser570*) p.(Cys998*) p.(Cys998*) p.(Cys998*) p.(Ala1556Pro) p.(Arg1465*)

Protein prediction

p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT p.Glu1569Glyfs/WT

ND ND

p.Leu148_Glu165del/WT ND p.Cys998*/WT p.Cys998*/WT p.Cys998*/WT ND ND

Protein effect based on RNA study

45, 11

This study

Ref.

Patient LCA-7 and LCA-25 are distantly related; LCA = Leber congenital amourosis; SLSN = Senior loken; CORS = Cerebello-oculo-renal syndrome; JBTS+retina = Joubert syndrome with retinal involvement, ND = not determined.

Sample ID Diagnosis Compound heterozygous Family A OT 809 LCA LCA-6 LCA LCA-7 LCA LCA-8 LCA LCA-24 LCA COR031/ CORS CORS1 SLS-2 SLSN SLS-3 SLSN 7 LCA Homozygous 1 LCA 2 LCA 738 LCA 848 LCA 258 LCA 419 LCA LEP LCA LCA-25 LCA 623 JBTS+ retina

Protein prediction

First allele

Table 2 Individuals harboring the c.4723A>T mutation in CEP290

Comment

This is the first report on long term follow-up in two patients with OT, showing the transition of a supposedly stationary cone dysfunction syndrome into a progressive degenerative disease, and thereby stressing the importance of studying the natural course of rare diseases. We also discovered that the genetic defect was located in CEP290, a gene primarily involved in syndromic and non-syndromic LCA. OT is a group of clinically and genetic heterogeneous disorders, since certain symptoms like nystagmus and photophobia are not present in all of them, and visual acuity may vary. In a few individuals, mutations were discovered in phototransduction genes suggesting an overlap with ACHM.17-19 Neuhann et al.25 described one OT patient and mentioned that a far relative in the same village suffered from sector retinal dystrophy. It is highly speculative to assume that this might have also been a case of progressive OT. Andersen et al.18 and Michaelides et al.26 reported on OT patients with subnormal rod specific response, a feature that our patients also displayed. Subnormal rod function has also been observed in patients with ACHM, due to CNGA3 and CNGB3 mutations. Today, we know that ACHM is a progressive diseases evolving into cone dystrophy. In ACHM patients, progressive loss of cones is observed on OCT in the macula after the first decade.27 There are no reports on progressive rod degeneration in ACHM. In our patients, the degeneration started in the inferior quadrants progressing towards the posterior pole , and was coupled by progressive visual field loss and deterioration of rod derived ERG responses. The cone function, at age 46, in our eldest patient was still relatively well preserved given the stable visual acuity and the preserved color vision. In the original report on OT by Van Lith, it was postulated that all three types of cones must be present but reduced in number. Michaelides and colleagues investigated this presumption and studied the cone photoreceptor mosaic with adaptive optics in four relatively young patients. It was found that in three out of four subjects indeed a sparse mosaic of cones remained at the fovea with no structure visible outside the fovea. These patients also showed decreased length of the outer segments on OCT. Only one patient seemed to have a normal cone mosaic. It is impossible to compare the OCTs of our youngest subject with the ones published by Andersen et al.18 and Michaelides et al.,24 since our patient had atrophic changes around the fovea and in the posterior pole. More to the periphery, however, three layers seemed visible. In individual II-1 imaging failed due to the nystagmus and photoaversion. There was a (double) ring sign observed on FAF in individual II-2 indicating the transitional zone between intact and absent photoreceptor inner and outer segments, a phenomenon also observed in other retinal dystrophies.28, 29 We tried to find an explanation for the cone dominated disease in these two patients since gene expression of CEP290 is seen in the connecting cilia of both photoreceptor cells. CEP290 plays a role in primary ciliary assembly30; 31 and has been shown to recruit small GTPase Rab8a (MIM#165040) to the centrosomes and cilia for ciliary membrane elongation. 30 The retinal architecture of CEP290-mutant retinas displayed clear remodeling in the rod-dominated periphery visualized by thickening of the inner retina, whereas the cone-dominated foveal region remained unaltered. The difference observed in cone and rod degeneration may indicate different functions in both cell types. 32 As the clinical examinations in our patients uncovered cone defects with late onset progressive peripheral degeneration one could argue that the cones are more vulnerable compared to rods due their higher metabolism. 33 The secondary impairment of rods as to CEP290 mutations is also seen in the Cnga3-/- mice and a zebrafish model, both models for cone degeneration. 34;35 It was hypothesized that the cell density plays a role in determining whether there will be secondary rod degeneration. Subsequent to cone degeneration, loss of rod photoreceptor cells occurs in retinal regions characterized by a low

145 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

3.2

density of rods, while the high rod density retinal regions remain intact. 35 A third hypothesis on secondary rod degeneration resides in aberrant gap junction coupling of the photoreceptors. 36 Gap junctions are crucial for photoreceptors survival31 and provides coupling between cones and rods, 38; 39 enabling transmission of signals among the photoreceptor terminals, which subsequently can be transmitted to the inner retina. Whether damage due to light exposure, as shown for rhodopsin mutations in rodents,40;41 may play a role in the second type of degeneration is speculative. Defects in CEP290 can cause a wide range of severe ciliary phenotypes, like Joubert syndrome (JBTS, MIM#610188),7 Senior LĂ¸ken syndrome(SLSN, MIM#610189)6, cerebello-oculo-renal syndrome (CORS, MIM#608091),15 Meckel-Gruber syndrome (MKS, MIM#611134), 8 a single case of Bardet Biedl syndrome (BBS, MIM#209900),9 LCA4 , and early-onset retinal degeneration14. The phenotype observed in our two subjects showed overlap with ACHM, but mutations in the ACHM genes were not detected. Up to date, 136 unique mutations are described in the 2,479 aa protein and limited genotype-phenotype correlations have been made.10; 42 These genotypes and subsequent phenotypes are recorded in the database CEP290base (http:// medgen.ugent.be/cep290base/index.php; http://grenada.lumc.nl/LOVD2/eye/home.php?select_ db=CEP290). Strikingly though, the deep intronic c.2991+1655A>G hypomorphic variant has been described to cause nonsyndromic LCA both in a homozygous state and in a compound heterozygous state together with null alleles.4 Littink et al. (2010)14 showed that c.451C>T produced two aberrant transcripts lacking exon 7 and exon 7-8, thereby escaping NMD. The skipping of exon 7, keeping the reading frame intact, leads to a deletion of 94 aa, which contains part of the 506 aa coiled-coil domain, but only represents a small fraction of the mutant 2,479 aa full length/normal CEP290 protein. In these patients, the mild c.2991+1655A>G variant was found on the other allele, thereby causing early onset severe retinal dystrophy. Leich et al (2008)9 described a single case in which a C-terminal homozygous p.(Glu1903*) mutation in CEP290, together with a hypomorphic variant in MKS3 (MIM#607361), are the cause of BBS. The strong genetic interaction between these genes, and the mutational load in closely interacting proteins are possible explanations to the clinical variability of the CEP290 genotypes. The identification of the hypomorphic nature of three CEP290 variants (c.442_495del, p.Leu148_ Glu165del; c.2991+1655A>G, r.2991_2992ins2991+1525_2991+1650; and c.4706_4812+1del, p.Glu1569Glyfs) due to nonsense-associated altered splicing in this and other studies,4; 14 warrants the question whether this could be an underappreciated phenomenon in CEP290associated diseases. Eligible exons should contain a multiple of 3 bps as their deletion would result in restoration of the open reading frame. Surprisingly, an analysis on all CEP290 exons showed that 27 out of 54 exons (50%) are in-frame, which, if this would be random, one would expect one-third of the exons to contain a multiple of 3 bps. In general, many genes evolve in evolution through intragenic duplications.43; 44 The products of such duplications are more likely to evolve in functionally relevant variants if the exons contain a multiple of 3 bps. For CEP290, this mechanism may have amplified the number of coiled coil domain modules. The c.4723A>T variant is a founder mutation in Belgium and France,11; 15; 45 which is in line with the ancestry of our patients as the mother originated from Belgium. Taking the northern European founder effect as a hypothesis we accordingly screened our ACHM and CD cohort on this mutation as well as the c.442_495del mutation, which was also found in a Dutch family. In our cohort of 23 ACHM and 145 CD individuals we did not identify additional persons carrying the c.4706_4812_1del or c.442_495del mutation. Previously, Littink et al. (2010)14 did not identify additional CRD or RP patients with c.442_495del. It seems that this combination of nonsense mutations, causing a dystrophy milder than LCA, has a very low prevalence.

146

Finally, our cohort was also tested for the prevalence of the most frequently found mutation in CEP290 causing nonsyndromic LCA, the deep intronic c.2991+1655A>G mutation causing a cryptic splice site with a premature stop codon. This screening did not yield any mutations in our cohort. A literature analysis revealed that the frequent mutation c.4706_4812+1del, is described in 16 cases, besides our family, of which nine homozygous cases (See Table 2).10; 11; 45; 46 In two cases abnormal respiratory cilia were observed.46 All cases were diagnosed with LCA, with the exception of one case with JBTS. We suspect that this exception may potentially be a carrier of a third (modifier) allele, potentially residing in one of the ciliary genes. In this study, we expanded phenotypes that can be caused by CEP290 mutations and it would be interesting to investigate whether other OT cases also carry mutations in this gene. Why genes that are expressed in both photoreceptor cells, can generate a cone- or rod-dominated disease is still unknown. With the use of NGS, we were able to identify the causative defect in this family, which hold great promises for elucidation of other subtle but distinct phenotypes in the coming years. Not only will NGS solve many phenotypic entities, it will also likely uncover more cases with multigenic inheritance pattern. The identification of current CEP290 variants as a cause of OT, due to the rare frequency, will on their own not shed more light on the progression of the phenotype in these individuals. Nevertheless, the identification of hypomorphic variants in CEP290 will raise new hypotheses on the occurrence of â&#x20AC;&#x2DC;milder than presumedâ&#x20AC;&#x2122; phenotypes due to CEP290 mutations, which also may be true for other genes. This study also stresses the importance of clinical observations and descriptions over longer period of time, which enables, even when imaging tools where less advanced, to follow the disease course. Also in presumed stationary phenotypes, clinical observations remain of great value and may elucidate potential progressive nature of disease.

Acknowledgements

This study was financially supported by the Foundation Fighting Blindness USA (grants BR-GE-0510-04890RAD, C-GE-0811-0545-RAD01) to A.I.d.H., and F.P.M.C., the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, the Gelderse Blinden Stichting, the Landelijke Stichting voor Blinden en Slechtzienden, the Stichting Blinden-Penning, the Stichting Macula Degeneratie fonds, the Rotterdamse Stichting Blindenbelangen (to F.P.M.C.) and the Stichting Wetenschappelijk Onderzoek Oogziekenhuis Rotterdam (to L.I.v.d.B.). None of the authors have any financial interest or conflicting interest to disclose. Supplemental table 1 Overlapping homozygous regions detected in both affected siblings by SNP microarray analysis Patient

Chromosome

Start position

End position

Size (Mb)

#SNPs

II-1, II-2

33,792,672

34,880,118

1,09

208

II-1, II-2

113,018,709

114,000,208

0,99

218

147 Oligocone trichromacy caused by hypomorphic nonsense mutations in CEP290

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References 1. Craige, B., Tsao, C.C., Diener, D.R., Hou, Y., Lechtreck, K.F., Rosenbaum, J.L., and Witman, G.B. (2010). CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J Cell Biol 190, 927-940. 2. Barbelanne, M., Song, J., Ahmadzai, M., and Tsang, W.Y. (2013). Pathogenic NPHP5 mutations impair protein interaction with Cep290, a prerequisite for ciliogenesis. Hum Mol Genet 22, 2482-2494. 3. Garcia-Gonzalo, F.R., Corbit, K.C., Sirerol-Piquer, M.S., Ramaswami, G., Otto, E.A., Noriega, T.R., Seol, A.D., Robinson, J.F., Bennett, C.L., Josifova, D.J., Garcia-Verdugo, J.M., Katsanis, N., Hildebrandt, F., and Reiter, J.F. (2011). A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet 43, 776-784. 4. den Hollander, A.I., Koenekoop, R.K., Yzer, S., Lopez, I., Arends, M.L., Voesenek, K.E., Zonneveld, M.N., Strom, T.M., Meitinger, T., Brunner, H.G., Hoyng, C.B., van den Born, L.I., Rohrschneider, K., and Cremers, F.P. (2006). Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am J Hum Genet 79, 556-561. 5. Chang, B., Khanna, H., Hawes, N., Jimeno, D., He, S., Lillo, C., Parapuram, S.K., Cheng, H., Scott, A., Hurd, R.E., Sayer, J.A., Otto, E.A., Attanasio, M., Oâ&#x20AC;&#x2122;Toole, J.F., Jin, G., Shou, C., Hildebrandt, F., Williams, D.S., Heckenlively, J.R., and Swaroop, A. (2006). In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse. Hum Mol Genet 15, 1847-1857. 6. Sayer, J.A., Otto, E.A., Oâ&#x20AC;&#x2122;Toole, J.F., Nurnberg, G., Kennedy, M.A., Becker, C., Hennies, H.C., Helou, J., Attanasio, M., Fausett, B.V., Utsch, B., Khanna, H., Liu, Y., Drummond, I., Kawakami, I., Kusakabe, T., Tsuda, M., Ma, L., Lee, H., Larson, R.G., Allen, S.J., Wilkinson, C.J., Nigg, E.A., Shou, C., Lillo, C., Williams, D.S., Hoppe, B., Kemper, M.J., Neuhaus, T., Parisi, M.A., Glass, I.A., Petry, M., Kispert, A., Gloy, J., Ganner, A., Walz, G., Zhu, X., Goldman, D., Nurnberg, P., Swaroop, A., Leroux, M.R., and Hildebrandt, F. (2006). The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. Nat Genet 38, 674-681. 7. Valente, E.M., Silhavy, J.L., Brancati, F., Barrano, G., Krishnaswami, S.R., Castori, M., Lancaster, M.A., Boltshauser, E., Boccone, L., Al-Gazali, L., Fazzi, E., Signorini, S., Louie, C.M., Bellacchio, E., International Joubert Syndrome Related Disorders Study, G., Bertini, E., Dallapiccola, B., and Gleeson, J.G. (2006). Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nat Genet 38, 623-625. 8. Baala, L., Audollent, S., Martinovic, J., Ozilou, C., Babron, M.C., Sivanandamoorthy, S., Saunier, S., Salomon, R., Gonzales, M., Rattenberry, E., Esculpavit, C., Toutain, A., Moraine, C., Parent, P., Marcorelles, P., Dauge, M.C., Roume, J., Le Merrer, M., Meiner, V., Meir, K., Menez, F., Beaufrere, A.M., Francannet, C., Tantau, J., Sinico, M., Dumez, Y., MacDonald, F., Munnich, A., Lyonnet, S., Gubler, M.C., Genin, E., Johnson, C.A., Vekemans, M., Encha-Razavi, F., and Attie-Bitach, T. (2007). Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome. Am J Hum Genet 81, 170-179. 9. Leitch, C.C., Zaghloul, N.A., Davis, E.E., Stoetzel, C., Diaz-Font, A., Rix, S., Alfadhel, M., Lewis, R.A., Eyaid, W., Banin, E., Dollfus, H., Beales, P.L., Badano, J.L., and Katsanis, N. (2008). Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome. Nat Genet 40, 443-448. 10. Coppieters, F., Lefever, S., Leroy, B.P., and De Baere, E. (2010). CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Hum Mutat 31, 1097-1108. 11. Perrault, I., Delphin, N., Hanein, S., Gerber, S., Dufier, J.L., Roche, O., Defoort-Dhellemmes, S., Dollfus, H., Fazzi, E., Munnich, A., Kaplan, J., and Rozet, J.M. (2007). Spectrum of NPHP6/

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CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype. Hum Mutat 28, 416. 12. Yzer, S., Hollander, A.I., Lopez, I., Pott, J.W., de Faber, J.T., Cremers, F.P., Koenekoop, R.K., and van den Born, L.I. (2012). Ocular and extra-ocular features of patients with Leber congenital amaurosis and mutations in CEP290. Mol Vis 18, 412-425. 13. McAnany, J.J., Genead, M.A., Walia, S., Drack, A.V., Stone, E.M., Koenekoop, R.K., Traboulsi, E.I., Smith, A., Weleber, R.G., Jacobson, S.G., and Fishman, G.A. (2013). Visual acuity changes in patients with leber congenital amaurosis and mutations in CEP290. JAMA ophthalmology 131, 178-182. 14. Littink, K.W., Pott, J.W., Collin, R.W.J., Kroes, H.Y., Verheij, J.B., Blokland, E.A., de Castro Miro, M., Hoyng, C.B., Klaver, C.C.W., Koenekoop, R.K., Rohrschneider, K., Cremers, F.P.M., van den Born, L.I., and den Hollander, A.I. (2010). A novel nonsense mutation in CEP290 induces exon skipping and leads to a relatively mild retinal phenotype. Invest Ophthalmol Vis Sci 51, 3646-3652. 15. Coppieters, F., Casteels, I., Meire, F., De Jaegere, S., Hooghe, S., van Regemorter, N., Van Esch, H., Matuleviciene, A., Nunes, L., Meersschaut, V., Walraedt, S., Standaert, L., Coucke, P., Hoeben, H., Kroes, H.Y., Vande Walle, J., de Ravel, T., Leroy, B.P., and De Baere, E. (2010). Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Hum Mutat 31, E1709-1766. 16. van Lith, G.H.M. (1973). General cone dysfunction without achromatopsia. In ed 10th ISCERG Symposium, P. J.T., ed. (Doc Ophthalmol Proc Ser ), pp 175â&#x20AC;&#x201C;180. 17. Vincent, A., Wright, T., Billingsley, G., Westall, C., and Heon, E. (2011). Oligocone trichromacy is part of the spectrum of CNGA3-related cone system disorders. Ophthalmic Genet 32, 107-113. 18. Andersen, M.K., Christoffersen, N.L., Sander, B., Edmund, C., Larsen, M., Grau, T., Wissinger, B., Kohl, S., and Rosenberg, T. (2010). Oligocone trichromacy: clinical and molecular genetic investigations. Invest Ophthalmol Vis Sci 51, 89-95. 19. Rosenberg, T., Baumann, B., Kohl, S., Zrenner, E., Jorgensen, A.L., and Wissinger, B. (2004). Variant phenotypes of incomplete achromatopsia in two cousins with GNAT2 gene mutations. Invest Ophthalmol Vis Sci 45, 4256-4262. 20. Marmor, M.F., Fulton, A.B., Holder, G.E., Miyake, Y., Brigell, M., and Bach, M. (2009). ISCEV Standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol 118, 69-77. 21. Littink, K.W., van Genderen, M.M., van Schooneveld, M.J., Visser, L., Riemslag, F.C., Keunen, J.E., Bakker, B., Zonneveld, M.N., den Hollander, A.I., Cremers, F.P., and van den Born, L.I. (2012). A homozygous frameshift mutation in LRAT causes retinitis punctata albescens. Ophthalmology 119, 1899-1906. 22. Miller, S.A., Dykes, D.D., and Polesky, H.F. (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16, 1215. 23. Frank, V., den Hollander, A.I., Bruchle, N.O., Zonneveld, M.N., Nurnberg, G., Becker, C., Du Bois, G., Kendziorra, H., Roosing, S., Senderek, J., Nurnberg, P., Cremers, F.P.M., Zerres, K., and Bergmann, C. (2008). Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome. Hum Mutat 29, 45-52. 24. Nagy, E., and Maquat, L.E. (1998). A rule for termination-codon position within introncontaining genes: when nonsense affects RNA abundance. Trends in biochemical sciences 23, 198-199. 25. Neuhann, T., Krastel, H., and Jaeger, W. (1978). Differential diagnosis of typical and atypical congenital achromatopsia. Analysis of a progressive foveal dystrophy and a nonprogressive oligo-cone trichromasy (general cone dysfunction without achromatopsia), both of which at first had been diagnosed as achromatopsia. Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie Albrecht von Graefeâ&#x20AC;&#x2DC;s archive for clinical and experimental

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ophthalmology 209, 19-28. 26. Michaelides, M., Rha, J., Dees, E.W., Baraas, R.C., Wagner-Schuman, M.L., Mollon, J.D., Dubis, A.M., Andersen, M.K., Rosenberg, T., Larsen, M., Moore, A.T., and Carroll, J. (2011). Integrity of the cone photoreceptor mosaic in oligocone trichromacy. Invest Ophthalmol Vis Sci 52, 4757-4764. 27. Thiadens, A.A., Somervuo, V., van den Born, L.I., Roosing, S., van Schooneveld, M.J., Kuijpers, R.W., van Moll-Ramirez, N., Cremers, F.P., Hoyng, C.B., and Klaver, C.C. (2010). Progressive loss of cones in achromatopsia: an imaging study using spectral-domain optical coherence tomography. Invest Ophthalmol Vis Sci 51, 5952-5957. 28. Scholl, H.P., Chong, N.H., Robson, A.G., Holder, G.E., Moore, A.T., and Bird, A.C. (2004). Fundus autofluorescence in patients with leber congenital amaurosis. Invest Ophthalmol Vis Sci 45, 2747-2752. 29. Escher, P., Tran, H.V., Vaclavik, V., Borruat, F.X., Schorderet, D.F., and Munier, F.L. (2012). Double concentric autofluorescence ring in NR2E3-p.G56R-linked autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci 53, 4754-4764. 30. Kim, J., Krishnaswami, S.R., and Gleeson, J.G. (2008). CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium. Hum Mol Genet 17, 3796-3805. 31. Tsang, W.Y., Bossard, C., Khanna, H., Peranen, J., Swaroop, A., Malhotra, V., and Dynlacht, B.D. (2008). CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease. Developmental cell 15, 187-197. 32. Cideciyan, A.V., Aleman, T.S., Jacobson, S.G., Khanna, H., Sumaroka, A., Aguirre, G.K., Schwartz, S.B., Windsor, E.A., He, S., Chang, B., Stone, E.M., and Swaroop, A. (2007). Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis. Hum Mutat 28, 1074-1083. 33. Miyazono, S., Shimauchi-Matsukawa, Y., Tachibanaki, S., and Kawamura, S. (2008). Highly efficient retinal metabolism in cones. Proc Natl Acad Sci U S A 105, 16051-16056. 34. Stearns, G., Evangelista, M., Fadool, J.M., and Brockerhoff, S.E. (2007). A mutation in the cone-specific pde6 gene causes rapid cone photoreceptor degeneration in zebrafish. J Neurosci 27, 13866-13874. 35. Haverkamp, S., Michalakis, S., Claes, E., Seeliger, M.W., Humphries, P., Biel, M., and Feigenspan, A. (2006). Synaptic plasticity in CNGA3(-/-) mice: cone bipolar cells react on the missing cone input and form ectopic synapses with rods. J Neurosci 26, 5248-5255. 36. Boudard, D.L., Tanimoto, N., Huber, G., Beck, S.C., Seeliger, M.W., and Hicks, D. (2010). Cone loss is delayed relative to rod loss during induced retinal degeneration in the diurnal cone-rich rodent Arvicanthis ansorgei. Neuroscience 169, 1815-1830. 37. Camacho, E.T., Colon Velez, M.A., Hernandez, D.J., Rodriguez Bernier, U., Van Laarhoven, J., and Wirkus, S. (2010). A mathematical model for photoreceptor interactions. Journal of theoretical biology 267, 638-646. 38. Smith, R.G., Freed, M.A., and Sterling, P. (1986). Microcircuitry of the dark-adapted cat retina: functional architecture of the rod-cone network. J Neurosci 6, 3505-3517. 39. Bloomfield, S.A., and Dacheux, R.F. (2001). Rod vision: pathways and processing in the mammalian retina. Progress in retinal and eye research 20, 351-384. 40. Organisciak, D.T., Darrow, R.M., Barsalou, L., Kutty, R.K., and Wiggert, B. (2003). Susceptibility to retinal light damage in transgenic rats with rhodopsin mutations. Invest Ophthalmol Vis Sci 44, 486-492. 41. Naash, M.L., Peachey, N.S., Li, Z.Y., Gryczan, C.C., Goto, Y., Blanks, J., Milam, A.H., and Ripps, H. (1996). Light-induced acceleration of photoreceptor degeneration in transgenic mice expressing mutant rhodopsin. Invest Ophthalmol Vis Sci 37, 775-782.

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42. Novarino, G., Akizu, N., and Gleeson, J.G. (2011). Modeling human disease in humans: the ciliopathies. Cell 147, 70-79. 43. Kondrashov, F.A., and Koonin, E.V. (2001). Origin of alternative splicing by tandem exon duplication. Hum Mol Genet 10, 2661-2669. 44. Strachan T, and A.P., R. (1999). Our place in the tree of life. In Human Molecular Genetics. New York, Wiley-Liss. 45. Brancati, F., Barrano, G., Silhavy, J.L., Marsh, S.E., Travaglini, L., Bielas, S.L., Amorini, M., Zablocka, D., Kayserili, H., Al-Gazali, L., Bertini, E., Boltshauser, E., Dâ&#x20AC;&#x2DC;Hooghe, M., Fazzi, E., Fenerci, E.Y., Hennekam, R.C., Kiss, A., Lees, M.M., Marco, E., Phadke, S.R., Rigoli, L., Romano, S., Salpietro, C.D., Sherr, E.H., Signorini, S., Stromme, P., Stuart, B., Sztriha, L., Viskochil, D.H., Yuksel, A., Dallapiccola, B., International, J.S.G., Valente, E.M., and Gleeson, J.G. (2007). CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndromerelated disorders. Am J Hum Genet 81, 104-113. 46. Papon, J.F., Perrault, I., Coste, A., Louis, B., Gerard, X., Hanein, S., Fares-Taie, L., Gerber, S., Defoort-Dhellemmes, S., Vojtek, A.M., Kaplan, J., Rozet, J.M., and Escudier, E. (2010). Abnormal respiratory cilia in non-syndromic Leber congenital amaurosis with CEP290 mutations. J Med Genet 47, 829-834. 47. Stone, E.M. (2007). Leber congenital amaurosis - a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson Memorial Lecture. Am J Ophthalmol 144, 791-811.

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Chapter 4 Novel mutated genes involved in cone dysfunctions

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Alberta A.H.J. Thiadens, Anneke I. den Hollander, Susanne Roosing, Sander B. Nabuurs, Renate C. Zekveld-Vroon, Rob W.J. Collin, Elfride de Baere, Robert K. Koenekoop, Mary J. van Schooneveld, Tim M. Strom, Janneke J.C. van Lith-Verhoeven, Andrew J. Lotery, Norka van Moll-Ramirez, Bart P. Leroy, L. Ingeborgh van den Born, Carel B. Hoyng, Frans P.M. Cremers, Caroline C.W. Klaver

American journal of human genetics 2009, 85, 240-247 154

Chapter 4.1 Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders

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Abstract

Cone photoreceptor disorders form a clinical spectrum of diseases which include progressive cone dystrophy (COD), and complete and incomplete achromatopsia (ACHM). The underlying disease mechanisms of autosomal recessive (AR)COD are largely unknown. Our aim was to identify causative genes for these disorders by genome-wide homozygosity mapping. We investigated 75 ACHM, 97 AR COD, and 20 early-onset AR COD probands, and excluded the involvement of known genes for ACHM and AR COD. Subsequently, we performed highresolution SNP analysis, and identified large homozygous regions spanning the PDE6C gene in one sibling pair with early-onset AR COD, and in one sibling pair with incomplete ACHM. The PDE6C gene encodes the cone a-subunit of cyclic huanosine monophosphate (cGMP) phosphodiesterase, which converts cGMP to 5â&#x20AC;&#x2122; GMP, and thereby plays an essential role in cone phototransduction. Sequence analysis of the coding region of PDE6C revealed homozygous missense mutations (p.R29W, p.Y323N) in both sibling pairs. Sequence analysis of 104 probands with AR COD and 10 probands with ACHM revealed compound heterozygous PDE6C mutations in three complete ACHM patients from two families. One patient had a frameshift mutation and a splice defect, the other two had a splice defect and a missense variant (p.M455V). Cross-sectional retinal imaging using optical coherence tomography (OCT) revealed a more pronounced absence of cone photoreceptors in patients with ACHM compared to early-onset AR COD. Our findings reveal PDE6C as a gene for cone photoreceptor disorders, and show that AR COD and ACHM constitute genetically and clinically overlapping phenotypes.

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Impairment or death of the cone photoreceptor cells is the clinical hallmark of cone disorders which have an estimated prevalence of 1:30,000-1:40,000.1; 2 Achromatopsia (ACHM) (MIM *600827) is a stationary congenital autosomal recessive cone disorder, characterized by low visual acuity (0.10-0.20 decimal Snellen equivalent), photophobia, nystagmus, and severe color vision defects. Patients with the complete ACHM subtype have no cone function on electroretinogram (ERG), whereas those with incomplete ACHM have residual cone function. Cone dystrophy (COD) (MIM *600827) is a progressive cone disorder in which patients may initially have normal cone function but develop progressive visual acuity loss, increasing photophobia, color vision disturbances, and diminished cone responses on ERG, usually in the first or second decade of life. The visual acuity of these patients generally worsens to legal blindness before the fourth decade of life. 3 The genetic basis of these disorders has been partly elucidated during the last decade. Three genes have been implicated in ACHM: CNGA3 (MIM *600053), CNGB3 (MIM *605080), and GNAT2 (MIM *139340). Mutations in these genes explain a large part of this disease, but a fraction still remains unsolved.4-7 For COD, several genes have been identified for the autosomal dominant3; 8; 9 and X-linked forms,10; 11 but little is known about the genetic causes of the most prevalent autosomal recessive (arCOD) form. Four genes have been implicated in this form: ABCA4 (MIM *601691),12 CACNA2D4 (MIM *608171)13 CNGB3,14 and KCNV2 (MIM *607604),15; 16 which together explain only ~10% of all cases. We aimed to identify disease genes for cone photoreceptor disorders, and investigated patients with ACHM and AR COD. Patients were ascertained from various ophthalmic centers in the Netherlands, Belgium, the United Kingdom and Canada. This study was approved by the local and national medical ethics committees and adhered to the tenets of the Declaration of Helsinki. All participants provided signed, informed consent for participation in the study, retrieval of medical records, and use of blood and DNA for research. All available data on visual acuity, color vision, fundus appearance, and ERGs were retrieved from medical charts, and updated by additional examinations including ocular coherence tomography (OCT) and fundus photography. Recent ERGs were performed according to the recommendations of the International Society for Clinical Electrophysiology of Vision.17 Patients could be stratified into three clinical categories: ACHM (n=75 probands), AR COD (n=97 probands), and a group who had clear documentation of progressive cone dysfunction, but already had impaired visual acuity in early childhood (early-onset AR COD, n=20 probands). We tested for the presence of known gene mutations in all probands in ABCA4 (A.A.H.J.T., unpublished data), and for the presence of CNGB3 variants in all probands by sequence analysis. 5; 7 When CNGB3 variants were absent, all ACHM patients were analyzed for CNGA3 and GNAT2 variants using sequence analysis. 5; 7 Finally, most of the AR COD patients were analyzed for KCNV2 variants by sequence analysis (A.A.H.J.T., unpublished data). These studies resulted in 116 probands, i.e. 11 ACHM, 85 AR COD, and 20 early-onset AR COD, with unknown etiology who form the basis of the present genetic analyses. We performed high-resolution SNP analysis (Affymetrix GeneChip Genome-Wide Human Array 5.0) on a subset of 76 patients of 64 families with autosomal recessive cone dysfunction from whom DNA was available. In 8 of these 64 families the parents were consanguineous. Genotyping was performed with Genotype Console software (Affymetrix), and regions of homozygosity were calculated with Partek Genomics Solution software (Partek). Large identity-by-descent (IBD) regions (i.e. > 5 Mb) were found in all consanguineous families and in 34 of the non-consanguineous probands. In these 34 non-consanguineous patients, we found a total of 57 IBD regions larger than 5 Mb, with an average size of 9 Mb (range 5 â&#x20AC;&#x201C; 31 Mb).

157 Homozygosity mapping reveals PDE6C mutations in cone disorders

4.1

Figure 1

22.2

22.3

23.1

23.3

23.2

24.1

24.3

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25.1

Family A

80,8

107,3 Mb

Family B

84,9

SNP_A-2260521

95,9 Mb

SNP_A-2003359

SNP_A-1892358

SNP_A-1804692

PDE6C

B. M1

ATG

M1: c.85C>T; p.R29W

WT Mutant

Lys Leu

Arg

Val

A A G T T G CG G G T G T

II-1 M1/M1

M2: c.257_258dupAG; p.L87Gfs*57

PDE6C

PDE6A

PDE6B

Gln

Arg Leu Ala

C A G AG GC T GG C C A G G C T G G

II-2 M1/M1

Glu

II-1 M4/M4

Glu

Intron

TAA

HDc

G A A G A G GT A A T G C

I-1 M4/+

M4: c.967T>A; p.Y323N Ile

Asp

Tyr

M5: c.1363A>G; p.M455V

Ile

Glu Met

A T T G A T T A C A T T A

Family B

I-2

M3: c.633G>C (splice defect)

GAF B

Family A I-1

GAF A

I-2 M4/+

I-1

II-2 M4/M4

Intron

T A T AA G G T A A GT T A A G CA

Family D

I-2

I-1 M3/+

II-1 M3/M5

II-1 M2/M6

Tyr Lys

Leu

A G G A A A T GC T CA T C

Family C

M6: c.2367+1delGTAAG (splice defect)

I-2 M5/+

II-2 M3/M5

Homo sapiens Bos taurus Canis lupus familiaris Mus musculus Rattus novergicus Rana pipens Danio rerio Homo sapiens Bos taurus Canis lupus familiaris Mus musculus Rattus novergicus Rana pipens Danio rerio Homo sapiens Bos taurus Canis lupus familiaris Mus musculus Rattus novergicus Rana pipens

Figure 1 Molecular genetic characterization of the PDE6C gene in four families with autosomal recessive cone photoreceptor disorders A) The 10q22-q25 region and the homozygous regions identified in patients of families A and B. The SNPs flanking the homozygous regions and their genomic positions, using the March 2006 UCSC genome build (hg18), are indicated. B) Protein and genomic structure of PDE6C. GAF: acronym derived from proteins in which these domains were initially identified (cGMPregulated mammalian phosphodiesterases, cyanobacterial adenylyl cyclases, and a formatehydrogen lyase transcriptional activator); HDc: domain with highly conserved histidines (H) and aspartic acids (D); cc, coiled-coil domain. The six mutations are depicted M1 through M6. C) Chromatograms showing the PDE6C mutations. D) Pedigrees of early-onset cone dystrophy (family A), incomplete achromatopsia (family B), and complete achromatopsia (families C and

158

D), and segregation analysis of the respective PDE6C variants. The parents of family A are not related to each other in the last three generations. BI-1 and BI-2 are first cousins. E) Evolutionary conservation of the altered amino acid residues in three families. PDE6A and PDE6C are rod and cone α-subunits respectively; PDE6B is the rod β-subunit. Using the Swiss-Prot database, we searched for their orthologues in as many species as possible, and included, with the exception of Danio rerio, those present in all species. White lettered residues on a black background are fully conserved between different species in all three subunits. White lettered residues on a grey background are conserved in most sequences. Black lettered residues with light grey background are similar to amino acid residues in other orthologues and homologues. The arginine residue at position 29 is fully conserved in all orthologues of both α-subunits (PDE6C and PDE6A), except for bovine and canine PDE6C. The aspartic acid residue in the GAF-A domain at position 211 is conserved or replaced by a functionally conserved glutamic acid residue in 19 of 20 orthologues and homologues. This observation strongly suggests that the predicted p.E211D substitution in family D is neutral and that the effect of c.633G>C on the splice site is pathologic. The tyrosine residue located in the GAF-B domain at position 323 is completely conserved among all three PDE6 subunits. The methionine residue at position 455 is conserved or substituted by a functionally conserved isoleucine in all PDE6 molecules. In a sibling pair from a consanguineous marriage (family B) and in a sibling pair from an nonconsanguineous family (family A), we identified large homozygous regions on chromosome 10 (11 Mb and 26 Mb, respectively) (Figure 1A). The common 26 Mb homozygous region in the sibs from family A was the largest homozygous region (SNP boundaries: SNP_A-2260521 and SNP_A-1892358); the second, third and fourth largest regions measured 8, 4.3 and 4.2 Mb. The overlapping 11 Mb homozygous region in the affected sibs of family B was the seond largest homozygous region (SNP boundaries: SNP_A-2003359 and SNP_A-1804692). The largest region in this family spanned 25 Mb; the third and fourth largest regions measured 10 and 6 Mb. The overlapping homozygous region in both families spanned the PDE6C gene but no other obvious candidate genes (Figure 1A; see also Supplemental table 1). These regions were not found in other patients. PDE6C encodes the cone a-subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase, an enzyme consisting of two α and two γ-subunits that is essential in the cone phototransduction cascade. It converts the second messenger cGMP to 5’ GMP during light exposure. This results in closure of the cGMP-gated ion-channel in the cone outer segment membrane leading to hyperpolarization of the cell.18 Because PDE6C appeared to be an excellent candidate for cone photoreceptor disorders, we performed direct sequencing of all 22 exons (Figure 1B). We detected homozygous missense mutations in exon 1 in both sibling pairs of family A (Figure 1B-D). The first proband (AII-1) and his brother (AII-2) carried a homozygous missense mutation (p.R29W) affecting a conserved residue just upstream of the first GAF (GAF-A) domain (acronym derived from proteins in which these domains were initially identified) (Figure 1B). The arginine residue at position 29 is fully conserved in all depicted orthologues of the cone and rod α-subunits (PDE6C and PDE6A), except for bovine and canine PDE6C (Figure 1E). The second proband (BII-1) and her brother (BII-2) carried the homozygous missense mutation p.Y323N (Figure 1C and 1D). This mutation affects a fully conserved residue located in the second GAF (GAF-B) domain (Figure 1B and 1E, mutation M4), a region of the protein which binds cGMP and regulates the activity of the enzyme.19 Protein homology modeling of the GAF-B domain on the basis of a recent GAF-A crystal structure19 shows that the p.Y323N mutation is located near the cGMP binding site. Altough not directly interacting with cGMP, Y323 neighbors D322 which is in direct

159 Homozygosity mapping reveals PDE6C mutations in cone disorders

4.1

contact with bound cGMP, as shown in Figure 2. D322 tightly interacts with cGMP by means of two hydrogen bonds, one originating from the peptide main chain and one originating from the aspartic acid side chain. Mutation of Y323 to N is likely to alter the local backbone conformation and mobility, and is also likely to affect D322 and its interaction with cGMP. Both the orientation as well as the mobility of the amino acids equivalent to D322 in other GAF domains have been suggested to be of crucial importance in determining affinity and selectivity of these domains for cGMP.19 We hypothesize that the p.Y323N mutation will probably alter the affinity of cGMP for the regulatory GAF-B domain. Figure 2 Molecular modeling of Y323 in the GAF-B domain of PDE6C The individual side chain orientations of D322 and Y323 are shown in magenta. A peptide backbone trace of the remainder of the model is shown, with beta-sheets colored orange and alpha-helices colored blue. The mutation site Y323 is located adjacent to D322. The latter amino acid is shown interacting with bound cGMP via two hydrogen bonds, one originating from the peptide main chain and one originating from the aspartic acid side chain. Hydrogen bonds are indicated by yellow dashed lines. Subsequently, we analyzed the remaining patients (n=114) for additional mutations in the PDE6C gene by direct sequencing of the coding region. Compound heterozygous mutations were detected in two additional probands. Patient CII-1 was compound heterozygous for a frameshift mutation (c.257_258dupAG; p.L87Gfs*57) in exon 1 and a 5-bp deletion (c.2367+1delGTAAG), which removes the first five nucleotides of intron 20 including the splice donor site (Figure 1C and 1D). An alternative splice donor site is predicted at position 2366 (splice prediction score 0.80, BGDP Splice Site Prediction by Neural Network).20 Use of this splice site would lead to a shift in the open reading frame and premature termination of the phosphodiesterase 6C protein (p.E790Sfs*12). Patient DII-1 and her affected sister (DII-2), revealed two missense variants (c.633G>C [p.E211D] and c.1363A>G [p.M455V]) (Figure 1C). The glutamic acid at position 211 is changed to the functionally conserved aspartic acid in five of six homologous rod PDE Î˛-subunits, and in frog PDE6C, strongly suggesting that the predicted amino acid change is a functionally neutral variant. The c.633G>C variant does however affect the last nucleotide of exon 2, and is predicted to completely abolish the splice donor site (BGDP Splice Site Prediction by Neural Network).20 As no alternative splice donor sites are predicted, the mutation may either result in removal of exon 2 from the mRNA, or in non-splicing of intron 2 (106 bp). In both options, this would result in a disruption of the open reading frame. The p.M455V mutation affects a conserved residue located just downstream of the GAF-B domain (Figure 1B and 1E). In those families in which DNA was available from parents (families B and D), mutations M3, M4, and M5 segregated as expected for pathologic variants (Figure 1D). None of the six PDE6C mutations were found in 180 ethnically matched controls. In four other unrelated patients with AR COD, we found four different heterozygous amino acid changes (c.413T>C [p.L138S]; c.696G>A [p.M232I]; c.2096A>G [p.E699G]; c.2477T>C [p.I826T]). In these patients, we did not find a second mutation on the other allele in the coding region of this gene, despite complete PDE6C sequencing. We considered the possibility of a digenic disease model, with mutations in both PDE6C and an interacting gene, and performed sequence analysis of the gene encoding the cone Îł-subunit of cGMP phosphodiesterase (PDE6H). We did not detect mutations in this gene.

160

p.R29W/ p.R29W

p.Y323N/ p.Y323N

AII-1

AII-2

BII-1

p.L87Gfs*57/ Comc.2367+ plete 1delGTAAG ACHM

c.633G>C/ p.M455V

CII-1

DII-1

DII-2

0.16

0.10

0.16

0.30

0.40

Severe

0.10

Yes

-1.5

-5

+10.5

-2

-10

-3.5

Severe

Yes

Severe

Mild Normal pigmentary changes

No foveal reflex

Normal

No foveal reflex

Normal

Mild Normal pigmentary changes

No foveal reflex

Mild Mild Normal pigmen- pigmentary tary changes changes

Pigmen- Pigmentary Normal tary changes changes

No foveal reflex

0.20

-11

Severe

Yes

0.16

Last exam

First exam

Last exam

First exam

Reduced

No responses

Last exam

No re- No responses sponses

Reduced

Abbreviations are as follows: BCVA, best corrected visual acuity; COD, cone dystrophy; ACHM, achromatopsia; ND, not determined

Complete ACHM

p.Y323N/ p.Y323N

BII-2

Incomplete ACHM

Early onset COD

PDE6C Diagmutations nosis

Patient

Cone ERG Periphery Last First exam exam

#SNPs

Age Age first last First exam exam exam

Macular appearance

Color vision defects

Refractive error Last Photo- Nystag- Spherical equivalent exam phobia mus

218

Visual Complaints

208

BVCA best eye

Table 1 Clinical findings in probands and affected relatives, stratified according to genotype

e (Mb)

Homozygosity mapping reveals PDE6C mutations in cone disorders

161

4.1

The four heterozygous variants in PDE6C were not detected in 180 ethnically matched control individuals. The missense variants p.M232I, p.E699A and p.I826T were predicted by SIFT software, to be tolerated; only variant p.L138S was predicted not to be tolerated. Moreover, the isoleucine at position 826 is substituted by a threonine in the mouse and rat orthologues, rendering this very likely a benign amino acid change. The patients carrying the p.L138S, p.E699A and p.I826T variants were part of the 5.0 Affymetrix SNP screening. A copy number variant analysis of the SNP data with Partek GS software did not reveal a deletion or duplication of twenty intragenic SNPs and five copy number variant probes evenly spaced across the PDE6C gene (Supplemental table 1). Though very small deletions encompassing single exons or part of the promotor cannot be excluded, these data strongly suggest that the heterozygous PDE6C missense variants represent rare benign sequence variants. Table 1 shows a summary of the clinical findings of all patients with PDE6C mutations on both alleles. Patients AII-1 and AII-2 carried a homozygous missense mutation, located just upstream of the GAF-A domain (Figure 1A). They showed an early-onset COD; their visual acuity and cone ERG progressively declined in early teens. Patients BII-1 and BII-2 had a homozygous missense mutation in the GAF-B domain, which presumably affects the interaction with cGMP. This sibling pair presented with an incomplete ACHM; the cone ERG responses were significantly reduced but measurable both in childhood as on recent examination, whereas the rod ERG parameters were normal. Patient CII-1, carrying a protein-truncating mutation and a splice defect, had the most severe cone phenotype. At four years of age, the visual acuity was 0.10, the cone ERG was non-recordable and the rod function was completely normal (Figure 3). Patients DII-1 and DII-2, each carrying a splice defect and a p.M455V missense mutation, were clinically diagnosed as complete ACHM because they exhibited a low visual acuity of 0.16 with nystagmus since early childhood and absent cone responses on ERG. Figure 3 Electroretinogram of patient CII-1 An ERG performed with an abbreviated protocol of standard ISCEV, performed at the age of four years, without sedation, is shown at top with normal control traces at bottom, for purposes of comparison. Note normal rod-specific response (top trace at left), with no significant contribution of cones to combined rod-cone response (bottom trace at left); dark-adapted oscillatory responses as seen on ascending limb of the b-wave are residual at most. The absence of cone-specific function was proven by the absence of cone-specific response to 30Hz flicker stimulation (at right). In the adult patients, we performed Heidelberg Spectralis optical coherence tomography (OCT) at the most recent ophthalmologic examination. The absence of the photoreceptor cell layer in the fovea, a site with predominantly cone photoreceptor cells, was apparent in all patients. A typical OCT picture for patients from family A (patient AII-2) is shown in Figure 4B; a characteristic OCT image for patients from families B and D (patient DII-1) is shown in Figure

162

4D. The area of absent cone photoreceptors in patient DII-1, which spans almost the entire fovea, is significantly larger than the lesion in patient AII-2 (Figure 4B and 4D). We hypothesize that the missense variant in family A, which is located outside functionally important domains, may have a less severe effect on the function of PDE6C than the mutations observed in the other families. In zebrafish, mutations in the ortholog of PDE6C are responsible for progressive cone photoreceptor degeneration.21; 22 An intron 11 splice acceptor site mutation, 21 and a p.M175R missense mutation in the GAF-A domain22 lead to rapid degeneration of cone photoreceptors soon after their formation. Unlike in humans, zebrafish photoreceptors are continuously generated at the retinal margin by a population of mitotic progenitor cells, so cone photoreceptors can be found at the periphery throughout the life of these mutant fish. The splice site mutation in the zebrafish also resulted in the degeneration of rod photoreceptors in the central part of the retina.21 In zebrafish with the p.M175R missense mutation, rods show an abnormal morphology and rhodopsin mislocalization, but otherwise function normally.22 These findings suggest that the effect of the zebrafish Pde6c p.M175R mutation is more cone-specific than that of the Pde6c splice defect.

4.1 Figure 4 Retinal phenotypes of early onset cone dystrophy and complete achromatopsia in families A and D A) Fundus photography of the right eye of patient AII-2, performed at age 51 years, showing myopic changes in the peripapillary region. In the macular region mild pigmentary changes are present. The arrow denotes the position of the OCT image in B. B) The Heidelberg Spectralis optical coherence tomography (OCT) of this patient reveals a serous detachment of the photoreceptor layer in the central fovea. The retinal pigment epithelium is intact, but the outer and inner segments of the photoreceptors are absent. The length of the lesion is ~500 Âľm. The OCT cross section is not fully perpendicular. C) Fundus picture of the left eye of patient DII-1, performed at age 37 years, showing mild pigmentary changes in the macula. The arrow denotes the position of the OCT image in D. D) The Heidelberg Spectralis OCT of this patient displays a large area of ~1300 Âľm with absent cone photoreceptors, which involves almost the entire fovea

163 Homozygosity mapping reveals PDE6C mutations in cone disorders

To test the involvement of rod photoreceptors in human patients, we repeated the ERG on the two oldest patients with PDE6C mutations (AII-1 and AII-2, 47 and 51 years old, respectively), and did not find abnormal rod responses. Normal rod responses were also observed in patients CII-1, DII-1, and DII-2. We concluded that rod involvement does not appear to be a major consequence of PDE6C mutations in humans, although some dysfunction of rods may still occur later in life. In conclusion, through homozygosity mapping of patients with cone photoreceptor disorders from non-consanguineous and consanguineous families, we identified another causative gene: PDE6C. PDE6C has a crucial role in the cone phototransduction cascade. Similar to the other disease genes in this cascade (CNGA3, CNGB, and GNAT2),4-6 PDE6C mutations result in a severe early-onset cone disorder. We show that early-onset COD, incomplete ACHM, and complete ACHM form a continuous spectrum of a similar etiology. The observed variability in phenotypes may be due to differences in the functional effects of PDE6C mutations. Our findings will improve diagnosis and counseling of patients and their families, and add another step to solving the etiology of cone photoreceptor disorders.

Acknowledgements

We thank the individuals and their families for participating in this study. We thank Riet BernaertsBiskop, Rob W.J. Collin, Christian F.H.A. Gilissen, Arjen Henkes, Michael P. Kwint, Hans Scheffer, Marloes Steehouwer, Saskia D. van der Velde-Visser, Joris A. Veltman, Nienke A.W. Wieskamp, and Marijke Zonneveld for their valuable advice or expert assistance. We are thankful to Jose S. Ramalho for providing the pEGFP-RAB28 construct. We thank other members of the European Retinal Disease Consortium (Elfride De Baere, Tamar Ben-Yosef, Alejandro Garanto, Robert K. Koenekoop, Bart P. Leroy, Alberta A.H.J. Thiadens) for providing valuable samples and expertise. This study was financially supported by the Foundation Fighting Blindness USA (grants BR-GE0510-04890RAD, C-GE-0811-0545-RAD01, and BR-GE-0510-0490-HUJ) to A.I.d.H, F.P.M.C., and D.S., the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, the Gelderse Blinden Stichting, the Landelijke Stichting voor Blinden en Slechtzienden, the Stichting BlindenPenning, the Stichting Macula Degeneratie fonds, and the Rotterdamse Stichting Blindenbelangen (to F.P.M.C. and C.C.W.K.), by the Yedidut Research Grant (to E.B.), a Netherlands Organization for Scientific Research (NWO Vidi-91786396 and Vici-016.130.664 to R.R.), and a BMBF grant (01GM1108A) to B.W. and S.K.. The work was supported by the German Ministry of Education and Research (01GR0802 and 01GM0867) and the European Commission 7th Framework Program (261123, GEUVADIS) to T.M.S.

Web Resources

Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim Mutation nomenclature: http://www.hgvs.org Splice splice prediction: http://www.fruitfly.org/seq_tools/splice.html UCSC human genome database build hg18, March 2006: http://www.genome.ucsc.edu Amino acid change prediction of pathogenicity: http://sift.jcvi.org PDE6 gene orthologues search Swiss-Prot: http://www.expasy.ch/tools/blast/

164

Homozygosity mapping reveals PDE6C mutations in cone disorders

165

Exon-intron structure of PDE6C and copy number probes in this genomic region based on the March 2006 UCSC genome build (hg18). The genomic structure of PDE6C is depicted on scale. In blue, 20 intragenic and two flanking single nucleotide polymorphisms (SNPs), and in red, the five intragenic copy number (CN) probes are shown that are present on the Affymetrix GeneChip Genome-Wide Human Array 5.0. Using Partek Genomics Suite software we analyzed the raw signal intensities for all probes shown above. For patients 25456 (heterozygous carrier of p.L138S), 36419 (heterozygous carrier of p.E699G), and 43627 (heterozygous carrier of p.I826T), the raw signal intensities were within the normal variation of the average signal intensities of 400 tested individuals, and never lower than 75%. We have not performed a genome-wide SNP array analysis for patient 50488, heterozygous carrier of the PDE6C variant p.M321I.

Supplemental figure 1 Exon-intron structure and copy number probes of PDE6C

4.1

Supplemental table 1 Homozygous regions for families A and B Chr.

Start

End

Sample ID(s)

Length region (Mb)

# SNPs

80,888,184

107,352,036

A II-1

26,5

4.399

48,125,240

56,227,770

A II-1

8,1

434

33,234,603

37,599,402

A II-1

4,4

271

89,229,236

93,489,863

A II-1

4,3

677

34,180,193

36,208,710

A II-1

2,0

214

188,650,371

190,654,845

A II-1

2,0

236

155,393,372

157,303,002

A II-1

1,9

359

102,266,106

103,917,101

A II-1

1,7

211

37,664,444

39,256,145

A II-1

1,6

217

39,393,635

40,642,718

A II-1

1,2

231

65,087,560

66,311,040

A II-1

1,2

258

126,936,549

127,965,642

A II-1

1,0

216

159,531,547

160,529,894

A II-1

1,0

215

42,822,997

43,692,360

A II-1

0,9

202

25,380,356

49,895,689

B II-1 and -2

24,5

1.394

84,963,195

95,977,303

B II-1 and -2

11,0

1.975

7,188,532

16,863,548

B II-1 and -2

9,7

2.164

19,088,173

25,421,520

B II-1 and -2

6,3

1.010

48,854,873

54,922,139

B II-1 and -2

6,1

534

2,293,782

5,834,807

B II-1 and -2

3,5

870

127,192,344

129,994,218

B II-1 and -2

2,8

674

40,825,939

42,234,514

B II-1 and -2

1,4

219

82,983,190

84,287,750

B II-1 and -2

1,3

210

26,233,321

26,715,651

B II-1 and -2

0,5

274

166

4.1

167 Homozygosity mapping reveals PDE6C mutations in cone disorders

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14. Michaelides, M., Aligianis, I.A., Ainsworth, J.R., Good, P., Mollon, J.D., Maher, E.R., Moore, A.T., and Hunt, D.M. (2004). Progressive cone dystrophy associated with mutation in CNGB3. Invest Ophthalmol Vis Sci 45, 1975-1982. 15. Thiagalingam, S., McGee, T.L., Weleber, R.G., Sandberg, M.A., Trzupek, K.M., Berson, E.L., and Dryja, T.P. (2007). Novel mutations in the KCNV2 gene in patients with cone dystrophy and a supernormal rod electroretinogram. Ophthalmic Genet 28, 135-142. 16. Wissinger, B., Dangel, S., Jagle, H., Hansen, L., Baumann, B., Rudolph, G., Wolf, C., Bonin, M., Koeppen, K., Ladewig, T., Kohl, S., Zrenner, E., and Rosenberg, T. (2008). Cone dystrophy with supernormal rod response is strictly associated with mutations in KCNV2. Invest Ophthalmol Vis Sci 49, 751-757. 17. Marmor, M.F., Fulton, A.B., Holder, G.E., Miyake, Y., Brigell, M., and Bach, M. (2009). ISCEV Standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol 118, 69-77. 18. Burns, M.E., and Baylor, D.A. (2001). Activation, deactivation, and adaptation in vertebrate photoreceptor cells. Annu Rev Neurosci 24, 779-805. 19. Martinez, S.E., Heikaus, C.C., Klevit, R.E., and Beavo, J.A. (2008). The structure of the GAF A domain from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding surface, and a large cGMP-dependent conformational change. J Biol Chem 283, 2591325919. 20. Reese, M.G., Eeckman, F.H., Kulp, D., and Haussler, D. (1997). Improved splice site detection in Genie. Journal of computational biology : a journal of computational molecular cell biology 4, 311-323. 21. Stearns, G., Evangelista, M., Fadool, J.M., and Brockerhoff, S.E. (2007). A mutation in the cone-specific pde6 gene causes rapid cone photoreceptor degeneration in zebrafish. J Neurosci 27, 13866-13874. 22. Nishiwaki, Y., Komori, A., Sagara, H., Suzuki, E., Manabe, T., Hosoya, T., Nojima, Y., Wada, H., Tanaka, H., Okamoto, H., and Masai, I. (2008). Mutation of cGMP phosphodiesterase 6alphaâ&#x20AC;&#x2DC;-subunit gene causes progressive degeneration of cone photoreceptors in zebrafish. MOD 125, 932-946.

4.1

169 Homozygosity mapping reveals PDE6C mutations in cone disorders

Susanne Roosing, Klaus Rohrschneider, Avigail Beryozkin, Dror Sharon, Nicole Weisschuh, Jennifer Staller, Susanne Kohl, Lina Zelinger, Theo A. Peters, Kornelia Neveling, Tim M. Strom, European Retinal Disease Consortium, L. Ingeborgh van den Born, Carel B. Hoyng, Caroline C.W. Klaver, Ronald Roepman, Bernd Wissinger*, Eyal Banin*, Frans P.M. Cremers*, Anneke I. den Hollander*

*These authors contributed equally.

American journal of human genetics 2013, 93(1):110-117 170

Chapter 4.2 Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal recessive cone-rod dystrophy

4.2

171

Abstract

The majority of the genetic causes of autosomal recessive (AR) cone-rod dystrophy (CRD) are currently unknown. A combined approach of homozygosity mapping and exome sequencing revealed a homozygous nonsense mutation (c.565C>T; p.Glu189*) in RAB28 in a German family with three siblings with AR CRD. Another homozygous nonsense mutation (c.409C>T; p.Arg137*) was identified in a family of Moroccan Jewish descent with two siblings affected by AR CRD. All five affected individuals presented with hyperpigmentation in the macula, progressive loss of the visual acuity, atrophy of the retinal pigment epithelium, and severely reduced cone and rod responses on the electroretinogram. RAB28 encodes a member of the Rab subfamily of the RAS-related small GTPases. Alternative RNA splicing yields three predicted protein isoforms with alternative C-termini, which are all truncated by the nonsense mutations identified in the AR CRD families in this report. Opposed to other Rab GTPases which are generally geranylgeranylated, RAB28 is predicted to be farnesylated. Staining of rat retina showed localization of RAB28 to the basal body and the ciliary rootlet of the photoreceptors. Analogous to the function of other RAB family members, RAB28 might be involved in ciliary transport in photoreceptor cells. This study reveals a crucial role for RAB28 in photoreceptor function, and suggests that mutations in other Rab proteins may also be associated with retinal dystrophies.

172

Cone-rod dystrophy (CRD, MIM *120970) is characterized by primary loss of cone photoreceptors and subsequent or simultaneous loss of rod photoreceptors. The symptoms include poor visual acuity, disturbances in color vision, decreased sensitivity of the central visual field, and photo aversion. Because rods are also involved, impairment of night vision and peripheral vision loss can occur. The fundus appearance can vary from normal to a bull’s eye maculopathy, or to marked atrophy of the macular region, with a variable degree of temporal pallor of the optic disc.1 Visual fields display a central scotoma and full-field electroretinography (ERG) records a progressive deterioration of cone-derived amplitude responses, which are more reduced than, or equally reduced as, rod-derived responses. CRD has an estimated prevalence of 1:30,000 to 1:40,000,1-3 and displays all types of Mendelian inheritance. Seven genes have been implicated in the autosomal recessive (AR) form, the most prevalent mode of inheritance, i.e., ABCA4 (MIM *601691),4 ADAM9 (MIM *602713),5 C8orf37 (MIM *614477),6 CERKL (MIM *608381),7 EYS (MIM *612424),8; 9 RPGRIP1 (MIM *605446),10 and TULP1 (MIM *602280)11. However, mutations in these genes explain only a minority of the cases.12 The goal in this study was to identify genes underlying AR CRD using a combined approach of homozygosity mapping and exome sequencing. Such an approach has proven to be very effective in the discovery of genetic defects in several rare autosomal recessive diseases.13-16 This study was approved by the local medical ethics committees and adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent prior to participation in the study. To localize the genetic defect in a German family with three siblings affected by CRD (Family A; Figure 1), the DNA of all three affected individuals was analyzed using Affymetrix GeneChip Human Mapping 250K SNP arrays (Affymetrix, Santa Clara, CA, USA) and regions of homozygosity were calculated with Partek Genomics Suite software (Partek, St. Louis, MO, USA). This analysis revealed a shared homozygous region of 4.68 Mb on chromosome 4 in all three affected siblings (see Supplemental Table 1) containing 15 genes. Subsequently, exome sequencing was performed in two out of three affected siblings (A-II:1 and A-II:2). Exome sequencing was performed on a SOLiD4 sequencing platform (Life Technologies, Carlsbad, CA, USA) and the exomes were enriched according to the manufacturer’s protocol using Agilent’s SureSelect Human All Exon v.2 Kit (50Mb), containing the exonic sequences of approximately 21,000 genes (Agilent Technologies, Inc., Santa Clara, CA, USA). LifeScope software v2.1 (Life Technologies, Carlsbad, CA, USA) was used to map color space reads along the hg19 reference genome assembly. The DiBayes algorithm, with high-stringency calling, was used for single-nucleotide variant calling. The small Indel Tool was used to detect small insertions and deletions. For the analyzed individuals, a total of 83,287,089 and 76,841,552 reads were uniquely mapped to the gene-coding regions, respectively, with a median coverage of 67.3x in person A-II:1 and 61.9x in person A-II:2. The analysis uncovered 39,561 variants for individual A-II:1 and 39,029 variants for individual A-II:2. Ninety-six non-synonymous exonic and canonical splice-site variants, occurring with a frequency of less than 1% in 1154 unrelated individuals (in-house exome database), were shared by both individuals (data not shown). No pathogenic variants in the genes known to be mutated in AR CRD (ABCA4, ADAM9, C8orf37, CERKL, EYS, RPGRIP1, and TULP1) were observed among the shared variants. Under the hypothesis of autosomal recessive inheritance, we did not identify two heterozygous variants in a single gene (>20-<80% variation) in both affected individuals and only a single homozygous (>80% variation) variant, p.Glu189*(c.565C>T [NM_001017979.2]), in RAB28 (MIM *612994). This variant is located in the largest shared homozygous region in family A (Figure 1A and Supplemental Table 1).

173 RAB28 mutations in cone-rod dystrophy

4.2

Figure 1 Chromosome 4 homozygous regions and RAB28 mutations in two families with AR CRD A) Chromosomal position of RAB28, and location of the homozygous regions identified in families A and B using homozygosity mapping. B) Genomic structure and mRNA variants of RAB28. Variant 1 (V1) encodes a 221 amino acid (aa) protein isoform (RAB28S, NM_001017979.2), variant 2 (V2) encodes a 220 aa protein isoform (RAB28L, NM_004249.3), and variant 3 (V3) encodes a predicted 204 aa protein (HST03798, NM_001159601.1). The location of the nonsense mutations found in families A (mutation M1) and B (mutation M2) are indicated. C) Sanger sequencing confirmed the segregation of the homozygous nonsense mutations M1 (c.565C>T; p.Glu189*) in family A and M2 (c.409C>T; p.Arg137*) in family B. D) Pedigrees of family A of German ancestry and family B of Moroccan Jewish ancestry, demonstrating the segregation of the nonsense mutations in RAB28.

174

Sanger sequencing confirmed the homozygous presence of c.565C>T in the three affected siblings (Figures 1C and 1D). The mutation was not identified in 176 ethnically matched controls, nor in the Exome Variant Server (EVS) database (release ESP6500). Primers to amplify all coding exons and their exon-intron boundaries were designed using Primer 3 software (Supplemental Table 2). Sanger sequencing of all coding exons of RAB28 in 468 unrelated CRD and 149 unrelated individuals with cone dystrophy did not identify additional likely pathogenic mutations. We assessed SNP data of >400 unrelated individuals with AR CRD, Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) through the European Retinal Disease Consortium,6; 17 and identified seven families with conspicuously large homozygous regions spanning RAB28. Sanger sequencing of RAB28 in these families revealed a homozygous nonsense mutation (c.409C>T; p.Arg137*) in a consanguineous family (family B) of Moroccan Jewish ancestry. The two affected siblings in family B (II:1 and II:2; Figure 1) shared four homozygous genomic regions (over 10 Mb) by whole-genome SNP analysis (Supplemental Table 1). The largest region was on chromosome 7 and the second largest region was on chromosome 4, the latter including 315 genes among which OPN1SW (MIM *613522), and RAB28. Mutation analysis of OPN1SW did not yield causative variants. Segregation analysis confirmed the presence of the homozygous nonsense mutation in RAB28 in both siblings with CRD (Figures 1C and 1D). This mutation was not identified in 118 ethnically matched controls, nor in the EVS database The clinical features of the affected individuals of both families are described in Table 1. Members of family A were diagnosed with CRD in the second decade of life, with rapidly deteriorating visual acuities and high myopia. At their last visit, fundoscopy showed hyperpigmention of the fovea and autofluorescence revealed a slight hyperfluorescent fovea (Figures 2A and 2B). Optical coherence tomography (OCT) displayed altered photoreceptors in the fovea whereas the peripheral photoreceptors are intact (Figure 2C). Color vision tests revealed defects in all axes, and visual field testing showed a central scotoma. On ERG photopic responses were non-detectable and scotopic responses reduced in all three siblings. The clinical presentation of both affected individuals of family B was comparable to that of family A. Individual B-II:1 presented with low vision since early childhood, with progressive deterioration of the visual acuity and high myopia. The retinal pigment epithelium showed foveal atrophy, hypofluorescence was observed in the fovea and a hyperfluorescent ring was noted around the fovea (Figures 2D and 2E, respectively). OCT imaging confirmed absence of photoreceptors in the central fovea, while the photoreceptor layer further out appears largely intact (Figure 2F). Color vision defects were noted in both siblings. Photopic ERG responses were non-detectable, and scotopic responses were moderately reduced. RAB28 encodes a member of the Rab (â&#x20AC;&#x2DC;Ras-related in brainâ&#x20AC;&#x2122;)18 subfamily of the RAS-related small GTPases. Rab GTPases are key regulators in trafficking of proteins, and function as molecular switches to regulate fusion steps in vesicle transport, vesicle budding, motility, tethering19 and membrane fission.20 Proper membrane-association and subcellular localization of Rab proteins requires post-translational prenylation of cysteine motif(s) at the carboxyl terminus, which for the majority of Rabs involves the attachment of a geranylgeranyl (20 carbons) anchor by geranylgeranyltransferase II (GGT2 or RabGGT). RAB28 is a rather distant relative of the at least 60 different Rabs, 21 standing out because of its limited similarity (32% amino acid identity) to other Rab GTPases and its specific amino acid motifs. 21-23 RAB28 expresses three mRNA variants encoding different RAB28 isoforms with alternative C-termini (see Figure 1B).22 Another unique feature of RAB28 is that isoforms 1 and 2 are predicted to be farnesylated at their CaaX-motifs, 22-25 which was experimentally proven for isoform 1 of RAB28.25 The third iso form does not contain a CaaX-motif and therefore cannot be prenylated.

175 RAB28 mutations in cone-rod dystrophy

4.2

Table 1 Summary of the clinical data of five individuals of two families associated with RAB28 mutations Refractive Age at error last visit at last visit

Family

Individual Ancestry Diagnosis

Age at diagnosis

A-II:1

German

CRD

14 years

RE -16D SE RE 20/320 LE -15.5D SE LE 20/200

Hyperpigmented foveal region

Hyperfluorescent Irreg area at foveal oute region thin layer

A-II:2

German

CRD

12 years

RE -19D SE LE -22D SE

RE 20/320 LE 20/50

Hyperpigmented foveal region

Hyperfluorescent Irreg area at foveal oute region thin layer

A-II:3

German

CRD

10 years

RE -6.5D SE LE -7.0D SE

RE 20/80 LE 20/60

Hyperpigmented foveal region

B-II:1

Moroccan Jewish

CRD

Early child- 33 hood

BE -11D SE

BE 20/125

Atrophy Ring of hyper(geographic fluorescence atrophy-like) in fovea. White without pressure in periphery. Tilted optic discs (LE>RE) with temporal pallor LE.

B-II:2

Moroccan Jewish

COD/CRD NA

Visual acuity at last visit Fundus

BE = both eyes, D = diopters, ERG = electroretinogram, OCT = optical coherence tomography, OS = left eye, RE = right eye, RPE = retinal pigment epithelium, SE = spherical equivalent

176

Autofluorescence

Atro layer regio

acuity visit Fundus

Autofluorescence

20 00

Hyperpigmented foveal region

Hyperfluorescent Irregular inner and area at foveal outer segments in fovea, region thinning of other retinal layers of fovea

D-15 Panel: tritan and Concentric reduc- Age 16: non-detectable cone defects in scotopic tion to 15-20 function and reduced rod lines degrees (III/4) responses

20 0

Hyperpigmented foveal region

Hyperfluorescent Irregular inner and area at foveal outer segments in fovea, region thinning of other retinal layers of fovea

D-15 Panel saturated: Concentric reduc- Age 13: non-detectable tritan defects, tion to 30-40 photopic and markedly desaturated: marked degrees (III/4) reduced scotopic ERG protan defects

0 0

Hyperpigmented foveal region

Diffuse errors

Atrophy Ring of hyper(geographic fluorescence atrophy-like) in fovea. White without pressure in periphery. Tilted optic discs (LE>RE) with temporal pallor LE.

Atrophy in fovea; retinal layers absent at foveal region

D-15 Panel: RE NA scotopic lines; LE Tritan and Deutan defect

Age 30: non-detectable cone responses under photopic response; rod responses under scotopic responses moderately reduced (30% below normal). Mixed cone-rod response moderately to severely reduced (~50-60% below normal)

D-15 Panel: Deutan defect and scotopic lines, more severe in OS

Age 30: non-detectable cone responses under photopic condition; rod responses under scotopic response at below normal in RE and within normal limits in OS. Mixed cone-rod response moderately reduced (~25-35% below normal)

OCT

Color vision

Visual field (Goldmann)

Central scotoma

ERG

Age 10: non-detectable cone responses under photopic conditions, rod responses under scotopic conditions slightly reduced; age 15: scotopic markedly reduced

4.2

177 RAB28 mutations in cone-rod dystrophy

Figure 2 A

Figure 2 Clinical presentation of CRD associated with RAB28 mutations A-C: Retinal imaging of the right eye of individual A-II:2 of family A at age 25. A) Fundus photography showed slight hyperpigmentation of the foveal region; B) Autofluorescence imaging revealed a slight hyperfluorescent leash at the fovea without striking pathology (central brightening is an artifact); C) Optical coherence tomography (OCT) displayed altered photoreceptors in the central fovea with an intact periphery. D-F: Retinal imaging of the right eye of individual B-II:1 of family B at age 33. D) Fundus photographs showed foveal atrophy with no marked peripheral changes; E). Autofluorescence imaging revealed a hyperfluorescent ring surrounding the hypofluorescent fovea; F) OCT scans confirm the presence of foveal atrophy while the photoreceptor layer is largely intact further out. Wide expression is found for variant 1 in contrast to variant 2, which has been reported to be primarily expressed in testis.22 Expression analysis in RNA samples from various human tissues showed highest expression of variant 1 in lung, bone marrow, retinal pigment epithelium (RPE) and kidney, wide and abundant expression of variant 2, and highest expression of variant 3 in heart, lung, bone marrow, retina, brain and RPE (Supplemental Figure 1). Staining of rat retina showed localization of RAB28 to the basal body and the ciliary rootlet of the photoreceptors(Figure 3 and Supplemental Figure 3). Both nonsense mutations identified in the CRD families in this report are found in the shared mRNA segment, and are therefore predicted to truncate all three RAB28 isoforms. To determine whether the RAB28 mRNA is subjected to nonsense-mediated decay (NMD), RT-PCR analysis was performed in RNA samples isolated from lymphocytes of affected individuals of families A and B using a primer set amplifying exons 2-3, a region that is shared by all three RAB28 isoforms, as well as three different primer sets to evaluate the expression of the three isoforms individually. RAB28 transcript was detected in affected members of both families, excluding complete loss of RAB28 transcript by NMD (Supplemental Figure 2).

178

4.2 Figure 3 Subcellular localization of RAB28 in rat retina Cryosections of rat photoreceptor cells from day P20 were assessed by immunohistochemistry using mouse polyclonal α-RAB28 antibodies raised against aa 1-96 of human RAB28. These antibodies recognize all RAB28 isoforms (Sigma-Aldrich, SAB1406812, validated as shown in Supplemental Figure 3). Affinity purified guinea pig-α-RPGRIP1L (SNC040) was used as a marker of the basal body of the connecting cilium, as shown previously.26; 27 (A) Rat retinal sections from P20 were stained with DAPI (blue, first panel), α-RAB28 (green, second panel) and α-RPGRIP1L (red, third panel). The merged image in the right panel shows the co-localization of RAB28 and RPGRIP1L. (B) Enlargement of the photoreceptor layer, demonstrating partial co-localization of RAB28 and RPGRIP1L at the base of the connecting cilium. (C) A higher magnification of the connecting cilium region. The retinal layers are retinal pigment epithelium (RPE), photoreceptor layer (Ph), outer nuclear layer (ONL), outer plexiform layer (OPL), inner plexiform layer (IPL), ganglion cell layer (GCL).

179 RAB28 mutations in cone-rod dystrophy

The exact role of RAB28 in cone and rod photoreceptors remains to be determined. The localization of RAB28 to the basal body and ciliary rootlet suggests a role in ciliary transport. Interestingly, RAB8 and RAB11, which have so far not been shown to be associated with disease, coordinate primary ciliogenesis, 28 and together with RAB3A and RAB6A are involved in rhodopsin transport from the photoreceptor inner to outer segments through the connecting cilium.29-32 RAB28 may have adopted a similar function with specific importance for the highly active vesicle-based transport of photoreceptor proteins through the connecting cilium. Identification of RAB28 mutations emphasizes the role of disrupted ciliary processes in the pathogenesis of CRD. In addition to RAB28, three proteins involved in CRD (C8orf37, RPGRIP1 and TULP1) have so far been shown to localize to the connecting cilia of photoreceptor cells, and approximately one third of the gene defects underlying retinal degeneration affect the connecting cilium structure and/or function in photoreceptors. 33 The observation that nonsense mutations in RAB28 cause a phenotype only in the eye may point to functional redundancy in other tissues. In conclusion, a combined approach of homozygosity mapping and exome sequencing identified nonsense mutations in RAB28 as a rare cause of AR CRD. This study reveals a crucial role for RAB28 in photoreceptor function, and suggests that mutations in other Rab proteins may also be associated with retinal dystrophies. The functional implications of RAB28 mutations remain unclear and warrant further investigation.

Acknowledgments

We thank the individuals and their families for participating in this study. We thank Riet Bernaerts-Biskop, Rob W.J. Collin, Christian F.H.A. Gilissen , Arjen Henkes, Michael P. Kwint, Hans Scheffer, Marloes Steehouwer, Saskia D. van der Velde-Visser, Joris A. Veltman, Nienke A.W. Wieskamp and Marijke Zonneveld for their valuable advice or expert assistance. We are thankful to Jose S. Ramalho for providing the pEGFP-RAB28 construct. We thank other members of the European Retinal Disease Consortium (Elfride De Baere, Tamar Ben-Yosef, Alejandro Garanto, Robert K. Koenekoop, Bart P. Leroy, Alberta A.H.J. Thiadens) for providing valuable samples and expertise. This study was financially supported by the Foundation Fighting Blindness USA (grants BR-GE-0510-04890RAD, C-GE-0811-0545-RAD01, and BR-GE-0510-0490-HUJ) to A.I.d.H, F.P.M.C. and D.S., the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, the Gelderse Blinden Stichting, the Landelijke Stichting voor Blinden en Slechtzienden, the Stichting Blinden-Penning, the Stichting Macula Degeneratie fonds, the Rotterdamse Stichting Blindenbelangen (to F.P.M.C. and C.C.W.K.), by the Yedidut Research Grant (to E.B.), a the Netherlands Organization for Scientific research (NWO Vidi91786396 and Vici-016.130.664 to R.R.), and a BMBF grant (01GM1108A) to B.W. and S.K.. The work was supported by the German Ministry of Education and Research (01GR0802 and 01GM0867) and the European Commission 7th Framework Program (261123, GEUVADIS) to T.M.S.. None of the authors have any financial interest or conflicting interest to disclose.

Web Resourses

Exome Variant Server (EVS) database, release ESP6500, http://evs.gs.washington.edu/EVS/ Primer 3, http://frodo.wi.mit.edu/ Online Mendelian Inheritance in Man (OMIM), http://www/omim.org/

180

Supplemental Figure 1 Expression profile of alternatively spliced mRNAs of RAB28 in various tissues

Primers were designed to amplify the three mRNA variants (Table S3) to verify the expression of adult total RNA from heart (H), testis (Te), skeletal muscle (Sk), spleen (Sp), liver (Li), thymus (Th), bone marrow (Bo), retina (R), placenta (Pl), brain (Br), retinal pigment epithelium (RPE), duodenum (D), and kidney (K). The size of the products was compared to a 100 bp ladder (M); (A) expression of mRNA with primers in exon 1-3 showing a 238 bp product in an overlapping region for all isoforms. (B) expression of mRNA variant 1 with primers located in exons 5-9 represented by the smaller product at 195 base pairs (bp); (C) expression of mRNA variant 2 with primers located in exons 5-8 as shown by a 177-bp product; (D) expression of mRNA variant 3 with primers located in exons 5-7 displaying a 152-bp product; (E) expression of GUSB, a ubiquitously expressed gene, in all tissues showing a 245-bp product.

4.2

181 RAB28 mutations in cone-rod dystrophy

Supplemental Figure 2 Expression profile of alternatively spliced mRNAs of RAB28 in family A and B

The occurrence of NMD in the affected individuals of families A (II-1, II-2, II-3, all homozygous mutants) and B (II-1 homozygous mutant, I-2 heterozygous carrier) was explored in RNA isolated from lymphocytes (primers are indicated in Table S3) and compared to controls. The size of the products was compared to a 100 bp ladder (M). (A) expression of mRNA with primers in exon 2-3 showing a 106 bp product in an overlapping region for all isoforms. (B) expression of mRNA variant 1 with primers located in exons 5-9 represented by the smaller product at 195 base pairs (bp); (C) expression of mRNA 2 with primers located in exons 5-8 as shown by a 177-bp product; (D) expression of mRNA 3 with primers located in exons 5-7 displaying a 152-bp product; (E) expression of GUSB, a ubiquitously expressed gene, in all individuals showing a 245-bp product. Supplemental Figure 3 Validation of the anti-RAB28 antibodies

Western blot showing expression of RAB28 in non-transfected HEK293-cells (NT; negative control), HEK293-cells transfected with pEGFP-C2 (GFP; positive control for GFP), and with pEGFPRAB28 (RAB28). The left panel revealed a band at ~53 kDa for the GFP-RAB28 fusion protein, correlating with its predicted size, using mouse polyclonal Îą-RAB28 antibodies. The right panel shows the detection of GFP using mouse monoclonal Îą-GFP (Roche, 11 814 460 001).

182

omozygous SNPs

Supplemental Table 1 Shared homozygous regions identified in Families A and B Ranking

Chr

Start position (hg19)

End position (hg19)

Size (Mb)

Homozygous SNPs

11,015,508

15,700,443

4.68

592

38,017,394

39,444,614

1.43

144

21,043,923

22,474,801

1.43

132

171,827,483

172,984,650

1.16

130

212,950,206

214,009,363

1.06

117

85,490,315

86,550,497

1.06

65,647,955

67,062,093

1.41

54,327,340

55,261,166

0.93

32,595,139

33,321,361

0.73

130,168,579

131,314,824

1.15

114,302,855

142,163,046

27.86

103

6,2222,138

21,764,761

15.54

31,518,595

44,239,557

12.72

16,051,902

28,492,600

12.44

5,774,674

14,304,168

8.53

Family A

Family B

Supplemental Table 2 Primer sequences for RAB28 Exon in RAB28

Forward

Reverse

Product size (bp)

Exon 1

gcaactacctcccttcctcc

agggctcggggtaacgag

270

Exon 2

ctgcttaaatttgcttgttgg

gcagccccaaaattttacac

314

Exon 3

taaaggcaaattgggctcag

tgcatttgggagtagatttgc

292

Exon 4

tgctagtaagcaatttaaatggaag

ccagacaccatcattttggg

411

Exon 5

tatagatgtggaaactgagtca

cattacgggtggtttatattcatt

507

Exon 6

gctatttttggaatcacctgct

tgggaaagaatttatgggtga

320

Exon 9 isoform 1

tccatcatcagaagttcagca

tgtgtggtcccaaagttgaa

400

Exon 8 isoform 2

tgcgtgtgtgtgtgagacat

ctggcagaggaccaattttt

397

Exon 7 isoform 3

ctgccacttccatttccagt

aaggctctgcagattccaaa

426

Supplemental Table 3 Primer sequences for RAB28 expression study RAB28 mRNA variants

Forward

Reverse

Product size (bp)

Exon 1-3

atgtcggactctgaggagga

tcccaaatttgaagggtaaca

238

Exon 2-3

ctcaagaaacttttgggaaaca

tcccaaatttgaagggtaaca

106

Isoform 1 (exon 5-9)

acttacggttttgccaggaa

tccttgacataggttcctggtt

195

Isoform 2 (exon 5-8)

acttacggttttgccaggaa

ccgggtacttcactatttctgc

177

Isoform 3 (exon 5-7)

acttacggttttgccaggaa

gaaatggccctgtgactgtt

204

GUSB

ctgtacacgacacccaccac

tacagataggcagggcgttc

245

183 RAB28 mutations in cone-rod dystrophy

4.2

References 1. Hamel, C.P. (2007). Cone rod dystrophies. Orphanet J Rare Dis 2, 7. 2. Michaelides, M., Hunt, D.M., and Moore, A.T. (2004). The cone dysfunction syndromes. Br J Ophthalmol 88, 291-297. 3. Thiadens, A.A.H.J., den Hollander, A.I., Roosing, S., Nabuurs, S.B., Zekveld-Vroon, R.C., Collin, R.W.J., De, B.E., Koenekoop, R.K., van Schooneveld, M.J., Strom, T.M., van LithVerhoeven, J.J., Lotery, A.J., van Moll-Ramirez, N., Leroy, B.P., van den Born, L.I., Hoyng, C.B., Cremers, F.P.M., and Klaver, C.C.W. (2009). Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders. Am J Hum Genet 85, 240-247. 4. Cremers, F.P., van de Pol, D.J., van Driel, M., den Hollander, A.I., van Haren, F.J., Knoers, N.V., Tijmes, N., Bergen, A.A., Rohrschneider, K., Blankenagel, A., Pinckers, A.J., Deutman, A.F., and Hoyng, C.B. (1998). Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardtâ&#x20AC;&#x2122;s disease gene ABCR. Hum Mol Genet 7, 355-362. 5. Parry, D.A., Toomes, C., Bida, L., Danciger, M., Towns, K.V., McKibbin, M., Jacobson, S.G., Logan, C.V., Ali, M., Bond, J., Chance, R., Swendeman, S., Daniele, L.L., Springell, K., Adams, M., Johnson, C.A., Booth, A.P., Jafri, H., Rashid, Y., Banin, E., Strom, T.M., Farber, D.B., Sharon, D., Blobel, C.P., Pugh, E.N., Jr., Pierce, E.A., and Inglehearn, C.F. (2009). Loss of the metalloprotease ADAM9 leads to cone-rod dystrophy in humans and retinal degeneration in mice. Am J Hum Genet 84, 683-691. 6. Estrada-Cuzcano, A.I., Neveling, K., Kohl, S., Banin, E., Rotenstreich, Y., Sharon, D., FalikZaccai, T.C., Hipp, S., Roepman, R., Wissinger, B., Letteboer, S.J., Mans, D.A., Blokland, E.A., Kwint, M.P., Gijsen, S.J., van Huet, R.A., Collin, R.W., Scheffer, H., Veltman, J.A., Zrenner, E., Consortium, E.R.D., den Hollander, A.I., Klevering, B.J., and Cremers, F.P. (2012). Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement. Am J Hum Genet 90, 102-109. 7. Tuson, M., Marfany, G., and Gonzalez-Duarte, R. (2004). Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26). Am J Hum Genet 74, 128-138. 8. Collin, R.W., Littink, K.W., Klevering, B.J., van den Born, L.I., Koenekoop, R.K., Zonneveld, M.N., Blokland, E.A., Strom, T.M., Hoyng, C.B., den Hollander, A.I., and Cremers, F.P. (2008). Identification of a 2 Mb human ortholog of Drosophila eyes shut/spacemaker that is mutated in patients with retinitis pigmentosa. Am J Hum Genet 83, 594-603. 9. Abd El-Aziz, M.M., Barragan, I., Oâ&#x20AC;&#x2122;Driscoll, C.A., Goodstadt, L., Prigmore, E., Borrego, S., Mena, M., Pieras, J.I., El-Ashry, M.F., Safieh, L.A., Shah, A., Cheetham, M.E., Carter, N.P., Chakarova, C., Ponting, C.P., Bhattacharya, S.S., and Antinolo, G. (2008). EYS, encoding an ortholog of Drosophila spacemaker, is mutated in autosomal recessive retinitis pigmentosa. Nat Genet 40, 1285-1287. 10. Hameed, A., Abid, A., Aziz, A., Ismail, M., Mehdi, S.Q., and Khaliq, S. (2003). Evidence of RPGRIP1 gene mutations associated with recessive cone-rod dystrophy. J Med Genet 40, 616619. 11. Roosing, S., van den Born, L.I., Hoyng, C.B., Thiadens, A.A., de Baere, E., Collin, R.W., Koenekoop, R.K., Leroy, B.P., van Moll-Ramirez, N., Venselaar, H., Riemslag, F.C., Cremers, F.P., Klaver, C.C., and den Hollander, A.I. (2013). Maternal uniparental isodisomy of chromosome 6 reveals a TULP1 mutation as a novel cause of cone dysfunction. Ophthalmology 120, 1239-1246. 12. Littink, K.W., Koenekoop, R.K., van den Born, L.I., Collin, R.W.J., Moruz, L., Veltman, J.A., Roosing, S., Zonneveld, M.N., Omar, A., Darvish, M., Lopez, I., Kroes, H.Y., van Genderen, M.M.,

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Hoyng, C.B., Rohrschneider, K., van Schooneveld, M.J., Cremers, F.P.M., and den Hollander, A.I. (2010). Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations. Invest Ophthalmol Vis Sci 51, 5943-5951. 13. Zangen, D., Kaufman, Y., Zeligson, S., Perlberg, S., Fridman, H., Kanaan, M., AbdulhadiAtwan, M., Abu Libdeh, A., Gussow, A., Kisslov, I., Carmel, L., Renbaum, P., and Levy-Lahad, E. (2011). XX ovarian dysgenesis is caused by a PSMC3IP/HOP2 mutation that abolishes coactivation of estrogen-driven transcription. Am J Hum Genet 89, 572-579. 14. Walsh, T., Shahin, H., Elkan-Miller, T., Lee, M.K., Thornton, A.M., Roeb, W., Abu Rayyan, A., Loulus, S., Avraham, K.B., King, M.C., and Kanaan, M. (2010). Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 87, 90-94. 15. Sirmaci, A., Walsh, T., Akay, H., Spiliopoulos, M., Sakalar, Y.B., Hasanefendioglu-Bayrak, A., Duman, D., Farooq, A., King, M.C., and Tekin, M. (2010). MASP1 mutations in patients with facial, umbilical, coccygeal, and auditory findings of Carnevale, Malpuech, OSA, and Michels syndromes. Am J Hum Genet 87, 679-686. 16. Gilissen, C., Arts, H.H., Hoischen, A., Spruijt, L., Mans, D.A., Arts, P., van Lier, B., Steehouwer, M., van Reeuwijk, J., Kant, S.G., Roepman, R., Knoers, N.V., Veltman, J.A., and Brunner, H.G. (2010). Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet 87, 418-423. 17. Ozg端l, R.K., Siemiatkowska, A.M., Yucel, D., Myers, C.A., Collin, R.W.J., Zonneveld, M.N., Beryozkin, A., Banin, E., Hoyng, C.B., van den Born, L.I., European Retinal Disease, C., Bose, R., Shen, W., Sharon, D., Cremers, F.P.M., Klevering, B.J., den Hollander, A.I., and Corbo, J.C. (2011). Exome sequencing and cis-regulatory mapping identify mutations in MAK, a gene encoding a regulator of ciliary length, as a cause of retinitis pigmentosa. Am J Hum Genet 89, 253-264. 18. Touchot, N., Chardin, P., and Tavitian, A. (1987). Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc Natl Acad Sci U S A 84, 8210-8214. 19. Stenmark, H., and Olkkonen, V.M. (2001). The Rab GTPase family. Genome Biol 2, REVIEWS3007. 20. Miserey-Lenkei, S., Chalancon, G., Bardin, S., Formstecher, E., Goud, B., and Echard, A. (2010). Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex. Nat Cell Biol 12, 645-654. 21. Pereira-Leal, J.B., and Seabra, M.C. (2001). Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 313, 889-901. 22. Brauers, A., Schurmann, A., Massmann, S., Muhl-Zurbes, P., Becker, W., Kainulainen, H., Lie, C., and Joost, H.G. (1996). Alternative mRNA splicing of the novel GTPase Rab28 generates isoforms with different C-termini. Eur J Biochem 237, 833-840. 23. Lee, S.H., Baek, K., and Dominguez, R. (2008). Large nucleotide-dependent conformational change in Rab28. FEBS Lett 582, 4107-4111. 24. Maurer-Stroh, S., and Eisenhaber, F. (2005). Refinement and prediction of protein prenylation motifs. Genome Biol 6, R55. 25. Maurer-Stroh, S., Koranda, M., Benetka, W., Schneider, G., Sirota, F.L., and Eisenhaber, F. (2007). Towards complete sets of farnesylated and geranylgeranylated proteins. PLoS Comput Biol 3, e66. 26. Arts, H.H., Doherty, D., van Beersum, S.E.C., Parisi, M.A., Letteboer, S.J.F., Gorden, N.T., Peters, T.A., Marker, T., Voesenek, K., Kartono, A., Ozyurek, H., Farin, F.M., Kroes, H.Y., Wolfrum, U., Brunner, H.G., Cremers, F.P.M., Glass, I.A., Knoers, N.V.A.M., and Roepman, R. (2007). Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome. Nat Genet 39, 882-888.

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27. Coene, K.L., Mans, D.A., Boldt, K., Gloeckner, C.J., van Reeuwijk, J., Bolat, E., Roosing, S., Letteboer, S.J., Peters, T.A., Cremers, F.P., Ueffing, M., and Roepman, R. (2011). The ciliopathyassociated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase. Hum Mol Genet 20, 3592-3605. 28. Knodler, A., Feng, S., Zhang, J., Zhang, X., Das, A., Peranen, J., and Guo, W. (2010). Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci U S A 107, 63466351. 29. Deretic, D., Huber, L.A., Ransom, N., Mancini, M., Simons, K., and Papermaster, D.S. (1995). Rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis. J Cell Sci 108 (Pt 1), 215-224. 30. Satoh, A.K., Oâ&#x20AC;&#x2DC;Tousa, J.E., Ozaki, K., and Ready, D.F. (2005). Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors. Development 132, 1487-1497. 31. Deretic, D. (1997). Rab proteins and post-Golgi trafficking of rhodopsin in photoreceptor cells. Electrophoresis 18, 2537-2541. 32. Moritz, O.L., Tam, B.M., Hurd, L.L., Peranen, J., Deretic, D., and Papermaster, D.S. (2001). Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. Mol Biol Cell 12, 2341-2351. 33. Estrada-Cuzcano, A.I., Roepman, R., Cremers, F.P., den Hollander, A.I., and Mans, D.A. (2012). Non-syndromic retinal ciliopathies: translating gene discovery into therapy. Hum Mol Genet 21, R111-124.

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187 RAB28 mutations in cone-rod dystrophy

Susanne Roosing, L. Ingeborgh van den Born, Riccardo Sangermano, Sandro Banfi, Robert K. Koenekoop, Marijke N. Zonneveld-Vrieling, Caroline C.W. Klaver, Janneke J.C. van Lith-Verhoeven, Frans P.M. Cremers*, Anneke I. den Hollander*, Carel B. Hoyng* *

These authors contributed equally.

Ophthalmology, In press 188

Chapter 4.3 Mutations in MFSD8, encoding a lysosomal membrane protein, are associated with non-syndromic cone dysfunction

4.3

189

Abstract

Purpose: This study aimed to identify the genetic defects in two families with autosomal recessive macular dystrophy with central cone involvement. Design: Case series Participants: Two families and a cohort of 244 individuals with various inherited maculopathies and cone disorders. Methods: Genome-wide linkage analysis and exome sequencing was performed in one large family with five affected individuals. In addition, exome sequencing was performed in the proband of a second family. Subsequent analysis of the identified mutations in 244 patients was performed by Sanger sequencing or restriction enzyme digestion. The medical history of individuals carrying the MFSD8 variants was reviewed and additional ophthalmic examinations were performed, including electroretinography (ERG), multifocal ERG (mfERG), perimetry, optical coherence tomography (OCT), fundus autofluorescence (FAF), and fundus photography. Main Outcome Measures: MFSD8 variants, age at diagnosis, visual acuity, fundus appearance, color vision defects, visual field, ERG, mfERG, FAF and OCT findings. Results: Compound heterozygous variants in MFSD8, a gene encoding a lysosomal transmembrane protein, were identified in two families with macular dystrophy with a normal or subnormal ERG, but reduced mfERG. In both families a heterozygous missense variant, p.Glu336Gln, was identified, which was predicted to have a mild effect on the protein. In the first family a protein-truncating variant (p.Glu381*) was identified on the other allele, and in the second family a variant (c.1102G>C) was identified which results in a splicing defect leading to skipping of exon 11 (p.Lys333Lysfs*3). The p.Glu336Gln allele was found to be significantly enriched in patients with maculopathies and cone disorders (6/488) compared to ethnically matched controls (35/18,682; p-value <0.0001), suggesting that it may act as a genetic modifier. Conclusions: In this study we identified variants in MFSD8 as a novel cause of non-syndromic autosomal recessive macular dystrophy with central cone involvement. Affected individuals showed no neurological features typical for variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a severe and devastating multisystem lysosomal storage disease previously associated with mutations in MFSD8. We propose a genotype-phenotype model in which a combination of a severe and a mild variant cause non-syndromic macular dystrophy with central cone involvement, while two severe mutations cause vLINCL.

190

Introduction

Inherited macular and cone diseases like achromatopsia (ACHM), cone dystrophy (COD) and cone-rod dystrophy (CRD) have an estimated worldwide prevalence of 1:30.000-1:40.000.1-3 Typically, individuals with ACHM present shortly after birth with significantly reduced visual acuity, severe photophobia, a congenital pendular nystagmus, and color vision defects in all color axes. Absent or residual cone responses with normal rod responses are measured with full-field electroretinography (ERG),4, 5 Optical coherence tomography (OCT) shows initial loss of inner- and outer cone segments with disruption of the ciliary layer, followed by the appearance of a bubble with cell loss in the cone photoreceptor layer. The end stage of ACHM is characterized by atrophy of the RPE. This degradation takes place in the second decade of life and progresses with age.6 Individuals with COD present with a normal cone function at birth, but develop progressive loss of cones and central vision during the first or second decade of life. Most individuals develop severe visual acuity loss before the fourth decade.7 Clinical features of COD are poor visual acuity, disturbances in color vision, a fundus appearance varying from normal, to a bullâ&#x20AC;&#x2122;s eye maculopathy, or total atrophy of the macular region, with a variable degree of temporal pallor of the optic nerve. The visual fields of these individuals show a central scotoma and ERG demonstrates progressive deterioration of the cone-derived photopic amplitude responses. Both diseases are characterized by loss of cone photoreceptors and a progressive visual decline, but CRD can be distinguished from COD by early involvement of rod photoreceptors. Besides the symptoms seen in COD, patients with CRD may also experience nyctalopia due to rod dysfunction.8 Both disorders have a high genetic heterogeneity and display all Mendelian inheritance forms. The most prevalent mode of inheritance is autosomal recessive (ar). Eight genes have been implicated in ar COD (ABCA4,9 CACNA2D4,10 CNGA3,11 CNGB3,12, 13 KCNV2,14 PDE6C, 3 PDE6H,15 TULP116). Three of these genes have initially been associated with ACHM, a form of cone-dysfunction that can develop into cone dystrophy. Eighteen genes have been associated to ar CRD (ABCA4,17 ADAM9,18 C8orf37,19 CDHR1,20, 21 CERKL,22 CNGB3, 23 CRB1, 24 CRX, 24 EYS, 25 FSCN2, 26 GUCY2D,27 KCNV2,14 PDE6C, 23 POC1B,28, 29 PROM1, 30 RAB28, 31 RPE65, 24 RPGRIP1, 23 and TULP116). In both disorders mutations in these genes explain disease in an estimated 21% (COD) and 25% (CRD) of patients.1;2 14, 32, 33 In the past few years, exome sequencing has shown its power to elucidate genetic defects in inherited retinal diseases.19, 31, 34, 35 The aim of this study was to identify novel causes of ar maculopathies and cone disorders using linkage analysis and next generation sequencing (NGS).

Methods

Subjects and clinical evaluation A large non-consanguineous Dutch family (Family A) with five family members and an isolated Dutch case (Family B) affected by macular dystrophy with central cone involvement were included in this study. An additional cohort of individuals with inherited maculopathies and cone disorders (ACHM, n=22; COD, n= 110; CRD, n= 112), the majority of which represent isolated cases, were ascertained from various ophthalmic centers in the Netherlands, Belgium, the United Kingdom, and Canada. Individuals were diagnosed with central cone disease if they showed a progressive decline of visual acuity, color vision disturbances, and reduced multifocal ERG. Individuals are diagnosed with ACHM when they showed shortly after birth a significantly reduced visual acuity, severe photophobia, a congenital pendular nystagmus, and color vision defects in the protan, deutan and tritan color axes. Persons were diagnosed with COD if they showed a progressive decline of visual acuity, color vision disturbances, and reduced cone amplitude responses on ERG, with normal rod responses for â&#x2030;Ľ5 years. Inclusion criteria for CRD were a progressive decline of visual acuity, color vision disturbances, and a reduction of both cone and rod ERG responses, with cones equally or more severely reduced. 36

191 MFSD8 mutations cause non-syndromic macular dystrophy

4.3

Figure 1

A I

Family A

d-Name (Mb) D4S406 (112.0) 1 D4S2989 (113,1) 2 MFSD8 (128,8) M1 1

5 6 + 4

3 4 M2

3 1 4 2 M2 M1

M1: c.1006G>C; p.Glu336Gln

3 1 4 2 M2 M1

8-10

6-7

3 4 M2

(M3/+)

2 3 1 4 2 M2 M1

II 1 2 M1

Family B

(M1/+)

7 8 +

3 5 6 +

1 2 M1

M2: c.1141G>T; p.Glu381*

M1/M3

11-13 3 7 8 +

3 4 M2

5 6 +

M3: c.1102G>C; p.Lys333Lysfs*3

Glu336Gln

Human MFSD8 Chimpanzee Mfsd8 Macaque Mfsd8 Rat Mfsd8 Mouse Mfsd8 Chicken Mfsd8 Cat Mfsd8 Dog Mfsd8 Cow Mfsd8 Opossum Mfsd8 Frog Mfsd8 Zebrafish Mfsd8 Fruitfly Mfsd8

321 321 321 321 321 321 321 321 321 321 321 321 321

VVIFLGVKLLSKKIGERAILLGGLIVVWVGF VVIFLGVKLLSKKIGERAILLGGLIVVWVGF VVIFVGVKLLSKKIGERAILLGGLIVVWVGF VLVFMGVKLLSKKIGERAILLGGFVVVWVGF VLVFMGVKLLSKKIGERAILLGGFVVVWVGF VIVFMVVKTLSKKTGERAILHGGLLIILIGF VIIFMGIKLLSKKIGERALLLGGLIIIWVGF LIIFMGIKLLSKKIGERAILLGGFIVVWVGF VIIFMGIKLLSKKIGERALLLGGLIIIWVGF VMVFMGIKILSKRAGERALLLGGLVVVFIGF VIVFLTVKILCKKTGERVLLLGGLAVIWIGF ILVFLVVKVVSARVGDRPLLLGGLILIFVGG LVTFVLIEPMCKIIAERYVLIWGGFSLMFLF

351 351 351 351 351 351 351 351 351 351 351 351 351

Figure 1 MFSD8 variants in two Dutch pedigrees with macular dystrophy with central cone involvement A) Segregation analysis in family A with five affected and eight non-affected family members demonstrated autosomal recessive inheritance of variants M1 (c.1006G>C; p.Glu336Gln) and M2 (c.1141G>T; p.Glu381*). Microsatellite marker positions are based on the UCSC human genome reference sequence, version hg19. In family B biallelism of M1 (c.1006G>C; p.Glu336Gln) and M3 (c.1102G>C; p.Lys333Lysfs*3) was confirmed by restriction fragment length analysis. B) Alignment of MFSD8 protein orthologs. The glutamic acid residue at position 336 is highly conserved among mammalian MFSD8 orthologs, but is not conserved in zebrafish. Identical amino acids are indicated in black boxes, and conserved residues are indicated in gray boxes. After identification of the genetic defect, the medical history of the affected individuals of families A and B was reviewed. Ophthalmologic examinations included best-corrected visual acuity (BCVA; Snellen chart), slit-lamp biomicroscopy, ophthalmoscopy, color vision testing (Hardy–Rand–Rittler color vision test and Lanthony Panel D-15 tests), and visual field testing using Goldmann kinetic perimetry (targets V-4e, and I-4e to I-1e). Fundus photographs centered on the macular area as well as on the four peripheral quadrants was performed using standard procedures. Electroretinography (ERG) was performed according to the extended protocol for the full-field ERG and multifocal ERG (mfERG) using 61 hexagons (central 28°) of the International Society for Clinical Electrophysiology of Vision (ISCEV). 37 For individual II-5 of family A and II-1 of family B spectral-domain optical coherence tomography (SD-OCT; Heidelberg Spectralis HRA+OCT, Heidelberg Engineering, Germany) was used to obtain cross-sectional images of the macular region through dilated pupils, one horizontally and one vertically (30° wide, 51 frames per line). For the same individuals fundus autofluorescence

192

(FAF) imaging (Heidelberg Spectralis HRA+OCT, Heidelberg Engineering, Germany) was performed of the macula at 488 nm wavelength using a 30° lens: a mean image out of 16 single images was calculated. In addition we performed a volume scan (20x15°, 37 lines, 36 frames per line) for all affected individuals. Individual A:II-2 was examined by a neurologist at age 48 due to a suspected optic neuropathy. Examinations included a cerebral computed tomography (CT) scan, measurement of evoked potentials, and an electroencephalogram (EEG). This study was approved by the Institutional Review Board, and adhered to the tenets of the declaration of Helsinki. The probands of both families provided written informed consent to perform exome sequencing. Molecular Genetic Analysis Blood samples were obtained of all probands, and when possible, their parents, affected and unaffected siblings. DNA was isolated from peripheral blood lymphocytes by standard procedures. Mutations in various genes previously implicated in inherited maculopathies and cone disorders, i.e. ABCA4, CNGA3, CNGB3, KCNV2, PDE6C and RAB28, were excluded by Sanger sequencing. Employing genomic DNA of four affected members of family A (II-1, II-2, II-3, II-4) genome-wide linkage analysis was carried out using 250K SNP arrays. Multipoint parametric linkage analysis was performed with Genehunter (Decode Genetics Reykjavik, Iceland) in the Easylinkage Plus software package (University of Würzburg, Germany) using the Marshfield genetic SNP map and the Caucasian allele frequencies. An autosomal recessive mode of inheritance was assumed since neither the parents nor the 10 children of the five affected siblings were affected. The diseaseallele frequency was estimated at 0.001. For all five affected members of family A and their available unaffected family members, microsatellite analysis was performed for selected markers to analyze the established linkage peaks on chromosomes 1, 2 and 4. Microsatellites D1S2779, D1S2813, D2S103, D2S143, D4S406 and D4S2989 were amplified with primers containing a M13 forward and reverse sequence tail. An additional polymerase chain reaction (PCR) was performed with a fluorescently labeled M13-forward primer and an unlabelled M13-reverse primer. PCR products were mixed with a fluorescent size marker (Applied Biosystems, Bleiswijk, the Netherlands) and samples were analyzed on a 3100 or 3730 DNA Analyzer (Applied Biosystems, Bleiswijk, the Netherlands). Fragment lengths were analyzed with GeneMapper software (Applied Biosystems, Bleiswijk, the Netherlands). Haplotypes were constructed based on the size of the alleles of the microsatellites. Subsequent exome sequencing was performed for probands of family A and of an unrelated family (family B) on a SOLiD4 sequencing platform (Life Technologies, Carlsbad, CA, USA). Exomes were enriched according to the manufacturer’s protocol using the Agilent SureSelect Human All Exon v.2 Kit (50Mb), which targets the exonic sequences of approximately 21,000 genes (Agilent Technologies, Inc., Santa Clara, CA, USA). LifeScope software v2.1 (Life Technologies, Carlsbad, CA, USA) was used to map color space reads along the hg19 reference genome assembly. The high-stringency calling DiBayes algorithm was used for single-nucleotide variant calling, and small insertions and deletions were detected using the small Indel Tool. Nonsense, frameshift, (canonical) splice site or missense variants with a nucleotide conservation score of PhyloP score >2.7 were selected for further analysis when presenting a frequency <0.5% in dbSNP and an in-house database containing exome sequencing variants of 2,095 individuals (4,190 alleles). Based on the assumption of autosomal recessive inheritance, potential compound heterozygous variants were selected when present on ≥20% variant reads, whereas homozygous variants were selected when present on ≥80% variant reads. Confirmation of identified variants and segregation analysis was performed using PCR and subsequent Sanger sequence analysis. All primers were designed with Primer3 software and are presented in Supplemental Table 1.

193 MFSD8 mutations cause non-syndromic macular dystrophy

4.3

Additional screening of patients and controls for the observed mutations in MFSD8 was performed by sequencing of exons 11 and 12 (892 basepairs (bp)). To prove the presence of the mutations on separate alleles in family B, digestion by HaeIII of the amplified product of exon 11 (589 bp) was performed. Mutant alleles carrying the c.1006G>C; p.Glu336Gln variant were digested into 333, 236 and 20 bp fragments, whereas the wildtype allele was digested into fragments of 569 and 20 bp. The undigested 569 bp product was subsequently Sanger sequenced to confirm the presence of c.1102G>C on the opposite allele. Using the same method 151 ethnically matched controls were evaluated for the presence of the missense variant p.Gly336Gln. EBVtransformed lymphocytes of affected individual B:II-1 were grown with or without cycloheximide to visualize the effect of the c.1102G>C mutation on the transcript level, and to determine possible degradation of nonsense-containing mRNAs by nonsense mediated decay (NMD).38 Reverse transcription with iScript (Biorad, Veenendaal, The Netherlands) was performed on total RNA (1 Âľg) to obtain cDNA. RT-PCR experiments were performed using 2.5 Âľl cDNA with forward primers in exon 10 or 11 and a reverse primer in exon 12, followed by Sanger sequencing of the obtained PCR products. Furthermore, the pathogenicity of the p.Gly336Gln mutation was evaluated using the Granthamand PhyloP-scores,39 SIFT,40 and Polyphen-2 (version 2.2.2; http://genetics.bwh.harvard.edu/ pph2/).

Results

Identification of MFSD8 variants in families A and B Genome-wide linkage analysis using DNA of four affected members of family A revealed three linkage peaks that were located on chromosome 1 (25.2 Mb; SNP_A-1923543-SNP_A-1910059; containing 206 genes), 2 (53.7 Mb; SNP_A-2027807-SNP_A-2039109; containing 327 genes) and 4 (29.7 Mb; SNP_A-1902084-SNP_A-2036896; containing 95 genes) with a LOD-score of 1.81. Using microsatellite marker analysis in all available individuals of family A, haplotypes at the linkage region on chromosome 4 were found to fully segregate with the disease phenotype (Figure 1A). Using Easylinkage a maximum LOD-score of 3.4 was determined, thereby reaching genomewide significance, whereas haplotypes at the regions on chromosome 1 and 2 did not segregate (not shown). To elucidate the gene defect in II-1 of family A, exome sequencing was performed. Under the hypothesis of autosomal recessive inheritance, we identified three potential compound heterozygous variants (>20% variation) and no homozygous (>80% variation) variants based on our sequence variant filtering criteria for II-1 of family A (Supplemental Table 2). Only one of these genes, Major Facilitator Superfamily Domain Containing 8 (MFSD8), which carried the compound heterozygous variants p.Glu381* and p.Glu336Gln, resides within the linkage region on chromosome 4. Segregation analysis of the variants using Sanger sequencing confirmed segregation within the family (Figure 1A). Exome sequencing in the proband (B:II-1) of a second family (Family B, figure 1B) revealed two heterozygous variants in the same gene, and also three homozygous variants in other genes (Supplemental Table 3). This isolated case is a heterozygous carrier of the same missense variant identified in family A, p.Glu336Gln, and of the variant c.1102G>C. The latter variant is predicted to alter a highly conserved amino acid (p.Asp368His) and in addition can affect splicing as it changes the last nucleotide of exon 11.41 Analysis of the effect of this variant at the transcript level revealed that it leads to skipping of exon 11, as the C residue could not be detected at position c.1102 (Figure 1C, Supplemental figure 1), indicating that only cDNA of the other mutant (p.Glu336Gln) allele could be amplified. The c.1102G>C variant thereby is predicted to result in a truncated protein (p.Lys333Lysfs*3). Since parental DNA was not available, segregation analysis could not be performed. However, due to the proximity of both variants within one amplicon, restriction analysis with HaeIII showed that the p.Glu336Gln and c.1102G>C variants lie on different alleles.

194

Figure 2 Clinical evaluation of macular dystrophy with central cone involvement caused by MFSD8 variants Aâ&#x20AC;&#x201C;C, Clinical characteristics of patient II-3 of family A. A) The fundus photograph showed retinal pigment epithelium (RPE) changes in the macula with a normal periphery. B) Fundus autofluorescence (FAF) revealed a hypofluorescent central area surrounded by a hyperfluorescent ring. C) Optical coherence tomography (OCT) displayed foveal atrophy and a generally thinned retina. D-F, Clinical characteristics of patient II-5 of family A. D) Fundus photographs showed a mild peripapillary atrophy with a granular aspect of macula. The periphery is without abnormalities E) FAF showed symmetrical central alterations at the level of the outer retinal layers F) OCT displayed relatively symmetrical central alterations at the level of the outer retinal layers. G-I, Clinical characteristics of patient II-1 of family B. H) Fundus photographs revealed a pale optic disc, attenuated vessels. Also, atrophy of the RPE in the posterior pole was noted with a normal appearance of (mid)peripheral RPE. H) FAF showed a decreased autofluorescence in the central macula and a perimacular mottled pattern of hypofluorescence) I) OCT displayed loss of retinal layers at the fovea, which are partially present, but show irregular outer retinal layers perifoveally and in posterior pole. Furthermore, an intact inner retina and thickened choroid were noted. The p.Glu381* and p.Lys333Lysfs*3 variants in families A and B were not identified in an in-house exome (4,190 alleles) database, in 12,006 alleles of the online databases (Exome Variant Server, http://evs.gs.washington.edu/EVS/, 6 November 2013), nor in 2,184 alleles of the 1000genomes

195 MFSD8 mutations cause non-syndromic macular dystrophy

4.3

project, www.1000genomes.org, 6 November 2013). Both mutations were previously described and are presumed to be loss-of-function mutations, causing variant late-infantile neuronal ceroid lipofuscinosis (vLINCL).41, 42 However, the missense mutation p.Glu336Gln identified in both families was also identified in 1/302 (0.3%) alleles of healthy ethnically-matched control individuals. Furthermore, the mutation was found in 9/4,190 alleles (0.21%) in our in-house exome database, and in 25/12,006 alleles (0.19%) of European Americans in the Exome Variant Server, but it was not identified in 2,184 alleles in the 1000genomes project. Screening of our inherited maculopathy and cone disorder cohort for the missense mutation p.Glu336Gln yielded four additional individuals carrying this mutation in a heterozygous state. Subsequent sequence analysis of the MFSD8 gene in these individuals did not identify a second variant on the other allele. The frequency of the p.Glu336Gln allele in 6/488 (1.2%) affected individuals with inherited maculopathies and cone disorders is significantly higher than in controls with a frequency of 35/18,682 (0.18%) alleles (P-value < 0.0001, Chi-square test, two-tailed). At the nucleotide level, c.1006G>C (p.Glu336Gln) is highly conserved with a PhyloP score of 5.53. The mutation is predicted to be possibly damaging with a score of 0.927 by Polyphen-2, but the glutamic acid to glutamine change at this position yields a low Grantham score of 29 and appears to be tolerated by SIFT. The wild-type glutamic acid residue is negatively charged, acidic and large, while the mutant glutamine residue is neutrally charged, and smaller in size. The glutamic acid residue at this position is conserved from human to fruitfly with the exception of zebrafish (Figure 1B) and it is located in the cytoplasmic region, two amino acids N-terminal to the 9th transmembrane domain of the protein. The mutant residue might disturb the core structure of this domain. Clinical features of patients of families A and B Table 1 summarizes the clinical findings of families A and B. The clinical images of the affected individuals are shown in Figure 2. Individual II-1 was diagnosed at the age of 65 with a visual acuity of 20/200 for both eyes. A macular bull’s eye was noted on fundoscopy with a relatively spared fovea and minor superior RPE defects. ERG responses were normal in photopic and scotopic testing, whereas mfERG indicated severely reduced responses. Individual II-2 (Figure 2D, E, F) presented at 53 years of age with a visual acuity of 100/200 in the right eye and 16/200 at the left eye. The fundus showed a subtle foveal change with an intact periphery. Color vision tests showed a red-green defect. Visual field testing revealed no abnormalities apart from a reduced central sensitivity in the macula. ERG was normal whereas mfERG showed severely reduced responses. Individual II-3 presented with a visual acuity of 1/60 in the right eye and 50/200 in the left eye at the age of 59 years. Fundoscopy showed diffuse macular retinal pigment epithelium (RPE) changes with a normal periphery and color vision tests showed red-green defects. FAF imaging showed a hypofluorescent central area surrounded by a hyperfluorescent ring and foveal atrophy. OCT studies showed a strikingly thinned fovea (188 µm juxtafoveal; 41 µm central fovea), with a complete loss of the inner segment/outer segment junction and loss of the outer nuclear layer. Both eyes show decreased central sensitivity on visual field analysis. The ERG has normal scotopic and reduced but within normal limits photopic responses, while mfERG revealed severely reduced responses. Individual II-4 of family A (Figure 2A, B, C) presented at the age of 29 with a visual acuity of <20/200 in the right eye and 100/200 in the left eye. Fundoscopy detected a small bull’s eye maculopathy with attenuated vessels and a relatively pale papillary rim. Color vision defects were identified in the red-green axis and the visual field appeared with reduced central sensitivity and a normal periphery. ERG responses appeared normal, while mfERG responses were severely reduced. Patient II-5 presented at the age of 53 years with a visual acuity of 125/200 in the right eye and 50/200 in the left eye, respectively. The fundus displayed mild peripapillary atrophy and a granular macula. Both eyes display

196

a decreased sensitivity for the red axis in color vision analysis and decreased central sensitivity on visual field testing. ERG responses lie within normal limits, although mfERG shows a significantly reduced central response (Figure 3). For this case also FAF and OCT examinations were performed, both indicating alterations at the outer retinal layers. EOG performed in all individuals of family A yielded normal results.

Figure 3

Figure 3 Multifocal electroretinogram of individual II-5 The mfERG of individual II-5 of family A visualized reduced responses. Individual A:II-2 was examined by a neurologist at age 48. Walking and sensibility were undisturbed. A cerebral CT scan suggested slight cortical atrophy. However, brainstem auditory evoked potentials (BAEP), somatosensory evoked potentials (SSEP) and visual evoked potentials (VEP) were normal, and normal variant EEG waves were observed. All affected individuals of family A at advanced age (range 54-71 years) have not experienced seizures throughout their lives, and have normal mental and psychomotor abilities. Individual II-1 of family B was diagnosed with cone dystrophy at the age of 27. His visual acuity deteriorated to counting fingers at the age of 40. The most recent recorded visual acuities were 2/60 and 1/60, respectively, with eccentric fixation. Through the years an enlargement of the central scotoma on Goldmann perimetry was documented. ERG was performed on nine different occasions, and showed normal rod function with mildly abnormal cone function on eight of these. On the ninth occasion, at the age of 60, the responses generated after dark adaptation seemed more reduced than under light adaption suggesting a tendency towards a rod-cone pattern, which might be explained by the progression of retinal atrophy towards the midperiphery. Color vision tested with the saturated version of the Panel D15 was normal at the age of 29 and developed into a deutan deficiency at the age of 42. Individual B:II-1 at age 62 has not experienced seizures throughout his life, and has normal mental and psychomotor abilities.

197 MFSD8 mutations cause non-syndromic macular dystrophy

4.3

Photopic and scotopic responses within normal limits. mfERG: significantly reduced responses central

rge al oma relantact hery Scotopic responses significantly reduced isolated rod and mixed reponses. BE photopic responses significantly reduced 30 Hz and single flash responses RE, responses within normal limits LE (latencies prolonged BE)

A:II-4 (66 yrs)

A:II-2 (71 yrs)

A:II-3 (67 yrs)

198

Myopic configuration of optic disc, peripapillary atrophy, RPE changes in macula, periphery no abnormalities (57 yrs) profound atrophy central in fovea

Optic discs normal, subtle changes in macula, no other abnormalities (50 yrs). Central RPE changes

Myopic optic discs with slightly attenuated vessels, macular bullâ&#x20AC;&#x2122;s eye with relatively spared fovea with surrounding atrophic ring (LE>RE), little RPE defects superior of macula, blonde aspect of fundus

Hypo-fluorescent central area surrounded by a hyperfluorescent ring

Autofluorescence

RE : 10/200 Papillary rim somewhat pale, Hyper-fluorescent LE : 10/200 attenuated arteries, macular small central area pigmentations, seems macular RE: SE bullâ&#x20AC;&#x2122;s eye (29 yrs). -8.0D Central RPE and choroid LE: SEatrophy, subjectively stable 7.0D (59 yrs)

BE: SE -6.0D

RE: 1/60 LE: 10/200

RE: 5/200 LE: 1/60

RE: SE -4.25D LE: SE -3.75D

RE: 20/200 LE: 20/200

Fundus

Foveal atrophy, thinned retina

OCT

Visual field (Goldmann)

Acquired redgreen defect with tendency to central ACHM BE (29 yrs)

BE highly variable with red-green defect (57 yrs)

Red-green defect, RE decreased red-sensitivity, LE scotopization (51 yrs) RE enlarged defect, LE stable (54 yrs)

Periphery normal (31 yrs)

RE inferior temporal defect in I2, decrease of central sensitivity and slightly enlarged blind spot, LE decrease of central sensitivity (57 yrs)

Decrease of central sensitivity, no other abnormalities (48 yrs)

Red-green defect Decrease of central blue-yellow defect sensitivity, (43 yrs) no other abnormalities (43 yrs)

Color vision

Photopic and scotopic responses normal (51 yrs) mfERG severely reduced responses

mfERG severely reduced responses (57 yrs), photopic responses subnormal 5%, scotopic responses normal (61 yrs)

Photopic and scotopic responses normal, mfERG severely reduced responses (51 yrs)

ERG

A:II-1 (65 yrs)

Visual Age at acuity diag(refracnosis tion) ERG

ease ntral tivity, her rmal-

Patient (age last examination)

dn)

Table 1 Clinical evaluation of macular dystrophy with central cone involvement due to MFSD8 variants

199

MFSD8 mutations cause non-syndromic macular dystrophy

B:II-1 (62 yrs)

RE: SE -4.0D LE:SE-3.25D

RE: CF LE: CF

RE: SE -8.0D LE: SE -7.0D

RE: 125/200 LE: 50/200 Symmetrical central alterations at the level of the outer retinal layers

Decreased auto-fluorescence in central macula, perimacular mottled pattern of hypo-fluorescence

Optic disc pallor, attenuated arterioles, RPE atrophy in posterior pole, normal appearance of (mid)peripheral RPE

Autofluorescence

Mild peripapillary atrophy, granular aspect of macula, periphery no abnormalities

Fundus

Visual field (Goldmann)

BE red-green Decrease of central defect with desensitivity, creased red-sensi- no other abnormalities tivity (37 yrs)

Color vision

Loss of retinal layers BE deutan anoma- BE large central scotoma at the fovea, partially ly with Panel D-15 with relative intact present, but irreg(42 yrs) periphery ular outer retinal layers perifoveally and in posterior pole, intact inner retina, thickened choroid

Relatively symmetrical central alterations at the level of the outer retinal layers

OCT

Scotopic responses significantly reduced isolated rod and mixed reponses. BE photopic responses significantly reduced 30 Hz and single flash responses RE, responses within normal limits LE (latencies prolonged BE)

Photopic and scotopic responses within normal limits.mfERG: significantly reduced responses central

ERG

ACHM = Achromatopsia; BE = both eyes; CF = counting fingers; D = diopters; EOG = electrooculography; ERG = electroretinogram; LE = left eye; mfERG = multifocal electroretinogram; NP = not performed; OCT = optical coherence tomography; RE = right eye; RPE = retinal pigment epithelium; yrs = years; SE = spherical equivalent

Visual Age at acuity diag(refracnosis tion)

A:II-5 (54 yrs)

Patient (age last examination)

4.3

Discussion

In this study, we describe two families with ar macular dystrophy with central cone involvement due to variants in the MFSD8 gene. MFSD8 variants have previously been described to cause vLINCL, an early-onset severe lysosomal storage disorder with intralysosomal accumulation of autofluorescent lipopigments known as ceroid-lipofuscin which presents with seizures and mental regression.43, 44 Other common initial symptoms are ataxia and poorly described visual impairment.43 The progression of the disease is rapid with neurological regression (motor and mental), loss of vision and seizures developing early in disease.43 The majority of individuals lose their capability to walk two years after disease onset and the outcome is fatal within the second decade.44 The diagnosis can be confirmed by electron microscopy of biopsied tissues such as skin, muscle and rectal biopsies, showing fingerprint profiles of the cytosomes, although ultrastructural abnormalities are not always seen in MFSD8-positive vLINCL patients.41, 45, 46 Very recently, a mouse model was described in which a lacZ gene-trap cassette was inserted between exons 1 and 2.47 Protein analysis revealed that residual Mfsd8 was present, probably due to a low degree of normal exon 1 to exon 2 splicing. Mutant mice showed neurological defects similar to NCL due to MFSD8 variants in humans. At 8.5 months of age, the retinae of these mice showed a severe degeneration, both in central and peripheral regions.47 As younger mice were not analyzed, a distinction between initial cone or rod degeneration was not made. MFSD8 is a transmembrane lysosomal protein41, 48, 49 and belongs to the major facilitator superfamily of transporter proteins. Therefore, it is presumed to function as a lysosomal transporter protein41, but its substrates remain to be identified. MFSD8 contains two N-glycosylation consensus sites, 12 transmembrane domains, and its N- and C-termini are cytosolic. A main dileucine-based motif at the N-terminus is presumed to direct MFSD8 to the lysosomes.41, 48, 49 Studies in gene-targeted mouse embryonic fibroblasts show that MFSD8 is asymmetrically double cleaved for proper function. 50 Analysis of two missense mutations demonstrates that the cleavage occurs in the luminal L9 loop, and the proteolytic cleavage is altered by these mutations. 50 The c.1102G>C mutation induces a splice defect and skipping of exon 11, resulting in a truncated protein (p.Lys333Lysfs*3). Nevertheless, a wildtype transcript lacking exon 11 was also seen in a control sample, suggesting that this event also occurs naturally, although more normal transcript (containing exon 11) was observed in the control sample compared to affected individual B:II-1. The p.Glu381* and p.Lys333Lysfs*3 variants likely result in nonsensemediated decay of the mRNA, and thus in severely reduced levels of the predicted proteins which lack the 137 and 184 carboxyterminal amino acid residues, respectively, with no residual function. Whereas MFSD8 mutations have previously been associated with vLINCL, the individuals described in this study have a non-syndromic macular dystrophy with central cone involvement due to MFSD8 variants. We describe two families, one with a macular dystrophy with central cone involvement (Family A) and one with a macular dystrophy with central cone involvement that developed into a cone-rod dystrophy (Family B). Affected individuals of family A initially experienced a drop in central vision, while photopic and scotopic responses were normal. Only slight pigmentary alterations and color vision abnormalities pointed towards a macular dystrophy. Due to their recent implementation as diagnostic tools, OCT and mfERG could not be performed during the early phases of the disease. At later stages OCT showed loss of central photoreceptors and mfERG was abnormal in affected individuals. In summary, the clinical details of all affected individuals of family A were consistent with a diagnosis of central cone disease as described by Pinckers, a term used for normal to subnormal ERG, but a (severely) reduced mfERG. 51

200

To date, no extraocular features present in vLINCL have been noticed in these persons. In view of the advanced age of the affected persons of families A and B at their last examination (range 54-71 years), these individuals are unlikely to develop other features of vLINCL. Clinical variation has been noted among other MFSD8-associated cases. In a single Dutch case with a homozygous p.Ala157Pro variant in MFSD8, a protracted course of the disease process was described.43 This substitution affects a highly conserved alanine residue located in the fourth cytosolic loop. This individual was diagnosed with visual failure at 11 years of age and only developed motor impairment, seizures and ataxia at the ages of 24, 25 and 28, respectively. Subsequently mental and speech regression was observed at 30 and 36 years, and this person became wheelchair bound at 39 years. Interestingly, the visual impairment clearly preceded the neurological defects. In almost all other younger vLINCL cases, the visual impairment was noted when the neurological defects were diagnosed, but it is very well possible that the initial retinal defects occurred earlier. The explanation for the phenotypic variability most likely resides in the genotype of these patients. Although several types of mutations have been found in MFSD8, the individuals with non-syndromic macular dystrophy with central cone involvement present a severe heterozygous mutation in combination with a heterozygous missense variant (p.Glu336Gln), which we hypothesize to yield a mild effect. The aggregate carrier frequency of this variant in the Caucasian population is 1/267 individuals. If this variant, i.e., the most frequent MFSD8 variant in public databases, had a severe effect on the function of the mutant protein it should have been previously found in multiple vLINCL cases, either in compound heterozygous or homozygous state. The fact that this has never been observed strongly suggests that it represents a hypomorphic variant that only causes cone dysfunction when it is present in combination with a severe variant. It seems logical to propose a threshold model in which the residual activity of MFSD8 suffices for its proper function in all organs except the eye. We therefore propose a mechanism in which the combination of a severe (truncating) mutation together with a mild variant is the cause of non-syndromic macular dystrophy with central cone involvement. Interestingly, the frequency of the p.Glu336Gln allele is significantly enriched in individuals with inherited maculopathies and cone disorders (6/488 alleles) compared to controls (35/18,682 alleles; P-value <0.0001). This suggests that this allele might act as a potential risk factor or modifier allele for such disorders. A similar disease mechanism involving mild and severe mutations has also been proposed for several other genes causing syndromic and non-syndromic retinal disease. Juvenile NCL or Batten disease, like vLINCL, belongs to the disease spectrum of the neuronal ceroid lipofuscinoses, which can be caused by mutations in CLN3. The onset of disease is between 5-8 years and results in premature death in the third decade of life. Sarpong et al. (2009) described a protracted mild phenotype of juvenile NCL in a large Lebanese pedigree segregating CLN3 mutations, which presents similarities to our cases of non-syndromic disease caused by MFSD8 mutations. 52 Analysis of the homozygous CLN3 p.Tyr199* variant in affected individuals indicated a nearly equal distribution of transcripts compared to control individuals, implying that the mRNA transcript is not degraded by nonsense mediated decay, nor affected by corrective splicing. Although severely affecting the structure of CLN3, it is assumed that residual function is retained as the mutant mRNA potentially encodes a truncated protein containing part of the catalytic domain. 52 In contrast, a common deletion of exons 7 and 8 in CLN3 shows a complete loss of function. 53 More recently, several cases with retinitis pigmentosa (RP), a single case of non-syndromic Leber congenital amaurosis (LCA), and a single case of CRD have been described to carry mutations in CLN3.54, 55 Some of these mutations have been described to have a mild effect or result in a protracted phenotype. Most of these cases are far above the

201 MFSD8 mutations cause non-syndromic macular dystrophy

4.3

age-threshold to develop neurological NCL features, with the exception of two unrelated individuals (age 9 and 10), who are too young to exclude the development of non-ocular features. Another hypomorphic genetic variant that is associated with inherited retinal disease is the most frequent intronic variant in CEP290 (c.2991+1655A>G), which has only been found (homozygous or compound heterozygous) in persons with LCA, whereas combinations of other (severe) CEP290 mutations can cause a broad range of syndromic phenotypes ranging from Senior L淡ken syndrome to the lethal Meckel Gruber syndrome phenotype. 56 The retinal phenotype in individuals with vLINCL has not been documented in detail, although post-mortem NCL studies revealed neuronal degradation starting in the photoreceptor outer segments, followed by the inner segments. Subsequently neuronal cell bodies of the retina degenerate, progressing towards the ganglion layer. The RPE cells containing lipopigments may proliferate and enter the depleted neuronal retinal layers.57 In the retina, lysosomes in the RPE play a role in the phagocytosis of photoreceptor outer segments. Kim et al (2013) reported that a noncanonical autophagy pathway, involving Atg5 and LC3 triggering phagosome fusion with the lysosome, is also involved in photoreceptor outer segment degradation. 58 Elevated lysosomal pH is also noticed in cultured RPE cells of the Abca4-/- mouse and human ARPE-19 cells exposed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen or chloroquine. After restoring the lysosomal pH by directly elevating the intracellular cAMP, an enhanced photoreceptor outer segment clearance was observed. This showed that an elevated lysosomal pH interfered with degradation of outer segments and the contribution to the A2E associated pathologies. 59 Recently, two general strategies were investigated towards treatment of individuals with NCL. The identification of a master regulator of lysosomal biogenesis and autophagy, transcription factor EB, has uncovered the adaptive behavior of lysosomes in response to environmental cues as starvation. A novel therapeutic strategy may be developed to modulate lysosomal function in human disease by targeting transcription factor EB.60 A second strategy to treat NCL is directed to suppress the reactive M端ller glia cells, which, due to photoreceptor degeneration induce rapid loss of vision and retinal function by releasing toxins. Dietary supplementation with immuno-regulating compounds, curcumin and docosahexaenoic acid, resulted in a reduced number of reactive M端ller glia and improved retinal function. Moreover, intake of docosahexaenoic acid also improved the retinal morphology by preservation of the photoreceptor layer thickness.61 Our hypothesis that non-syndromic retinal degeneration is caused by relatively mild MFSD8 deficiencies could imply that only a minor increase of protein function, or suppression of M端ller cell activation, could result in a significant therapeutic effect. In conclusion, employing linkage analysis and exome sequencing, mutations in MFSD8 were identified as a novel cause of non-syndromic macular dystrophy with central cone involvement. This finding highlights the genetic overlap between lysosomal storage disorders and nonsyndromic inherited retinal dystrophies, which opens new avenues for treatment by targeting lysosomal dysfunction. Acknowledgements We thank Eveline Kersten and Alejandro Garanto for their valuable advice or expert assistance. This work was supported by the Foundation Fighting Blindness USA BR-GE-05100489-RAD (to A.I.d.H.) and C-GE-0811-0545-RAD01 (to F.P.M.C.), Prof. Dr. H.J. Flieringa Foundation SWOO, the Rotterdam Eye Hospital (to L.I.v.d.B., F.P.M.C., A.I.d.H.), the European Union Seventh Framework Programme [FP7-People-2012-ITN] under grant agreement 317472 (EyeTN) (to A.I.d.H. and F.P.M.C.), the Italian Telethon Foundation (to S.B.) and the Foundation Fighting Blindness Canada (to R.K.K.). None of the authors have any financial interest or conflicting interest to disclose.

202

Supplemental figure 1 Analysis of the c.1102G>C mutation in individual II-1 of family B

CHX

Exon 11 - Exon 12 C1 MQ BII-1 -

CHX

400 300 200 100 -

400 300 200 -

GUSB

Exon 10 - Exon 12 C1 BII-1 -

+ - + exon11 - Δ exon 11

100 -

exon 11

exon 12

AAATACAGTGGGAAGATTTGCACAATAATT

exon 10

exon 12

GTTGCTTTCCAAAAAATTTGCACAATAATT

BII-1 - CHX + exon 11

exon 11

BII-1 - CHX Δ exon 11

exon 12

exon 10

AAATACAGTGGGAAGATTTGCACAATAATT

exon 12

GTTGCTTTCCAAAAAATTTGCACAATAATT

C1 - CHX Δ exon 11

C1 - CHX + exon 11

The effect of mutation M3 (c.1102G>C) on the transcript level was determined using forward primers in exon 10 and 11 and a reverse primer in exon 12. A) Primers in exon 11 to exon 12 determined the presence of the wildtype allele in individual B:II-1. B) Using primers in exon 10 to exon 12 it was shown that the c.1102G>C mutation affecting the last nucleotide of exon 11, induces a splice defect and skipping of exon 11, predicted to result in a truncated protein (p.Lys333Lysfs*3). A transcript lacking exon 11 was also seen in the control sample (C1), suggesting that this event also occurs naturally, although more normal transcript (containing exon 11) was observed in the control sample compared to affected individual B-II:1. The ratio of normal transcript containing exon 11 and the transcript lacking exon 11 in individual B-II:1 is quantified as 1:4, whereas in the control this ratio is 1:1. C) Sequence analysis of the abundant cDNA product in the CHX treated samples shows the exon transition from 11 to 12. The C residue was not detected at position c.1102 of individual B-II:1, indicating that only cDNA of the other mutant (p.Glu336Gln) allele could be amplified. D) The right panel shows sequences of the aberrant product from exon 10 to 12, skipping exon 11.

203 MFSD8 mutations cause non-syndromic macular dystrophy

4.3

Supplemental Table 1 MFSD8 gene primers Exon

Forward

Reverse

Product size (bp)

exon 1

tcaactcccacgtttcagc

aggtagaggtggcttccagc

213

exon 2

gatgggagaaagcaggtgtc

ggaaaggaaccagtcccaac

184

exon 3

tctgtgcatgtcaatggtagc

caaagtttgctagggcttgg

652

exon 4

taaaatccatgccacggttc

gcacaataaagcatatggtcac

308

exon 5

gcgctaatggctgtttaacg

ggaggtctccctatgttaccc

985

exon 6

ggtccattaattgatgcatttg

tttcctccctttcccttcag

410

exon 7

ccccatcaagaaccaattcc

cttcaaagaccttccttttctg

511

exon 8

gtaggggaaaggcatggaag

aagatcttccccaatcaccc

639

exon 9

tgggcgtggtagtacatctg

tgcacaaggcagagattttg

851

exon 10

aaaaggcatgcacattcttg

ttttcctggtatatccatttgc

337

exon 11-12

tttgctgtgaaagatctgagc

ccacaatgccactcctacaac

892

exon 11 (alternative)

tttgctgtgaaagatctgagc

agctgatgtaaggaactggg

589

exon 12 (alternative)

caatttcccaaaatacagtg

ccacaatgccactcctacaac

583

exon 13

agggcttcagcagacagtaag

tcaccgcaattgtctagcag

512

exon 10-12

acaatggatatgtatgcctgg

cagctgatgtaaggaactgg

391

exon 11-12

tgctattctactgggaggac

cagctgatgtaaggaactgg

260

GUSB

ctgtacacgacacccaccac

tacagataggcagggcgttc

245

mRNA variants

Supplemental Table 2 Variants after stringent filtering for family A

Gene

Genomic

Protein

% Frequency varia- SNP In houseGran- SNP Coverage tion frequency database PhyloP tham identity

Compound heterozygous MFSD8

c.1141C>A

p.Glu381*

57.7

-1

2.544

1000

MFSD8

c.1006C>G

p.Glu336Gln

0.1776

0.46

5.543

MUC6

c.4232G>A

p.Thr1411Met

55.5

-1

1.282

MUC6

c.1532A>G

p.Leu511Pro

37.8

-1

2.342

XDH

c.1274G>C

p.Ser425Cys

0.0879

0.23

3.245

112

XDH

c.364T>A

p.Met122Leu

-1

0.15

5.075

204

rs150418024

rs138649664

Supplemental Table 3 Variants after stringent filtering for family B Gene name

Genomic

Protein

% Frequency varia- SNP In houseGran- SNP Coverage tion frequency database PhyloP tham identity

Homozygous CREB3L2 300GGTG>A p.Thr100del ITIH6

2261G>A

SHCBP1 14G>A

100

-1

1.812

1000

rs66593747, rs111266186, rs41306886

p.Thr754Met

0.4898

0.23

-0.001

p.Ser5Leu

-1

-0.339

145

p.Arg445Trp

44.4

-1

0.369

101

Compound heterozygous CENPT

1333G>A

CENPT

782G>A

p.Thr261Met

44.4

-1

0.08

0.261

MFSD8

1102C>G

p.Asp368His/p.? 34

-1

5.543

MFSD8

1006C>G

p.Glu336Gln

43.9

0.1776

0.46

5.543

rs150418024

4.3

205 MFSD8 mutations cause non-syndromic macular dystrophy

References 1.Michaelides, M., Hunt, D.M., and Moore, A.T. (2004). The cone dysfunction syndromes. Br. J. Ophthalmol. 88, 291-297. 2. Hamel, C.P. (2007). Cone rod dystrophies. Orphanet J. Rare Dis. 2, 7. 3. Thiadens, A.A., den Hollander, A.I., Roosing, S., Nabuurs, S.B., Zekveld-Vroon, R.C., Collin, R.W., De, B.E., Koenekoop, R.K., van Schooneveld, M.J., Strom, T.M., et al. (2009). Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders. Am. J. Hum. Genet. 85, 240-247. 4. Kohl, S., Baumann, B., Broghammer, M., Jagle, H., Sieving, P., Kellner, U., Spegal, R., Anastasi, M., Zrenner, E., Sharpe, L.T., et al. (2000). Mutations in the CNGB3 gene encoding the betasubunit of the cone photoreceptor cGMP-gated channel are responsible for achromatopsia (ACHM3) linked to chromosome 8q21. Hum. Mol. Genet. 9, 2107-2116. 5. Thiadens, A.A., Slingerland, N.W., Roosing, S., van Schooneveld, M.J., van Lith-Verhoeven, J.J., van Moll-Ramirez, N., van den Born, L.I., Hoyng, C.B., Cremers, F.P., and Klaver, C.C. (2009). Genetic etiology and clinical consequences of complete and incomplete achromatopsia. Ophthalmology 116, 1984-1989 e1981. 6. Thiadens, A.A., Somervuo, V., van den Born, L.I., Roosing, S., van Schooneveld, M.J., Kuijpers, R.W., van Moll-Ramirez, N., Cremers, F.P., Hoyng, C.B., and Klaver, C.C. (2010). Progressive loss of cones in achromatopsia: an imaging study using spectral-domain optical coherence tomography. Invest. Ophthalmol. Vis. Sci. 51, 5952-5957. 7. Michaelides, M., Hardcastle, A.J., Hunt, D.M., and Moore, A.T. (2006). Progressive cone and cone-rod dystrophies: phenotypes and underlying molecular genetic basis. Surv. Ophthalmol. 51, 232-258. 8. Thiadens, A.A., Phan, T.M., Zekveld-Vroon, R.C., Leroy, B.P., van den Born, L.I., Hoyng, C.B., Klaver, C.C., Roosing, S., Pott, J.W., van Schooneveld, M.J., et al. (2012). Clinical course, genetic etiology, and visual outcome in cone and cone-rod dystrophy. Ophthalmology 119, 819-826. 9. Michaelides, M., Chen, L.L., Brantley, M.A., Jr., Andorf, J.L., Isaak, E.M., Jenkins, S.A., Holder, G.E., Bird, A.C., Stone, E.M., and Webster, A.R. (2007). ABCA4 mutations and discordant ABCA4 alleles in patients and siblings with bullâ&#x20AC;&#x2122;s-eye maculopathy. Br. J. Ophthalmol. 91, 16501655. 10. Wycisk, K.A., Zeitz, C., Feil, S., Wittmer, M., Forster, U., Neidhardt, J., Wissinger, B., Zrenner, E., Wilke, R., Kohl, S., et al. (2006). Mutation in the auxiliary calcium-channel subunit CACNA2D4 causes autosomal recessive cone dystrophy. Am. J. Hum. Genet. 79, 973-977. 11. Wissinger, B., Gamer, D., Jagle, H., Giorda, R., Marx, T., Mayer, S., Tippmann, S., Broghammer, M., Jurklies, B., Rosenberg, T., et al. (2001). CNGA3 mutations in hereditary cone photoreceptor disorders. Am. J. Hum. Genet. 69, 722-737. 12. Kellner, U., Wissinger, B., Kohl, S., Kraus, H., and Foerster, M.H. (2004). Molecular genetic findings in patients with congenital cone dysfunction. Mutations in the CNGA3, CNGB3, or GNAT2 genes. Ophthalmologe 101, 830-835. 13. Michaelides, M., Aligianis, I.A., Ainsworth, J.R., Good, P., Mollon, J.D., Maher, E.R., Moore, A.T., and Hunt, D.M. (2004). Progressive cone dystrophy associated with mutation in CNGB3. Invest. Ophthalmol. Vis. Sci. 45, 1975-1982. 14. Littink, K.W., Koenekoop, R.K., van den Born, L.I., Collin, R.W., Moruz, L., Veltman, J.A., Roosing, S., Zonneveld, M.N., Omar, A., Darvish, M., et al. (2010). Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations. Invest. Ophthalmol. Vis. Sci. 51, 5943-5951. 15. Kohl, S., Coppieters, F., Meire, F., Schaich, S., Roosing, S., Brennenstuhl, C., Bolz, S., van Genderen, M.M., Riemslag, F.C., European Retinal Disease, C., et al. (2012). A nonsense muta-

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tion in PDE6H causes autosomal-recessive incomplete achromatopsia. Am. J. Hum. Genet. 91, 527-532. 16. Roosing, S., van den Born, L.I., Hoyng, C.B., Thiadens, A.A., de Baere, E., Collin, R.W., Koenekoop, R.K., Leroy, B.P., van Moll-Ramirez, N., Venselaar, H., et al. (2013). Maternal uniparental isodisomy of chromosome 6 reveals a TULP1 mutation as a novel cause of cone dysfunction. Ophthalmology 120, 1239-1246. 17. Cremers, F.P., van de Pol, D.J., van Driel, M., den Hollander, A.I., van Haren, F.J., Knoers, N.V., Tijmes, N., Bergen, A.A., Rohrschneider, K., Blankenagel, A., et al. (1998). Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardtâ&#x20AC;&#x2122;s disease gene ABCR. Hum. Mol. Genet. 7, 355-362. 18. Parry, D.A., Toomes, C., Bida, L., Danciger, M., Towns, K.V., McKibbin, M., Jacobson, S.G., Logan, C.V., Ali, M., Bond, J., et al. (2009). Loss of the metalloprotease ADAM9 leads to conerod dystrophy in humans and retinal degeneration in mice. Am. J. Hum. Genet. 84, 683-691. 19. Estrada-Cuzcano, A.I., Neveling, K., Kohl, S., Banin, E., Rotenstreich, Y., Sharon, D., FalikZaccai, T.C., Hipp, S., Roepman, R., Wissinger, B., et al. (2012). Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement. Am. J. Hum. Genet. 90, 102-109. 20. Ostergaard, E., Batbayli, M., Duno, M., Vilhelmsen, K., and Rosenberg, T. (2010). Mutations in PCDH21 cause autosomal recessive cone-rod dystrophy. J. Med. Genet. 47, 665-669. 21. Henderson, R.H., Li, Z., Abd El Aziz, M.M., Mackay, D.S., Eljinini, M.A., Zeidan, M., Moore, A.T., Bhattacharya, S.S., and Webster, A.R. (2010). Biallelic mutation of protocadherin-21 (PCDH21) causes retinal degeneration in humans. Mol. Vis. 16, 46-52. 22. Auslender, N., Sharon, D., Abbasi, A.H., Garzozi, H.J., Banin, E., and Ben-Yosef, T. (2007). A common founder mutation of CERKL underlies autosomal recessive retinal degeneration with early macular involvement among Yemenite Jews. Invest. Ophthalmol. Vis. Sci. 48, 5431-5438. 23. Huang, L., Zhang, Q., Li, S., Guan, L., Xiao, X., Zhang, J., Jia, X., Sun, W., Zhu, Z., Gao, Y., et al. (2013). Exome sequencing of 47 chinese families with cone-rod dystrophy: mutations in 25 known causative genes. PLoS One 8, e65546. 24. Henderson, R.H., Waseem, N., Searle, R., van der Spuy, J., Russell-Eggitt, I., Bhattacharya, S.S., Thompson, D.A., Holder, G.E., Cheetham, M.E., Webster, A.R., et al. (2007). An assessment of the apex microarray technology in genotyping patients with Leber congenital amaurosis and early-onset severe retinal dystrophy. Invest. Ophthalmol. Vis. Sci. 48, 5684-5689. 25. Littink, K.W., van den Born, L.I., Koenekoop, R.K., Collin, R.W., Zonneveld, M.N., Blokland, E.A., Khan, H., Theelen, T., Hoyng, C.B., Cremers, F.P., et al. (2010). Mutations in the EYS gene account for approximately 5% of autosomal recessive retinitis pigmentosa and cause a fairly homogeneous phenotype. Ophthalmology 117, 2026-2033, 2033 e2021-2027. 26. Zhang, Q., Li, S., Xiao, X., Jia, X., and Guo, X. (2007). The 208delG mutation in FSCN2 does not associate with retinal degeneration in Chinese individuals. Invest. Ophthalmol. Vis. Sci. 48, 530-533. 27. Ugur Iseri, S.A., Durlu, Y.K., and Tolun, A. (2010). A novel recessive GUCY2D mutation causing cone-rod dystrophy and not Leberâ&#x20AC;&#x2122;s congenital amaurosis. Eur. J. Hum. Genet. 18, 1121-1126. 28. Durlu, Y.K., Koroglu, C., and Tolun, A. (2014). Novel Recessive Cone-Rod Dystrophy Caused by POC1B Mutation. JAMA Ophthalmol. 29. Roosing, S., Lamers, Ideke J.C., de Vrieze, E., van den Born, L.I., Lambertus, S., Arts, Heleen H., Peters, T.A., Hoyng, C.B., Kremer, H., Hetterschijt, L., et al. Disruption of the Basal Body Protein POC1B Results in Autosomal-Recessive Cone-Rod Dystrophy. The American Journal of Human Genetics. 30. Pras, E., Abu, A., Rotenstreich, Y., Avni, I., Reish, O., Morad, Y., Reznik-Wolf, H., and Pras, E. (2009). Cone-rod dystrophy and a frameshift mutation in the PROM1 gene. Mol. Vis. 15,

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4.3

1709-1716. 31. Roosing, S., Rohrschneider, K., Beryozkin, A., Sharon, D., Weisschuh, N., Staller, J., Kohl, S., Zelinger, L., Peters, T.A., Neveling, K., et al. (2013). Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy. Am. J. Hum. Genet. 93, 110-117. 32. den Hollander, A.I., Black, A., Bennett, J., and Cremers, F.P. (2010). Lighting a candle in the dark: advances in genetics and gene therapy of recessive retinal dystrophies. J. Clin. Invest. 120, 3042-3053. 33. Roosing, S., Thiadens, A.A., Hoyng, C.B., Klaver, C.C., den Hollander, A.I., and Cremers, F.P. (2014). Causes and consequences of inherited cone disorders. Prog. Retin. Eye Res. 34. Zeitz, C., Jacobson, S.G., Hamel, C.P., Bujakowska, K., Neuille, M., Orhan, E., Zanlonghi, X., Lancelot, M.E., Michiels, C., Schwartz, S.B., et al. (2013). Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness. Am. J. Hum. Genet. 92, 67-75. 35. Audo, I., Bujakowska, K., Orhan, E., Poloschek, C.M., Defoort-Dhellemmes, S., Drumare, I., Kohl, S., Luu, T.D., Lecompte, O., Zrenner, E., et al. (2012). Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness. Am. J. Hum. Genet. 90, 321-330. 36. Thiadens, A.A., and Klaver, C.C. (2010). Genetic testing and clinical characterization of patients with cone-rod dystrophy. Invest. Ophthalmol. Vis. Sci. 51, 6904-6905. 37. Marmor, M.F., Fulton, A.B., Holder, G.E., Miyake, Y., Brigell, M., and Bach, M. (2009). ISCEV Standard for full-field clinical electroretinography (2008 update). Doc. Ophthalmol. 118, 69-77. 38. Nagy, E., and Maquat, L.E. (1998). A rule for termination-codon position within introncontaining genes: when nonsense affects RNA abundance. Trends Biochem. Sci. 23, 198-199. 39. Pollard, K.S., Hubisz, M.J., Rosenbloom, K.R., and Siepel, A. (2010). Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 20, 110-121. 40. Sim, N.L., Kumar, P., Hu, J., Henikoff, S., Schneider, G., and Ng, P.C. (2012). SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res. 40, W452-457. 41. Siintola, E., Topcu, M., Aula, N., Lohi, H., Minassian, B.A., Paterson, A.D., Liu, X.Q., Wilson, C., Lahtinen, U., Anttonen, A.K., et al. (2007). The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter. Am. J. Hum. Genet. 81, 136-146. 42. Aiello, C., Terracciano, A., Simonati, A., Discepoli, G., Cannelli, N., Claps, D., Crow, Y.J., Bianchi, M., Kitzmuller, C., Longo, D., et al. (2009). Mutations in MFSD8/CLN7 are a frequent cause of variant-late infantile neuronal ceroid lipofuscinosis. Hum. Mutat. 30, E530-540. 43. Kousi, M., Siintola, E., Dvorakova, L., Vlaskova, H., Turnbull, J., Topcu, M., Yuksel, D., Gokben, S., Minassian, B.A., Elleder, M., et al. (2009). Mutations in CLN7/MFSD8 are a common cause of variant late-infantile neuronal ceroid lipofuscinosis. Brain 132, 810-819. 44. Topcu, M., Tan, H., Yalnizoglu, D., Usubutun, A., Saatci, I., Aynaci, M., Anlar, B., Topaloglu, H., Turanli, G., Kose, G., et al. (2004). Evaluation of 36 patients from Turkey with neuronal ceroid lipofuscinosis: clinical, neurophysiological, neuroradiological and histopathologic studies. Turk. J. Pediatr. 46, 1-10. 45. Aldahmesh, M.A., Al-Hassnan, Z.N., Aldosari, M., and Alkuraya, F.S. (2009). Neuronal ceroid lipofuscinosis caused by MFSD8 mutations: a common theme emerging. Neurogenetics 10, 307-311. 46. Stogmann, E., El Tawil, S., Wagenstaller, J., Gaber, A., Edris, S., Abdelhady, A., Assem-Hilger, E., Leutmezer, F., Bonelli, S., Baumgartner, C., et al. (2009). A novel mutation in the MFSD8 gene in late infantile neuronal ceroid lipofuscinosis. Neurogenetics 10, 73-77. 47. Damme, M., Brandenstein, L., Fehr, S., Jankowiak, W., Bartsch, U., Schweizer, M., HermansBorgmeyer, I., and Storch, S. (2014). Gene disruption of Mfsd8 in mice provides the first animal model for CLN7 disease. Neurobiol. Dis. 65, 12-24.

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48. Steenhuis, P., Herder, S., Gelis, S., Braulke, T., and Storch, S. (2010). Lysosomal targeting of the CLN7 membrane glycoprotein and transport via the plasma membrane require a dileucine motif. Traffic 11, 987-1000. 49. Sharifi, A., Kousi, M., Sagne, C., Bellenchi, G.C., Morel, L., Darmon, M., Hulkova, H., Ruivo, R., Debacker, C., El Mestikawy, S., et al. (2010). Expression and lysosomal targeting of CLN7, a major facilitator superfamily transporter associated with variant late-infantile neuronal ceroid lipofuscinosis. Hum. Mol. Genet. 19, 4497-4514. 50. Steenhuis, P., Froemming, J., Reinheckel, T., and Storch, S. (2012). Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7. Biochim. Biophys. Acta 1822, 1617-1628. 51. Pinckers, A. (1984). Clinical color vision examination. In Colour Vision Deficiencies VII, G. Verriest, ed. (Springer Netherlands), pp 171-179. 52. Sarpong, A., Schottmann, G., Ruther, K., Stoltenburg, G., Kohlschutter, A., Hubner, C., and Schuelke, M. (2009). Protracted course of juvenile ceroid lipofuscinosis associated with a novel CLN3 mutation (p.Y199X). Clin. Genet. 76, 38-45. 53. Chan, C.H., Mitchison, H.M., and Pearce, D.A. (2008). Transcript and in silico analysis of CLN3 in juvenile neuronal ceroid lipofuscinosis and associated mouse models. Hum. Mol. Genet. 17, 3332-3339. 54. Wang, X., Wang, H., Sun, V., Tuan, H.F., Keser, V., Wang, K., Ren, H., Lopez, I., Zaneveld, J.E., Siddiqui, S., et al. (2013). Comprehensive molecular diagnosis of 179 Leber congenital amaurosis and juvenile retinitis pigmentosa patients by targeted next generation sequencing. J. Med. Genet. 50, 674-688. 55. Wang, F., Wang, H., Tuan, H.F., Nguyen, D.H., Sun, V., Keser, V., Bowne, S.J., Sullivan, L.S., Luo, H., Zhao, L., et al. (2014). Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements. Hum. Genet. 133, 331-345. 56. den Hollander, A.I., Roepman, R., Koenekoop, R.K., and Cremers, F.P.M. (2008). Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog. Retin. Eye Res. 27, 391419. 57. Anderson, G.W., Goebel, H.H., and Simonati, A. (2013). Human pathology in NCL. Biochim. Biophys. Acta 1832, 1807-1826. 58. Kim, J.Y., Zhao, H., Martinez, J., Doggett, T.A., Kolesnikov, A.V., Tang, P.H., Ablonczy, Z., Chan, C.C., Zhou, Z., Green, D.R., et al. (2013). Noncanonical autophagy promotes the visual cycle. Cell 154, 365-376. 59. Liu, J., Lu, W., Reigada, D., Nguyen, J., Laties, A.M., and Mitchell, C.H. (2008). Restoration of lysosomal pH in RPE cells from cultured human and ABCA4(-/-) mice: pharmacologic approaches and functional recovery. Invest. Ophthalmol. Vis. Sci. 49, 772-780. 60. Settembre, C., Fraldi, A., Medina, D.L., and Ballabio, A. (2013). Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat. Rev. Mol. Cell Biol. 14, 283-296. 61. Mirza, M., Volz, C., Karlstetter, M., Langiu, M., Somogyi, A., Ruonala, M.O., Tamm, E.R., Jagle, H., and Langmann, T. (2013). Progressive retinal degeneration and glial activation in the CLN6 (nclf) mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation. PLoS One 8, e75963.

209 MFSD8 mutations cause non-syndromic macular dystrophy

4.3

210

Chapter 5 General discussion

211

212

Chapter 5.1 Novel disease mechanisms in cone dysfunction

5.1

213

214

Function of novel disease genes

The genetic studies described in this thesis have lead to the discovery of several new genes involved in CD: PDE6C (Chapter 4A), RAB28 (Chapter 4B) and MFSD8 (Chapter 4C). In addition, several genes previously implicated in other retinal dystrophies were found to be involved in CD: TULP1 (Chapter 3A) and CEP290 (Chapter 3B). New gene findings contributed to an increased knowledge on the disease mechanisms in CD and can potentially result in the elucidation of novel pathways involved in the development of CD. Thus far, genes causing CD were found in major pathways of the visual system like the phototransduction cascade, retinoid cycle, transport processes, disk membrane morphogenesis, and synaptic transduction (Chapter 1.2; review). Mutations in PDE6C were identified in ACHM cases by a combination of homozygosity mapping and a positional candidate gene approach. PDE6C was considered a strong candidate gene due to its crucial role in cone phototransduction, and due to functional evidence of rapid cone degeneration in zebrafish with a Pde6c defect.1 PDE6C defects lead to CD due to the absence or reduced activity of the Îąâ&#x20AC;&#x2122; subunit causing reduced hydrolysis of cGMPresulting in the reduction of loss of the catalytic activity.2; 3 As a consequence the phototransduction cascade is interrupted and the cone photoreceptor remains in the dark state.2 The identification of mutations in three proteins localizing to the connecting cilium in this thesis (CEP290, RAB28 and TULP1) stresses the role of disrupted ciliary processes in the pathogenesis of IRDs, and may indicate that many other ciliary proteins can be defective in persons with CDs. This process is highly important for the transport of proteins from the inner segment towards the outer segment and vice versa; which is crucial for the development and homeostasis of cone photoreceptors. The exact function of some newly identified CD-associated proteins, such as RAB28, is currently unknown and requires further studies, such as protein localization and interaction studies, as well as in vivo analyses in animal models. Interestingly, the identification of MFSD8 mutations in this thesis pinpoints lysosomal dysfunction as a novel disease mechanism in non-syndromic CD. Syndromic CD has previously been found to be due to lysosomal dysfunction through LAMP2 defects observed in Danon disease.4 More recently more cases of nonsyndromic retinal dystrophy (RP, LCA and CRD) have been described to be caused by a defect in the lysosomal protein CLN3. 5; 6 Further studies are warranted to determine the exact role of the transmembrane protein MFSD8 in the lysosomes, and how variants in this gene result in non-syndromic photoreceptor dysfunction.

Uniparental isodisomy

The association of CD with TULP1 mutations was facilitated by the discovery of an individual with uniparental disomy (UPD), which is a rare phenomenon that occurs approximately in 1:5000 or even less individuals.7 This event can lead to the inheritance of two copies of a chromosome carrying a recessive mutation from the same parent, resulting in disease. Similar to our case, the majority of the cases seem to be associated with an advanced maternal age. 8 Only seven other cases of maternal UPD of chromosome 6 have been described previously.9-15 Of these, several cases were diagnosed with intrauterine growth retardation, which was also suggested for our case. Other retinal dystrophies in which UPD was involved were caused by mutations in MERTK, RPE65 and USH2A on chromosome 1, 1 and 2, respectively.16-18 UPD could play a role in more cases of CD, which can be identified through dedicated homozygosity mapping or homozygous calls in WES.

Hypomorphic mutations in syndromic disease genes in CD

Strikingly, two novel CD-associated genes identified in this thesis, CEP290 and MFSD8, have previously been implicated in multi-systemic syndromes. Based on the mutations identified in these genes, we hypothesize that combinations of severe and mild variants, or the combination

215 Novel disease mechanisms in cone dysfunction

5.1

of two moderately severe variants, can result in CD because of residual protein activity, while loss-of-function mutations lead to more severe, multi-system syndromes. The phenotypic spectrum of CEP290 mutations was previously established to be broad, ranging from earlyonset non-syndromic retinal dystrophies (Leber congenital amaurosis or early-onset retinitis pigmentosa) to syndromes involving neurological and renal disorders (Senior-LĂ¸ken, Joubert syndrome, Bardet-Biedl syndrome), to Meckel-Gruber syndrome leading to prenatal lethality.19 Although it remains difficult to establish an exact genotype-phenotype correlation for CEP290 mutations in these different diseases, 20 we were able to add another phenotype to the CEP290 disease spectrum. In this thesis we describe two compound heterozygous truncating mutations in CEP290 that cause CD. Usually, truncating mutations result in a shortened protein or the absence of protein, since the aberrant transcripts may be degraded by the process of nonsense mediated decay (NMD). NMD serves as a protective mechanism against translation of mRNAs containing a premature termination codon likely resulting in a truncated protein with dominant-negative or deleterious gain-of-function properties.21 However, studies on mutational mechanisms have unraveled cases of truncating mutations undergoing nonsenseassociated altered splicing (NAS). This mechanism is presumed to correct the translated protein by excluding one or more exons from the mature mRNA when a premature termination codon is recognized, leading to a restoration of the open-reading frame.22 Using RNA studies it was demonstrated that both RNAs carrying truncating CEP290 mutations identified in CD (p.(Arg151*) and p.(Lys1575*)) undergo NAS, in both cases leading to a restoration of the open reading frame. The p.(Arg151*) mutation was previously described in non-syndromic earlyonset retinal dystrophy, 23 whereas p.(Lys1575*), with one exception, was described to cause LCA when found in a homozygous state.20; 24 In a compound heterozygous state this variant was described to cause LCA, Senior LĂ¸ken or cerebello-oculo-renal syndrome, depending on the severity of the second allele.20; 24-27 Although the hypomorphic character of p.(Arg151*) and p.(Lys1575*), p.Leu148_Glu165del/wildtype and p.Glu1569Glyfs/wildtype respectively, provide new insights into the genotype-phenotype correlation of CEP290 mutations, it remains unclear how these two mutations cause oligocone trichromacy rather than a pan-retinal, early-onset retinal dystrophy previously associated with these mutations. The second unexplained event is the occurrence of initial cone defects in these individuals, with progressive rod defects later in life. Though, a distinct function for CEP290 in cones and rods has been suggested. Potentially the skipped exons harbor essential functional domains for proper function in the cones, whereas rod function is longer retained. As the clinical examinations in these individuals uncovered a progressive peripheral degeneration one could argue that the cones are more vulnerable compared to rods due their higher metabolism. In this thesis, I also established a novel genotype-phenotype correlation for MFSD8 by the discovery of mutations in this gene in families with CD. Defects in the lysosomal transmembrane protein MFSD8 were previously associated with variant-late infantile neuronal ceroid lipofuscinosis (vLINCL), an early-onset severe syndromic lysosomal disorder with fatal outcome within in the second decade of life.28; 29 Although visual impairment is the first feature in most persons with vLINCL, whole exome sequencing (WES) unexpectedly enabled us to associate MFSD8 mutations to a non-syndromic CD phenotype. Although we cannot completely exclude that these individuals may develop extra-ocular features in the future, their advanced age (current ages 55-73) demonstrates a tremendous phenotypic difference compared to vLINCL. Persons diagnosed with vLINCL usually present two rare severe (truncating) mutations, while in the non-syndromic CD individuals presented in this thesis were found to carry a rare severe mutation in combination with a predicted mild mutation. The truncating mutations in this study were previously described to cause vLINCL in combination with other severe mutations. 30; 31 The second allele, classified by us as mild, was not described previously in vLINCL cases and was present in 0.19% of the alleles in the Caucasian population.

216

5.1

217 Novel disease mechanisms in cone dysfunction

218

Chapter 5.2 Considerations in future cone dysfunction disease gene identification studies

5.2

219

220

The studies described in this thesis have important implications for future studies directed towards disease gene identification in CD. Future studies, in most cases, will be based on WES as it is most time- and cost-effective. Often, it is not easy to pinpoint the genetic defect(s) among the large number of variants that are identified by WES. However, several lessons can be learned from the gene identification studies we present in this thesis. First of all, the identification of mutations in three genes encoding proteins localized in or near the connecting cilium (CEP290, RAB28 and TULP1) demonstrates that genes encoding ciliary proteins are excellent candidate genes for CD. Upcoming gene identification approaches should thus take such genes into consideration. These studies could include experiments to unravel protein-protein interactions of the known ciliary CD-associated proteins, which may not only give clues about its functional network in the connecting cilium but also enable the identification of novel candidate genes. Alternatively, candidate gene directed searches can be performed in online ciliary databases based upon functional relevance or interactions with known CD proteins. Second, our results show that it is critical when analyzing WES data to consider genes associated with various retinal phenotypes as candidate genes for CD. Different functional effects of variants in known CD genes can lead to unexpected phenotypes. For example, an effect on splicing, as demonstrated in this thesis for CEP290 mutations, which lead to the skipping of exons carrying a truncating mutation, can also be induced by missense or silent mutations disrupting an exonic splice enhancer and thereby affecting the process of splicing. The mechanism behind this oligocone trichromacy seems to be caused by NAS, preserving an intact protein lacking the in-frame exon 36. Although the exact mechanism of NAS has not yet been revealed, 22; 32 it potentially enables us to explain multiple milder or unexpected phenotypes of disease in the future. The potential effect of all types of variants on splicing should thus be taken into account when analyzing WES data. Third, a group of genes previously described to be involved in various syndromes unexpectedly are associated with with non-syndromic CD. In this thesis, we detected mutations in MFSD8 as a cause of non-syndromic CD due to the combination of a severe and a mild mutation. Several other genes associated with syndromes have been involved in retinal dystrophies, like CEP290 for which in this thesis another phenotype has been added to the disease spectrum. In addition, mutations in the CLRN1 gene cause Usher syndrome as well as non-syndromic RP, 33 defective IQCB1 underlying Senior-LĂ¸ken disease can also lead to non-syndromic LCA, 34 and mutations in BBS1 provoke Bardet-Biedl syndrome or non-syndromic RP. 35 The mild p.Met380Arg allele found in BBS1 is described in 21/4,014 (0.52%) alleles in RP probands versus 8/3,648 (0.22%) control alleles. Interestingly, the occurrence of this hypomorphic BBS1 mutation as a low-frequency variant in the population is in line with our finding in MFSD8. In our cases, the p.Glu336Gln variant in MFSD8 is found 1/302 (0.3%) in ethnically matched control alleles, in 6/2,604 alleles (0.23%) of the in-house exome database, 25/12,006 alleles (0.19%) in the Exome Variant Server and 1/1000 (0.1%) in the 1000genomes project. The high prevalence in control individuals argues to increase the filter to <2% in dbSNP and in-house database to allow the identification of mild variants which can occur relatively frequent in the general population as long as they are not associated with a retinal phenotype in a homozygous state. The highest frequency of a mild genetic variant associated with an IRD is the c.2588G>C (p.Gly863Ala/DelGly863) variant in ABCA4, which was found heterozygously in ~3% of Dutch controls and (in a compound heterozygous state) in 1/3 of persons with Stargardt disease. 36; 37 The involvement of MFSD8 in non-syndromic CD reveals an unexpected group of lysosomal genes with possible involvement in CD. vLINCL belongs to a family of neuronal ceroid lipofuscinosis (CLN) which can be due to variants in 14 members of CLN proteins of which

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at least eight are associated with visual impairment. 38 Visual impairment is the first symptom in the majority of these, but as the disease progresses over time and other features occur, therefore minor attention is assigned the retinal phenotype. For instance, it is unknown if the degeneration occurs in a cone-rod or rod-cone pattern. As mutations in CLN3 were recently described to cause non-syndromic RP and LCA, 5; 6 we hypothesize that the other CLNs are potential candidate genes to cause non-syndromic CD, but also rod-dominated retinal disease.

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223 Considerations in future cone dysfunction disease gene identification studies

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Chapter 5.3 Evolving genomic technologies

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225

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WES facilitates disease gene discovery Throughout the years, with the development of improved technological approaches, the rate of gene discoveries has grown linearly (Figure 1). Whereas the majority of genes has been discovered using traditional positional cloning approaches which made use of genetic linkage studies and candidate genes, already six genes have been discovered in the last two years with the implementation of WES.

Figure 1 Gene discovery for CD throughout the years The large number of genes underlying IRD is illustrative of the high heterogeneity, and the number of disease genes is anticipated to increase when taking into account all currently unsolved cases (Figure 2). In 2014, the majority of all CD cases remain unsolved and the expectations are that these will largely be explained by rare gene findings, as the latest gene discoveries are represented by small numbers of families (Chapter 1.2).39

Implementation of WES in diagnostics

Since a few years, WES has been integrated in the diagnostic screening methods for individuals with inherited disease. This was initiated with a targeted approach with probe arrays targeting including all coding exons, noncoding exons, and untranslated regions of 111 known blindness genes.41 This method resulted in the detection of causal variants in 36 RP probands in a recessive, dominant or X-linked inheritance pattern. As these patients were derived from a larger group of families pre-screened for variants in several RP genes, the corrected yield was ~54%. Surprisingly, three out of 28 (10.7%) isolated cases showed a de novo causative mutation, having a large effect on the subsequent genetic counseling.41 The targeted approach was quickly replaced by the current WES in diagnostics. Although this routine implies a filtering step show-

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ing only variants within the boundaries of previously assigned genes, in this case genes causing IRD. Especially in heterogeneous diseases like IRD, WES can be time-saving and cost-effective as all kinds of genetic variation at base-pair resolution can be identified throughout the human genome in a single experiment compared to traditional Sanger sequencing which would cost many years and millions of Euros. WES provides an unbiased analysis when a clinical diagnosis may be incomplete as non-ocular features such as late onset of kidney dysfunction, may appear later in life. In addition, the age of the investigated individual can influence the final diagnosis, the end stage of some diseases tend to overlap significantly, like CRD and rod-cone dystrophies.42 Finally, identification of the genetic defect can help to determine the pattern of inheritance, in particular in isolated cases which can be due to mutations in genes implicated in autosomal dominant, recessive or X-linked genes, some of which can be de novo.41 WES may also identify multigenic causes of IRDs. In the era of Sanger sequencing but also in WES, it may occur that cases with additional features are incorrectly addressed to defects in a single gene as other genes will not be analyzed after the gene defect has been elucidated. One would continue the investigation for the involvement of multiple genes in exceptions to these, i.e. families which include differences in phenotypes or features. The involvement of a second gene may be unmasked faster by analysis if the entire genome, however, in most cases additional functional analysis are required to proof the additive or protective effect of a second gene. Moreover, the direct analysis of an entire exome would prevent reporting mutations of uncertain pathogenicity to the patient, while mutations outside the filter could immediately result in convincing mutations as the cause of the IRD in the individual. Furthermore, the analysis of the entire exome of an affected individual makes it possible to investigate in a single experiment all possible combinations of genetic modifiers which result in phenotypic variability.

Uncovering non-coding mutations by transcriptomics and wholegenome sequencing

Although WES facilitates the discovery of rare variants, elucidation of complex and unexpected modes of inheritance, still a subset of pathogenic mutations is still missing. Excellent examples are the deep intronic mutations in CEP29043 and USH2A,44 which result in the â&#x20AC;&#x2DC;exonificationâ&#x20AC;&#x2122; of intronic sequences containing stop mutations. To identify mutations explaining genetically unsolved cases, transcriptomics offers a powerful approach in studying the transcriptional landscape of a specific cell or tissue. Recently, transcriptome analysis on RNA from human donor eyes was used to perform deep mRNA sequencing of the ABCA4 mRNA, uncovering 19 alternative splice products of ABCA4, the most frequently mutated IRD gene.45 Sequencing these alternative exons in individuals with STGD1 revealed five deep-intronic variants with an effect on splicing by creating novel splice sites. Furthermore, human retinal tissue was shown to carry 79,915 novel alternative splice events and over a hundred novel genes were elucidated. It was presumed that 15-36% of these will be transcribed into coding transcripts, as they contain an open reading frame.46 Transcriptomics may not be applicable in all cases as the affected tissue must be analyzed and compared to tissue in the healthy situation. One option could be performing transcriptomics on blood cells, however gene expression profiles may differ in each tissue or potentially completely absent. In these cases other cell systems have to be used to study IRD defects. An example is the differentiation of induced pluripotent stem cells (iPSC) into photoreceptor precursor cells and/or retinal pigment epithelium. This is possible by reprogramming patientâ&#x20AC;&#x2122;s fibroblasts or keratinocytes (derived from a skin biopsies) into iPSCs, which can be subsequently differentiated to the preferred cell type for further studies.47 Transcriptomics will be performed in parallel to whole genome sequencing (WGS) which reveals almost all (~ 3 million) genomic variants . The huge challenge resides in the confirmation of the pathogenic effect of intronic variants, which occasionally is still troublesome with

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exonic mutations due to a lack of family members for segregation analysis or discrepancies of in silico prediction programs.42 Besides the identification of exonic and intronic variants in an individual genome, WGS can robustly identify heterozygous deletions, duplications, inversions, and intronic variants.48 The issues regarding confirmation of pathogenicity may be partially diminished by testing large patient cohorts through collaborative efforts. In addition, the causality of novel variants can be tested through cellular and animal models.

Figure 2 Genetic heterogeneity in inherited retinal disease Clinical diagnoses are indicated by colored circles. Genes in the overlapping areas represent genes in which mutations can lead to multiple phenotypes. COD/CRD: cone and cone-rod dystrophy; CVD: color vision defects (ACHM, blue cone monochromatism, oligocone trichromacy, tritanopia); MD: macular degeneration; ERVR/EVR: erosive and exudative vitreoretinopathies. Adapted from updated figure from Berger et al. 2013.40 Future research in the identification of novel genes as a cause of CD could be facilitated by the expansion of public databases currently containing data of thousands of human genomes. Using the exome variant server (12,006 alleles), 1000genomes project (2,184 alleles), an in-house database (5,812 alleles) together with panels with DNA of ethnically matched control samples (384 alleles), we are able to generate a fair estimate of the allele frequency of specific variants. The ongoing growth of these public and local databases will allow more reliable assessments of allele frequencies of candidate variants. Potentially, this can be done in a more origin-specific fashion by detailed registration of the individualsâ&#x20AC;&#x2122; background such as place of birth from the individuals and their parents, and thereby determine the origin of a specific variant. In a similar way, with the expansion of human genome data, the knowledge on the effect of variants is likely to grow. This knowledge can be used as training sets for in silico programs and

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are likely to determine the effect of other/similar candidate variants with higher levels of confidence. Better than in silico analyses would be to perform functional approaches to determine the effect of each variant or assess the gene function to draw a conclusion on the pathogenicity of this particular variant. Clinicians can facilitate the process of gene identification by performing detailed clinical examinations of ocular and non-ocular features. Detailed clinical examination of a patient prior to genetic studies allows geneticists to anticipate on possible novel candidate genes. Analysis of DNA of the patientsâ&#x20AC;&#x2122; parents and affected and unaffected siblings allows to determine the pathogenicity of a variant, a possible unexpected inheritance pattern and the recurrence risk in siblings or offspring. Exome sequencing for all affected individuals for a family quickly excludes the majority of variants as the assumption is that all affected members from a family carry the same gene defect(s). Therefore only variants present in both individuals have to be investigated.

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231 Evolving genomic technologies

Susanne Roosing, Rob W. J. Collin, Anneke I. den Hollander, Frans P. M. Cremers*, Anna M. Siemiatkowska*

*These authors contributed equally.

Journal of medical genetics 2014, 51:143-151 232

Chapter 5.4 Prenylation defects in inherited retinal diseases

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233

Summary

Many proteins depend on posttranslational prenylation for a correct subcellular localisation and membrane anchoring. This involves the covalent attachment of farnesyl or geranylgeranyl residues to cysteines residing in consensus motifs at the C-terminal parts of proteins. Retinal photoreceptor cells are highly compartmentalised and membranous structures, and therefore it can be expected that the proper function of many retinal proteins depends on prenylation, which has been proven for several proteins that are absent or defective in different inherited retinal diseases (IRDs). These include proteins involved in the phototransduction cascade, such as GRK1, the phosphodiesterase 6 subunits and the transducin Îł subunit, or proteins involved in transport processes, such as RAB28 and retinitis pigmentosa GTPase regulator (RPGR). In addition, there is another class of general prenylation defects due to mutations in proteins such as AIPL1, PDE6D and rab escort protein-1 (REP-1),which can act as chaperones for subsets of prenylated retinal proteins that are associated with IRDs. REP-1 also is a key accessory protein of geranylgeranyltransferase II, an enzyme involved in the geranylgeranylation of almost all members of a large family of Rab GTPases. Finally, mutations in the mevalonate kinase (MVK) gene, which were known to be principally associated with mevalonic aciduria, were recently associated with nonsyndromic retinitis pigmentosa. We hypothesise that MVK deficiency results in a depletion of prenyl moieties that affects the prenylation of many proteins synthesized specifically in the retina, including Rabs. In this review, we discuss the entire spectrum of prenylation defects underlying progressive degeneration of photoreceptors, the retinal pigment epithelium and the choroid.

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Introduction

Many proteins require posttranslational modification to function in several cellular processes. Protein prenylation is a posttranslational lipid modification which allows protein-protein interactions and ensures correct cellular localisation of the protein via covalent attachment of one or two prenyl groups (farnesyl or geranylgeranyl).49-52 Prenyl residues belong to a large family of chemicals consisting of terpenoids. In animals, these compounds may contain from one to six isoprene units, each containing a 5-carbon backbone; higher polyterpenoids are present in bacteria and plants (e.g. rubber, consisting of long chains of isoprene subunits). Farnesyl and geranylgeranyl residues consist of three and four C5 isoprene units, respectively. Protein farnesylation and geranylgeranylation may provide lipid residues necessary to anchor the respective proteins to cell membranes or intracellular membranous structures. The covalent attachment of prenyl groups occurs through a thioether bond to one or two cysteines at, or in close proximity of, the carboxy-terminus of the protein. More than 100 proteins, collectively called the prenylome, are involved in the molecular pathways producing these lipid moieties, or serve as potential substrates for prenylation, 53 PRENbase (http://mendel.imp.ac.at/PrePS/PRENbase/, date accessed 22 October 2013 54). In vision-related phenotypes, the first prenylation defects were identified in persons with choroideremia (CHM). Mutations in the choroideremia CHM gene that encodes Rab escort protein-1 (REP-1), a subunit of the geranylgeranyltransferase II (GGTase II; also called Rab geranylgeranyltransferase) protein complex, were found to cause progressive atrophy of the choroid, retinal pigment epithelium (RPE) and photoreceptors, resulting in visual impairment that eventually leads to blindness. 55; 56 Since then, variants in genes encoding prenylated proteins, chaperones of prenylated protein, or other proteins involved in prenylation, were discovered to underlie inherited retinal diseases (IRDs). Herewith, we will summarise the knowledge on these proteins and hypothesise on the underlying molecular mechanisms that may be common in these diseases.

Prenylation

Prenoid synthesis pathway Farnesyl and geranylgeranyl residues are synthesized from acetyl coenzyme A via the mevalonate pathway, which was discovered in the early 1950s. 57 This process, also called the terpenoid or isoprenoid backbone pathway, is a crucial mechanism of creating sterols, including cholesterol, ubiquinone, squalene and dolichol, which play key roles in various metabolic pathways. Noteworthy, unlike in plants, protozoa and bacteria, where the so-called MEP/DOXP pathway represents an alternative to obtain these compounds, in higher organisms these molecules must exclusively be synthesized through the mevalonate pathway (Supplemental Figure 1). Starting with two molecules of acetyl-coenzyme A, the terpenoid synthesis continues to mevalonic acid via 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). HMG-CoA reductase is a ratelimiting enzyme of the pathway. Hence, it has been targeted as a crucial phase to reduce the endogenous cholesterol synthesis in patients with hypercholesterolaemia. 58 Ever since then, HMG-CoA inhibitors (statins) have been successfully used for diminishing cholesterol levels. The product of the next step, mevalonic acid, is converted into phosphomevalonate by mevalonate kinase (MVK). At the end of this pathway, the farnesyl and geranylgeranyl residues are synthesized directly from isoprenyl diphosphate and subsequently attached to various proteins by prenyltransferases in the process called prenylation, which provides posttranslational modification essential for membrane attachment and protein-protein interactions. Prenylation process The prenyltransferases represented by farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase-I), consist of two subunits, an Îą and Î˛ subunit; in FTase and GGTase-I the Îą sub235 Prenylation defects in inherited retinal diseases

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unit is encoded by the same gene, FNTA (farnesyltransferase, CAAX box, α; Figure 1). All genes encoding the α subunits of the prenyltransferases are members of the tetratricopeptide repeat family, which includes proteins that are functionally relevant in the process of transcription, transport, co-chaperoning, control of the cell cycle and phosphorylation. 59 The β subunits, although less thoroughly investigated, are hypothesized to function in lipid anchor attachment or catalytic activity involving polyisoprenes. In general, the obtained products are processed to essential substances like cholesterol, steroid hormones or vitamin D and equivalents to these. The CaaX prenyltransferases, FTase and GGTase-I, can catalyse the modification of peptides and substrate proteins containing the Ca1a2X-motif (where C = cysteine, a1a2 = two aliphatic amino acids, being more flexible in a1, X = C, S, Q, A, M, T, H, V, N, F, G or I for FTase and L, F, I, V, M for GGTase-I).60; 61 Subsequently, CaaX endoprotease causes removal of the ‘aaX’ residue and methyl esterification of the carboxyl-group at the terminal cysteine.

Figure 1 Overview of proteins taking part in or being dependent on prenylation Schematic representation of the proteins which are prenylated and proteins taking part in prenylation process in the retina. Mutations in some of their corresponding genes were discovered to cause choroideremia (CHM), cone-rod dystrophy (CRD), congenital stationary night blindness (CSNB), Joubert syndrome (JS), Leber congenital amaurosis (LCA), mevalonate kinase deficiency (MKD), retinal disease (RD) and retinitis pigmentosa (RP). Proteins for which knockout mice displayed retinal disease are marked with a hash (#). Alpha (α) and beta (β) subunits of the prenylation enzymes encoded by different genes are depicted in different shades of grey. The third prenyltransferase, called non-CaaX-prenyltransferase, Rab geranylgeranyltransferase or geranylgeranyltransferase-II (GGTase-II; used hereafter), recognises Rab (member RAS oncogene family) proteins with a CC, CXC, CCX, CCXX, CCXXX or CXXX termination sequence, where methyl esterification only occurs in the CXC residues. In contrast to

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the CaaX-prenyltransferases, the heterodimer GGTase-II is unable to prenylate any peptide without the use of an accessory REP as a substrate.62; 63 The gene encoding the α subunit of GGTase-II (RABGGTA, Rab geranylgeranyltransferase, α subunit) was found to be mutated in the gunmetal mouse, a model of Hermansky-Pudlak syndrome;64 however, thus far no mutation in this gene was identified in humans with this syndrome. In the retina, the cyclic GMP phosphodiesterase 6α subunit, transducin γ subunit, rhodopsin kinase and ceroid lipofuscinosis neuronal 3 protein are substrates for FTase, whereas the cGMP phophodiesterase 6β subunit and X-linked retinitis pigmentosa (RP) GTPase regulator (RPGR) are substrates for GGTase-I. These proteins will be discussed below.

Genes implicated in general prenylation-associated retinal diseases

Rab escort protein-1 The first retinal prenylation defect was identified when protein-truncating mutations in, or deletions of CHM , the gene encoding REP-1, a chaperone for Rabs and a crucial accessory protein of GGTase-II, were found to be the cause of X-linked choroideremia. 55; 65 In this disease, there is a progressive mid-peripheral atrophy of the retina, RPE and the choroid, which results in night blindness, tunnel vision and eventually blindness. REP-1 is known to bind unprenylated Rabs and presents them to the GGTase-II heterodimer for geranylgeranylation (Figures 1 and 2). 56; 66 Despite the ubiquitous expression of CHM, only the retina and choroid are affected in male patients with mutations in this gene. This is explained by the compensation of geranylgeranylation of most Rab GTPases by REP-2 in all tissues except the eye. Indeed, REP-2 shows functional overlap with REP-1.67 Possibly, REP-1 preferentially prenylates a subset of retinal Rabs through a higher substrate affinity compared with REP-2. Larijani et al.68 proved that in cells of choroideremia patients, Rab27a accumulated in an unprenylated state due to differences in its ability to bind REP1 and REP2. The REP2-Rab27a complex was shown to have a lower affinity for GGTase II compared with REP1-Rab27a. Defects in intracellular membrane traffic pathways, including melanosome transport and phagosome processing, were described for the defective REP-1 protein.69 REP-1 was also shown to be essential for geranylgeranylation of RAB27A in lymphoblasts isolated from choroideremia patients, meaning that defective REP1 results in an accumulation of unprenylated RAB27A, specifically in the RPE.70 Missense mutations in RAB27A result in a dysfunction in melanosome transport and regulation, as RAB27A cannot recruit the effector MyRip (Myosin VIIA and Rab-interacting protein) that regulates melanosome transport in the RPE in complex with RAB27A and MYO7A.71; 72 These mutations cause Griscelli syndrome, which is characterised by cutaneous albinism and immunodeficiency.73 Both of these features are not described in choroideremia patients, and thereby provide no clue as to the crucial cellular defect(s) underlying this disease. Several preclinical studies have been carried out to develop a therapy for choroideremia. Contrary to males with choroideremia that only show a chorioretinal defect, male Chm knockout mice are not viable due to trophoblast defects at embryogenesis.74 To overcome this early lethality, Tolmachova and colleagues developed conditional knockout models in which Rep-1 was exclusively depleted either in the neural retina or in the RPE. In both models, defects were seen in the neural retina or RPE, respectively, suggesting cell-autonomous pathologies.75 This disease therefore requires a therapeutic approach preferably targeting both cell types. HIV-based lentiviral vectors have been used for in vivo CHM gene transfer studies.76 Rep-1deficient mice received a subretinal injection with a vector that expresses CHM under the control of the elongation factor-1 α promoter and showed a specific transduction of the RPE for 6 months. Functional restoration was confirmed by a reduced number of unprenylated Rabs. A second therapeutic strategy involves the use of adeno-associated virus (AAV) serotype 2-based vectors to transduce both photoreceptor cells and the RPE. In conditional knockout mice, it showed efficient RPE transduction and compared to the lentiviral approach allowed

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more potent transduction of neuroretinal cells.76; 77 The AAV2 vector, expressing REP-1 under the control of CMV-enhanced chicken β-actin promoter with a Woodchuck hepatitis virus post-translational regulatory element to enhance transgene expression vector, was able to provide efficient, functional and non-toxic transgene expression in diseased and healthy RPE and photoreceptor cells.78; 79 Currently, the first gene augmentation clinical trials using this vector are ongoing (clinicalTrials.gov Identifier: NCT01461213). No adverse effects of subretinal injections have been noticed in two groups consisting of three patients each.80 Aryl-hydrocarbon-interacting protein-like 1 Aryl-hydrocarbon-interacting protein-like 1 (AIPL1) functions as a chaperone for farnesylated proteins (Figures 1 and 2).81 AIPL1 has been found to be mutated in persons with autosomalrecessive Leber congenital amaurosis (LCA) or autosomal-dominant cone-rod dystrophy.82 AIPL1 is expressed in rods and cones in the early stages of retinal development,83 whereas in later stages expression is restricted to the rod photoreceptor cells.84 In Aipl1-/- mice, an LCA model, rapid rod degeneration is described, followed by cone degeneration.85; 86 Kirschman et al described a slower development of cone-rod degeneration in the absence of Aipl1, which was restricted to cone photoreceptor cells,87 This protein interacts with DnaJ (Hsp40) homologue, subfamily A, member 2 (DNAJA2), γ-transducin 81 and the α and β subunits of rod phosphodiesterase 88 in the presence of FTase. In the light of newly published research, it may be hypothesized that, similarly to chaperoning PDE6 subunits, 86 AIPL1 may function as a chaperone of other retinal Rab GTPases or proteins crucial in cone photoreceptors. Prenyl binding protein δ PrBP/δ, encoded by the phosphodiesterase 6D, cGMP-specific, rod, delta (PDE6D) gene, facilitates intracellular trafficking.89 PrBP/δ was shown to interact in vitro with RPGR,90 the prenyl side chains of GRK1 and the PDE6α and PDE6β subunits.91 Due to its ability to solubilise prenylated proteins, a role in transport was suspected.92 Interestingly, through its binding to carboxy-terminal prenyl groups of the phototransduction proteins PDE6α and PDE6β, PrBP/δ dissociates the PDE6αβγγ complex from the rod segment membrane discs. Likewise, it is thought that PrBP/δ can dissociate RAB13 from the membrane.93 As RAB13 is also predicted to be farnesylated, PrBP/δ may also interact with other prenylated proteins, such as RAB28.94 Pde6d-/- mice present with a slowly progressive rod-cone dystrophy,83 thereby making it an excellent candidate gene to be mutated in human retinal degeneration. Very recently, mutations in this gene were discovered to result in Joubert syndrome.95 It was also shown that PrBP/δ function is required for ciliary targeting of INPP5E. INPP5E is also a farnesylated protein, and the loss of its CaaX box was earlier proven to cause MORM (mental retardation, truncal obesity, retinal dystrophy, and micropenis syndrome), with features similar to Bardet Biedl syndrome.96; 97 Mevalonate kinase As mentioned above, MVK is an enzyme catalysing the transformation of mevalonic acid into phosphomevalonate. In a group of disorders called mevalonate kinase deficiency (MKD), this reaction is significantly hindered due to mutations in the MVK gene that encodes this enzyme. The metabolic block results in two distinct phenotypes of MKD: mevalonic aciduria (MEVA) or hyper-immunoglobulin D and periodic fever syndrome (HIDS). Both diseases may result from the same mutations within MVK; however, the clinical outcome is different.98; 99 MEVA is a severe condition characterised by progressive cerebellar ataxia, psychomotor retardation, and recurrent febrile crises, the latter usually being the only symptom occurring in HIDS. Several additional symptoms of MKD include abdominal complications and retinal dystrophy. Being a rare disorder, MKD has only been described in approximately 300 cases worldwide, 7 of which

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were diagnosed with retinal degeneration.100-102 Recently, we reported that mutations in MVK cause late-onset nonsyndromic RP, which may be accompanied by mild MKD features.103 It was previously hypothesized that neuronal degeneration observed in MEVA could be a result of toxic mevalonic acid levels in the brain. The same process may also underlie photoreceptor degeneration.101 However, the spectrum of disorders associated with MVK mutations is wide and it is difficult to assess the role of external factors in this process. Recently, in an attempt to lower the toxic levels of mevalonate, lovastatin and simvastatin were applied in MKD. However, the results were ambiguous. The administration of lovastatin in patients with classic MEVA caused severe crisis (high fever, acute myopathic changes, diarrhoea) and the trial had to be terminated.104 Conversely, in HIDS, the administration of simvastatin resulted in reduced excretion of mevalonic acid and a decreased number of febrile days.105 In patients displaying retinal dystrophy these medications have not yet been tested. It has also been hypothesized that the unique isoprenoid metabolism in photoreceptors is responsible for MVK-associated retinal degeneration.100 Intriguingly, the same mutations that have previously been described to cause MEVA were shown to result in MKD displaying predominantly the RP phenotype. Clearly, we still lack fundamental knowledge on the action of other proteins, often referred to as the ‘genetic background’. It may be hypothesized that mutations in other proteins may rescue the severe neurological phenotype, perhaps by alleviating the toxicity of mevalonic acid in these tissues, while the retina would remain affected. The contribution of epigenetic and/or environmental factors should also be taken into consideration. For example, an enhanced expression of a downstream enzyme may set the prenylated residues to a level acceptable in neurons. However, it may be still insufficient for the correct function of photoreceptors, which possess many membranous structures and therefore are more dependent on prenylated proteins.

Prenylated proteins mutated in nonsyndromic retinal diseases

Guanine nucleotide-binding protein subunit gamma, Tγ The γ chain of transducin is encoded by GNGT1 (guanine nucleotide binding protein gamma transducing activity polypeptide 1). Transducin is a membrane-bound heterotrimer containing α, β and γ subunits, and in its inactive state it is bound to a guanosine diphosphate (GDP) molecule. Activation by metarhodopsin II results in binding of guanosine triphosphate (GTP) and detachment of the α subunit from the membrane, which results in the activation of PDE6. The remaining subunits (called Tβγ) then diffuse to the inner photoreceptor segment. A CaaX motif is present on the α and γ subunits of rod transducin, but only the γ subunit has been found to be farnesylated.106 This modification is neither necessary for rhodopsin binding nor for the interaction with the α subunit. During dark adaptation dislocated transducin subunits must return to the outer segment for rod sensitivity restoration.107 It was established that this process may be mediated by the farnesyl residue;108 the potential Tγ partners include PrBP/δ and phosducin. In the study by Lebanova et al.109, knockout of the gene encoding the transducin γ subunit in mice resulted in severe down regulation of α and β subunits of PDE6, despite normal mRNA levels. The mice also underwent fairly rapid photoreceptor degeneration. In contrast, Kolesnikov et al.110 described no retinal degeneration during the early stages of postnatal life. GNGT1 has previously been suggested to play a role in human retinal degeneration disorders,111 but thus far no mutations have been found in this gene in human IRDs,112 which may be due to its relatively small size (225 bp open- reading frame) and the associated low mutation frequency. Mutations in the gene encoding the transducin α subunit, GNAT1, were identified in Nougarettype congenital stationary night blindness. These findings, along with the lack of photoreceptor degeneration in one of the mouse models, suggest that mutations in GNGT1 may be involved in a similar disease.

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Phoshodiesterase 6α and 6β subunits Several proteins essential for the process of vision require farnesyl and geranylgeranyl residues for proper functionality. Among them, rod cyclic guanosine monophosphate (cGMP)specific phosphodiesterase 6 (PDE6) consists of catalytic α and β subunits, two inhibitory γ subunits, and a δ subunit. This enzyme is crucial for the phototransduction pathway. Briefly, in response to a photon, rhodopsin is activated and stimulates GTP synthesis (Figure 2). In turn, a G-protein, transducin, is activated and displaces the inhibitory γ subunits thus activating PDE6. This phosphodiesterase is involved in the reduction of cGMP concentration, which leads to closure of cGMP-gated channels. As a direct result of ensuing transient hyperpolarisation, at the synapse glutamate release is then reduced and the signal travels via the optic nerve to the brain. PDE6 is anchored to the membrane through isoprenyl residues attached to the C-termini of PDE6α and PDE6β. Mammalian PDE6A was proven to contain one farnesyl residue; in comparison, PDE6B is geranylgeranylated.113 These modifications are thought to facilitate membrane attachment.114 The prenyl moieties of PDE6 subunits were recently shown to bind AIPL1, the aforementioned chaperone protein involved in LCA.115 PDE6 subunits form a complex containing prenyl-binding protein delta (PrBP/δ), which is highly expressed in photoreceptors. Mutations in both PDE6A and PDE6B (phosphodiesterase 6A, cGMP-specific, rod, α and β, respectively), encoding the α and β subunits, are responsible for a small subset of autosomalrecessive RP cases.116; 117 RAB28, member RAS oncogene family Rab GTPases are key regulators in trafficking of proteins and function as molecular switches to regulate fusion steps in vesicle transport, vesicle budding, motility, tethering,118 and membrane fission.119 The human genome contains more than 60 Rab genes,120; 121 and for at least 16 different Rab proteins the structure in either the active (GTP-bound) or inactive (GDP-bound) state has been elucidated.122-124 The first prenylated small Rab GTPase demonstrated to be involved in retinal disease is RAB28, as protein-truncating mutations in RAB28 cause cone-rod dystrophy.125 Although the exact role of RAB28 in cone and rod photoreceptors remains to be determined, a unique feature is that isoforms 1 and 2 of RAB28 are predicted to be farnesylated at their CaaX-motifs, 54; 126-128 in contrast to the large majority of other RAB GTPases that are geranylgeranylated by GGTase-II. The farnesylation was experimentally proven for isoform 1 of RAB28. Other Rabs, like Rab8 and Rab11, are found to coordinate primary ciliogenesis.84 Together with Rab3a and Rab6, they are involved in rhodopsin transport through the connecting cilium to the outer segments of photoreceptor cells.129-132 Interestingly, a single mutation in the RAB3Ainteracting molecule, RIMS1, was associated with dominant cone-rod dystrophy (Figure 1).133 RAB28 is also localised to the basal body and ciliary rootlet, which suggests a role in ciliary transport (Figure 2).125 In addition, Rab17 and Rab23 were found to be essential for ciliogenesis, but are not present in the cilium.134 The disease mechanism underlying photoreceptor degeneration due to protein-truncating mutations in RAB28 remains to be elucidated. The retina-specific phenotype is surprising in view of the wide expression of this gene in the human body. Either there is functional redundancy outside the eye through the action of other Rabs or RAB28 may be involved in the transport of crucial retina-specific proteins.

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Figure 2 General overview of visual phototransduction and ciliary transport Depicted is a scheme of rod phototransduction and transport process crucial for vision. Prenylated proteins, as well as proteins involved in the prenylation process discussed in this review are marked in colours: prenylated proteins are in orange, while chaperones are in green. Farnesyl and geranylgeranyl residues are depicted as a red and blue tags, respectively. PrBP/δ is depicted by delta (Pδ) in a green balloon. PDE6 subunits are marked as alpha (Pα), beta (Pβ), gamma (Pγ); transducin subunits are marked as (Tα), beta (Tβ), gamma (Tγ); and GGTase-II subunits are marked as GGTα and GGTβ; activated rhodopsin (a), phosphorylation (P), gua-

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nosine monophosphate (GMP) and its cyclic counterpart (cGMP), guanosine-5’-triphosphate (GTP), guanosine diphosphate (GDP). Activation of rhodopsin with a photon is indicated by a lightning symbol. PC – photoreceptor. Retinitis pigmentosa GTPase regulator Another prenylated protein involved in retinal dystrophy is RPGR, a ubiquitously expressed protein. RPGR can act as a GTP-GDP exchange factor for RAB8,135 a protein involved in ciliary transport and rhodopsin trafficking to the photoreceptor outer segment.136 Since some disease-causing RPGR mutations do not affect this GTP-GDP exchange function, RPGR may have additional roles in the photoreceptors. The geranylgeranylation of RPGR is required for its correct localisation in the Golgi apparatus,137 but the lack of this isoprenylation does not seem to disrupt the retinal function.138 Several splice variants are found in different tissues, and the RPGR ORF15 isoform is crucial for normal functioning of the retina. Mutations in this part of RPGR were shown to be involved in the majority of X-linked retinal dystrophy cases.139; 140 However, the RPGR ORF15 transcript lacks the CaaX prenylation signal at its C-terminus. Therefore, although both transcripts are expressed in photoreceptors, it remains unclear whether this modification is necessary for retinal function. G protein-coupled receptor kinase 1 G protein-coupled receptor kinases specifically recognise and phosphorylate the numerous G protein-coupled receptors after the release of bound G proteins. Rhodopsin kinase (GRK1), which belongs to this group and regulates cellular responses. It is activated allosterically by its own substrate, the light-activated form of rhodopsin. Membrane association of GRK1 occurs after its farnesylation. Since in PDE6D knockout mice GRK1 is absent in the photoreceptor outer segments, it is suggested that PrBP/δ is required for its correct trafficking.83 GRK1 mutations are a cause of Oguchi disease, a form of stationary night blindness with prolonged dark adaptation. Since GRK1 is crucial for visual processes, it is hypothesized that in cones GRK7 compensates for the loss of GRK1 activity, and hence patients with mutations in GRK1 only display a relatively mild phenotype.141; 142 Ceroid lipofuscinosis neuronal 3 Mutations in ceroid lipofuscinosis, neuronal 3 (CLN3), encoding a lysosomal transmembrane protein, are a cause of Batten disease. Vision loss often is the first clinical feature in Batten disease, preceding severe and progressive neurodegeneration leading to death in the second decade of life.143 Recently, a mutation has been described in a patient with nonsyndromic LCA. 5 This previously described mutation (p.Y199*) was found to result in a milder, protracted phenotype due to the escape of nonsense-mediated decay, hypothetically leaving a partially functional protein.144 The CLN3 protein is described to be farnesylated,145 and it has been shown that mutations in the CLN3-binding motif of FTase display normal trafficking, but result in a functionally impaired protein.146

Are there overlapping mechanisms of disease due to defects in the retinal prenylome?

The photoreceptors are highly compartmentalised and membranous structures, and therefore it can be expected that many retinal proteins depend on prenylation for a proper subcellular localisation and trafficking. This is true for several proteins found to be mutated in different IRDs. The mechanisms underlying retinal degeneration due to defects in the prenylated proteins described above are not fully understood, but can be inferred from their known roles in the phototransduction cascade (GNGT1, PDE6A, PDE6B), transport processes close to or at the connecting cilium (RAB28, RPGR), or lysosomes (CLN3). Apart from these ‘gene-specific’

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defects, mutations affecting AIPL1 and PrBP/δ exert a more general effect as both are chaperones for a subset of prenylated retinal proteins. Defects in the corresponding genes thereby may affect the function of the respective proteins with which they normally interact. REP-1 does not only act as a chaperone for RABs but it also is a key accessory protein of GGTase-II, which is involved in the geranylgeranylation of almost all RABs. It is thus far not understood how REP-1 deficiency, despite the presence of a functional homologue REP-2 (encoded by choroideremia-like gene, CHML) that is also widely expressed, results in a specific chorioretinal disease. The same is true for the surprising recent finding that mutations in mevalonic kinase, which were known to be principally associated with MEVA, were associated with nonsyndromic RP. We hypothesise that MVK deficiency results in a depletion of prenyl moieties, which affects the prenylation of many proteins synthesized specifically in the retina. It stands to reason that the prenylated proteins illustrated in Figures 1 and 2 would most prominently be affected, though some of the associated retinal phenotypes differ from the RP phenotype observed in persons with MVK deficiencies. Assuming that the depletion of isoprenyl moieties underlies RP in MKD, there may be an overlap with another ‘general’ prenylation defect, that is the underprenylation of one or several RABs due to mutations in the CHM gene. Clearly, future studies are needed to shed light on these issues.

Concluding remarks

The mechanisms underlying retinal dystrophies due to variants in prenylated proteins in the retina are straightforward as each of these proteins, maybe except for RAB28, plays a unique function in the retina. Similar to other geranylgeranylated RABs, the farnesylated RAB28 is synthesized widely in human tissues. Nevertheless, it apparently fulfils a crucial function in the transport of as yet unknown molecules. Possibly, it also is involved in the shuttling of key molecules towards the connecting cilium, such as rhodopsin. The chaperone proteins AIPL1 and PrBP/δ play diverse roles in the assembly and transport of several key protein complexes, but the full spectra of their function are not yet known. Mutations in CHM result in X-linked choroideremia and MVK variants are associated with a late-onset form of autosomal-recessive RP in a small subset of MKD cases. There is a conspicuous phenotypic overlap between these two diseases. Although choroideremia is characterised by the degeneration of all posterior cell layers (neural retina, RPE, choroid), and RP is characterised by the dystrophy of the neural retina and RPE, their similarity is striking. Both display a midperipheral-to-centre degeneration, primarily affecting rods, preserving the macular region for a long time. Both result from general prenylation defects, although at different levels. MKD potentially leads to a widespread shortage of prenoids, which include farnesyl and geranylgeranyl moieties. CHM mutations, on the other hand, cause an insufficiency of GGTase-II to geranylgeranylate the large majority of RABs. We hypothesise that the comparable phenotypes result from depletion of prenylated RABs. Many studies suggest that the retina displays specific requirements for proteins possessing lipid moieties, most probably due to the membranous structure of the photoreceptors. The retina therefore may be particularly susceptible to underprenylation of proteins, which would give rise to retinal symptoms before (if at all) affecting other tissues. Several genes implicated in IRDs encode proteins that either require prenylation or are crucial in the prenylation process. Mutations in these genes may result in diminished or abolished protein synthesis, but can also cause loss of function and accumulation of inactive prenylated proteins, which may be toxic to photoreceptor cells. The recent finding of LCA-specific features in a patient with CLN3 variants may also point to a disease mechanism involving the accumulation of toxic molecules in retinal lysosomes. Many IRD genes are being explored as targets for gene augmentation therapy, and it may be

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hypothesized that targeting the prenylation process could provide a more generic treatment. However, in view of the diversity of the proteins and cellular processes in question, the development of a more generalised therapy does not seem plausible at the moment. Although a general prenylation therapy seems incredibly challenging, steps are currently being made to devise a therapy directed to specific geranylgeranyl or farnesylation defects. One of the deficiencies that may be addressed is retinal degeneration caused by the loss of MVK functionality. Although it was proven that patients with RP and mild MKD symptoms display the same, severe decrease in MVK activity compared with MEVA patients, it may be hypothesized that boosting the concentration of substrates for prenyl synthesis either by direct supplementation or by targeting one of the downstream enzymes in mevalonate pathway would increase the overall supply of prenyl residues and rescue the phenotype. However, it is very difficult to predict how this would affect the target cells. FTase inhibitors have been previously used as a target for decreasing prenylation in treatment of some cancer types.147-150 Nevertheless, the K-RAS protein that is involved in some neoplasms appears to be geranylgeranylated in the absence of FTase.151 Therefore, a similar method involving both farnesyltransferase and geranylgeranyltransferase inhibitors was investigated, but the dual prenylation of proteins caused the agent to exhibit dose-limited toxicities in the clinic and were withdrawn for further investigation.152; 153 Considering all of the above issues, stimulating the synthesis of prenyl residues in the retina may cause undesirable side effects, such as neoplastic growth. It is also possible that manipulating these levels will have no effect at all since there are multiple feedback loops in the isoprenoid pathway, and increased concentration of one of the compounds may result in inhibition rather than stimulation of prenyl synthesis. Targeting the inhibitors of prenyltransferases was already tested in other genetic disorders. In progeria, a disease of rapid aging, farnesyltransferase inhibitors have shown their potential in vitro by blocking the expression of the defective farnesylated protein laminin A (or progerin), as well as in vivo with respect to longevity, body weight and bone integrity phenotypes.154 In retinal diseases, for example in case of a malfunctioning chaperone, it is conceivable that a prenylated protein might accumulate at an improper location and result in retinal degeneration. However, general inhibition of either farnesylation or geranylgeranylation would probably result in underprenylation of other targets. Therefore, it is not certain whether such therapies should be considered. At this point, gene augmentation therapy still presents the best option for treatment of such diseases. Although the eye has excellent properties for the development of treatment, the bottleneck will remain that each prenylation defect requires a different therapeutic approach, posing significant challenges for the future.

Acknowledgements

This study was financially supported by the Netherlands Organisation for Scientific Research (TOP-grant 91209047, to F.P.M.C., H. Kremer, and A.I.d.H.), Foundation Fighting Blindness USA (grants BR-GE-0510-04890RAD to A.I.d.H., and C-GE-0811-0545-RAD01 to F.P.M.C., the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, the Gelderse Blinden Stichting, the Landelijke Stichting voor Blinden en Slechtzienden, the Stichting Blinden-Penning, the Stichting Macula Degeneratie fonds, the Rotterdamse Stichting Blindenbelangen (to F.P.M.C.). None of the authors have any financial interest or conflicting interest to disclose.

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Supplementary Figure 1 Mevalonate pathway

5.4 The mevalonate pathway (HMG-CoA reductase pathway/isoprenoid pathway), is a crucial cellular metabolic route for the production of dimethylallyl diphosphate and isopentenyl diphosphate. These compound are the basis for the biosynthesis of molecules used in diverse processes, e.g. protein prenylation, hormone synthesis, protein anchoring, and N-glycosylation. It is also a part of steroid biosynthesis.

245 Prenylation defects in inherited retinal diseases

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258

Summary

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The leading causes of hereditary blindness worldwide are retinal disorders, which primarily affect the photoreceptors. An affected person with cone dysfunction (CD) has difficulties perceiving colors and seeing details. In this thesis I mainly focused on early-onset cone-dominated phenotypes leading to severe visual impairment, such as achromatopsia (ACHM), cone dystrophy (COD) and cone-rod dystrophy (CRD). CDs can be inherited in all Mendelian forms of inheritance, however, in this thesis I focused on those inherited in an autosomal recessive fashion. Although important steps are being made, there are currently limited therapeutic options to inhibit, stop, or reverse the degenerative disease process. In order to develop effective therapeutic approaches the genetic etiology of CD needs to be elucidated. Throughout the years, dozens of genes have been found to cause CD, but still significant percentages of CD cases are unexplained. Mutations in the 5 genes implicated in ACHM explain approximately 93% of the cases. On the contrary, only 21% of autosomal recessive CRD cases (17 genes) and 25% of autosomal recessive COD (8 genes) have been elucidated. In this thesis I aim to elucidate novel genetic causes for CD using homozygosity mapping and exome sequencing, to increase the molecular genetic knowledge of CDs and open new avenues for generating therapeutic approaches. Chapter 1 provides a comprehensive background on CD, the topic of this thesis. In Chapter 1.1 a general introduction is given on the human eye and its properties which enable color vision. Subsequently, in Chapter 1.2 insight is provided in homozygosity mapping and exome sequencing which are currently used to identify novel gene defects. Finally, Chapter 1.3 reviews the current knowledge about the clinical phenotypes representing CD, the molecular processes in which the 42 CD-associated genes are involved, an update on animal models with cone defects and current therapeutic approaches. Chapter 2 describes the identification of pathogenic CNGA3 and CNGB3 mutations in COD, while previous studies only showed involvement of these genes in stationary ACHM. The underlying mechanism how different phenotypes can be caused by similar mutations remains unexplained. Chapter 3 describes novel genotype-phenotype correlations for two genes, TULP1 and CEP290. Mutations in these genes were known to cause inherited retinal disease, but have not previously been associated with CD. Chapter 3.1 describes the identification of an uncommon phenomenon of maternal uniparental isodisomy of chromosome 6 in a patient with CD. Subsequent sequencing of all retinal disease associated genes on chromosome 6 led to the identification of a homozygous TULP1 mutation, which was also identified in another unrelated case with CD. In Chapter 3.2 exome sequencing in two siblings with oligocone trichromacy enabled the identification of compound heterozygous nonsense mutations in CEP290, which were demonstrated to have a hypomorphic effect. These mutations caused a relatively mild phenotype in these siblings, while CEP290 mutations are known to cause a spectrum of diseases ranging from early-onset retinal dystrophy to Meckel-Gruber syndrome. Chapter 4 describes the identification of three novel genes associated with CD. Chapter 4.1 reports the identification of PDE6C as a new disease associated gene for ACHM and earlyonset COD. Several ACHM families and one COD family carried mutations in PDE6C, a gene expressed specifically in the cone photoreceptor cells. These mutations were discovered using homozygosity mapping and provided evidence for a genetic overlap between ACHM and COD. In Chapter 4.2 we used exome sequencing to unravel the genetic defect in two CRD families. Both families showed truncating mutations in the farnesylated small GTPase RAB28, localized at the base of the connecting cilium, as the cause of their retinal disease. Chapter 4.3

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describes compound heterozygous mutations in MFSD8 in a large Dutch family with central cone disease, identified by linkage analysis and exome sequencing. Mutations in this gene are known to cause variant late infantile neuronal ceroid lipofuscinosis, a severe neurodegenerative disorder. We hypothesize that neuronal ceroid lipofuscinosis is caused by two severe mutations, while central cone disease in this family is caused by a severe mutation on one allele and a mild variant on the other allele. In line with this hypothesis, the same mild variant was found in combination with another severe mutation in a second Dutch family with a CRD. In Chapter 5 the main findings and implications of this thesis are discussed. First, in Chapter 5.1 the efficiencies of the genomic technologies are described and the advantages of implementing exome sequencing as well as the disadvantages of the current approach in routine diagnostics. Chapter 5.2 highlights the observations that hypomorphic mutations, in combination with more severe variants, can be associated with phenotypes limited to the eye, whereas combinations of truncating mutations cause a range of more severe, often syndromic, phenotypes. Chapter 5.3 provides a review on prenylated proteins that are associated with retinal dystrophies, such as RAB28. In addition, chaperones guiding prenylated proteins to their proper subcellular location are discussed (i.e. AIPL1, PrBP/Î´), which can be defective in persons with a retinal dystrophy. Finally, a possible overlap is hypothesized for the underlying retina dystrophy-associated disease mechanisms due to MVK defects (retinitis pigmentosa) or CHM defects (chorioderemia). In both diseases it is possible that a crucial set of RAB GTPases shows geranylgeranyl deficiencies which result in intracellulair transport function abnormalities and subsequently retinal degeneration.

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Samenvatting

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De belangrijkste oorzaken van erfelijke blindheid zijn retinale aandoeningen, die voornamelijk de fotoreceptoren aantasten. Een persoon met kegel dysfunctie (CD) heeft problemen met het waarnemen van kleuren en details. In dit proefschrift heb ik onderzoek verricht naar CDs die op jonge leeftijd ontstaan en tot ernstige visuele beperkingen leiden, zoals achromatopsie (ACHM), kegel dystrofie (COD) en kegel-staaf dystrofie (CRD). CDs kunnen via alle Mendeliaanse vormen overerven, echter, dit proefschrift concentreert zich op de vormen die op de autosomaal recessieve wijze worden overgeërfd. Alhoewel er belangrijke stappen zijn gezet in het onderzoek naar behandelingen, zijn er momenteel weinig therapeutische mogelijkheden.. Om effectieve therapieën te kunnen ontwikkelen is het belangrijk om de genetische etiologie van CD te ontrafelen. De afgelopen jaren zijn er tientallen genen gevonden die CD kunnen veroorzaken, maar nog steeds blijft een groot deel van alle CD gevallen onverklaard. Ongeveer 93% van ACHM wordt verklaard door mutaties in 5 genen. Bij autosomaal recessieve CRD en COD werden echter 21% (17 genen) en 25% (8 genen) van de families genetisch opgelost. Het doel van mijn proefschrift is om nieuwe genetische oorzaken voor CD te vinden door middel van homozygotie mapping en exoom sequencing, om hiermee de moleculair genetische kennis over CDs te vergroten waardoor nieuwe therapeutische mogelijkheden ontwikkeld kunnen worden. Hoofdstuk 1 omvat een uitgebreide achtergrond over CD, het onderzoeksthema van dit proefschrift. In Hoofdstuk 1.1 wordt een algemene introductie gegeven over het menselijk oog en de eigenschappen die kleurzien mogelijk maken. Vervolgens wordt in Hoofdstuk 1.2 inzicht gegeven in homozygotie mapping en exoom sequencing, geavanceerde genetische technieken die gebruikt kunnen worden om nieuwe gendefecten te identificeren. Tenslotte volgt in Hoofdstuk 1.3 een overzicht van de huidige kennis van het klinisch beeld van CD, de moleculaire processen waarbij de 42 CD-geassocieerde genen zijn betrokken, diermodellen met kegel defecten en de huidige therapeutische mogelijkheden voor CD. Hoofdstuk 2 beschrijft de identificatie van mutaties in CNGA3 en CNGB3 bij CD, terwijl eerdere studies alleen betrokkenheid van deze genen lieten zien bij stationaire ACHM. Het is nog onduidelijk hoe deze verschillende ziekten veroorzaakt kunnen worden door vergelijkbare mutaties. Hoofdstuk 3 toont nieuwe genotype-fenotype correlaties aan voor twee genen, TULP1 en CEP290. Het was reeds bekend dat mutaties in deze genen erfelijke retinale aandoeningen veroorzaken, echter, ze waren niet eerder geassocieerd met CD. Hoofdstuk 3.1 beschrijft een zeldzaam fenomeen van maternale uniparentale isodisomie van chromosoom 6 in een persoon met CD. Sequentie analyse van alle retinale dystrofie geassocieerde genen gelegen op chromosoom 6, leidde tot de identificatie van een homozygote TULP1 mutatie, die vervolgens ook bij een niet-verwante persoon met CD werd gevonden. In Hoofdstuk 3.2 heeft exoom sequencing bij een broer en zus met een bijzondere vorm van ACHM geleid tot de identificatie van samengestelde heterozygote stopmutaties in CEP290, waarvan gedemonstreerd werd dat deze een hypomorf effect hebben. Deze mutaties veroorzaakten een relatief milde ziekte bij deze broer en zus, terwijl het bekend is dat CEP290 mutaties een spectrum van aandoeningen variërend van een ernstige retinale dystrofie tot afwijkingen niet verenigbaar met het leven. Hoofdstuk 4 beschrijft de vondst van mutaties in drie nieuwe genen die CD veroorzaken. Hoofdstuk 4.1 rapporteert de bevinding van PDE6C als een nieuw ziekte-geassocieerd gen voor ACHM en COD. Drie ACHM families en één COD familie dragen mutaties in PDE6C, een gen dat specifiek in de kegel fotoreceptoren tot expressie komt. Deze mutaties werden gevonden middels homozygotie mapping en tonen een genetische overlap aan tussen ACHM

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en COD. In Hoofdstuk 4.2 werd exoom sequencing gebruikt om het genetisch defect in twee CRD families te ontrafelen. Beide families droegen stopmutaties in het RAB28 gen, welke codeert voor een gefarnesyleerd GTPase dat in het netvlies gelocaliseerd is nabij het connecting cilium. Hoofdstuk 4.3 beschrijft de ontdekking van samengestelde heterozygote mutaties in het MFSD8 gen in een grote familie met CD door middel van koppelingsonderzoek en exoom sequencing. Het was al bekend dat mutaties in dit gen infantiele neuronale ceroid lipofuscinose, een ernstige neurodegeneratieve aandoening, veroorzaken. Onze hypothese is dat neuronale ceroid lipofuscinose in MFSD8 wordt veroorzaakt door twee ernstige mutaties, terwijl CD wordt veroorzaakt door een ernstige mutatie op het ene allel en een milde variant op het andere allel. Deze hypothese werd bevestigd doordat in een tweede familie met CD dezelfde milde variant gevonden werd in combinatie met een andere ernstige mutatie. In Hoofdstuk 5 worden de belangrijkste bevindingen en implicaties van dit proefschrift bediscussieerd. In Hoofdstuk 5.1 worden de efficiëntie van de genomische technologieën beschreven, de voordelen van implementatie van exoom sequencing, en de nadelen van de huidige aanpak in de routine diagnostiek. Hoofdstuk 5.2 belicht de observatie dat hypomorfe mutaties in combinatie met een ernstigere variant geassocieerd kunnen worden met een ziekte die zich beperkt tot het oog, terwijl combinaties van stopmutaties een spectrum van ernstigere, syndromale ziekten kunnen veroorzaken. Hoofdstuk 5.3 biedt een overzicht over geprenyleerde eiwitten die geassocieerd zijn met retinale dystrofie, zoals RAB28. Daarnaast worden chaperonnes van geprenyleerde eiwitten bediscussieerd (o.a. AIPL1, PrBP/δ) die defect kunnen zijn in personen met dystrofie van de retina. Tenslotte wordt een mogelijke overlap voorgesteld van de ziektemechanismen die ten grondslag liggen aan retina dystrofie-geassocieerde ziekten als gevolg van MVK defecten (retinitis pigmentosa) of CHM defecten (choroideremie). Bij beide ziekten is het mogelijk dat een cruciale set van RAB GTPases geranylgeranyl deficiënties vertonen, waardoor intracellulaire transportfuncties uitvallen hetgeen leidt tot retinale dystrophie.

265 Samenvatting

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Eenvoudiger gezegd

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Retinale aandoeningen zijn de belangrijkste oorzaken van erfelijke blindheid, die met name de fotoreceptoren aantasten. De mens kent twee typen fotoreceptoren: de kegeltjes en de staafjes. Het beeld dat op het netvlies valt wordt door de kegeltjes en staafjes omgezet in een elektrisch signaal naar de hersenen. Bij een persoon met kegel dysfunctie werken de kegel fotoreceptoren niet goed, waardoor er problemen ontstaan in het waarnemen van kleuren en details. In de studies beschreven in dit proefschrift heb ik onderzoek verricht naar kegel dysfuncties die op jonge leeftijd ontstaan en tot ernstige visuele beperkingen leiden, zoals achromatopsie, kegel dystrofie en kegel-staaf dystrofie. Dit proefschrift concentreert zich op kegel dysfuncties die op een recessieve wijze overerven. Bij recessieve overerving zijn beide gezonde ouders drager van het gendefect. Als de gendefecten van beide ouders doorgegeven worden aan hun kind, dan zal hij/zij kegel dysfunctie ontwikkelen. De afgelopen jaren zijn er afwijkingen in tientallen genen gevonden die kegel dysfunctie kunnen veroorzaken, maar nog steeds zijn de genetische oorzaken van een groot aantal personen met kegel dysfunctie onbekend. Het doel van mijn promotieonderzoek was om nieuwe genetische oorzaken van kegel dysfunctie te vinden door middel van de nieuwste genetische methoden. Door de moleculair genetische kennis over kegel dysfuncties te vergroten kan de diagnose verbeterd worden, en kunnen in de toekomst nieuwe behandelingen ontwikkeld worden. Hoofdstuk 1 omvat een uitgebreide achtergrond over kegel dysfunctie, het onderwerp van dit proefschrift. Er wordt een algemene introductie gegeven over het menselijk oog en de eigenschappen die kleuren zien mogelijk maken. Vervolgens wordt inzicht gegeven in geavanceerde genetische technieken die gebruikt kunnen worden om nieuwe gendefecten op te sporen. Tenslotte volgt in het laatste deel een overzicht van de huidige kennis van het klinisch beeld van kegel dysfunctie, de moleculaire processen waarbij de 42 kegel dysfunctie genen betrokken zijn, diermodellen met kegel defecten, en de huidige therapeutische mogelijkheden voor kegel dysfunctie. Hoofdstuk 2 beschrijft de identificatie van afwijkingen in de CNGA3 en CNGB3 genen bij kegel dystrofie, een progressieve ziekte waarbij de kegel functie steeds verder afneemt. Eerdere studies toonden alleen betrokkenheid van deze genen aan bij achromatopsie, een ernstige oogziekte die vanaf de geboorte aanwezig is maar niet of nauwelijks steeds erger wordt. Het is nog onduidelijk hoe deze verschillende ziekten veroorzaakt kunnen worden door genetische afwijkingen die niet veel van elkaar verschillen. Hoofdstuk 3 bestaat uit twee artikelen waarin gendefecten in twee genen, TULP1 en CEP290, worden beschreven in personen met kegel dysfunctie. Het was reeds bekend dat afwijkingen in deze genen erfelijke retinale aandoeningen veroorzaken, echter, ze waren niet eerder geassocieerd met kegel dysfunctie. Hoofdstuk 4 beschrijft in drie artikelen de vondst van afwijkingen in drie nieuwe genen die kegel dysfunctie veroorzaken. In deze artikelen toonden we aan dat gendefecten in PDE6C, RAB28 en MFSD8 achromatopsie, kegel dystrofie en kegel-staaf dystrofie kunnen veroorzaken. Het vinden van afwijkingen in deze genen biedt nieuwe perspectieven voor de ontwikkeling van behandelingen voor patiënten met kegel dysfunctie. In Hoofdstuk 5 worden de belangrijkste bevindingen en implicaties van dit proefschrift bediscussieerd. Deze omvatten onder andere de doeltreffendheid van de gebruikte genomische technieken, de voordelen van het aflezen van alle humane genen (ook wel ‘exome sequencing’ genoemd), de nadelen van de huidige aanpak in de routine diagnostiek, en de observatie dat verschillende genafwijkingen kunnen leiden tot verscheidene oogaandoeningen. Tenslotte

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wordt een mogelijke overlap voorgesteld van de ziektemechanismen die ten grondslag liggen aan een aantal retinale aandoeningen.

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List of publications

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Roosing, S., van den Born, L.I., Sangermano, R., Banfi, S., Koenekoop, R.K., Zonneveld-Vrieling, M.N., Klaver, C.C., van Lith-Verhoeven, J.J., Cremers, F.P.#, den Hollander, A.I.#, Hoyng, C.B.#, 2014. Mutations in MFSD8, encoding a lysosomal membrane protein, are associated with nonsyndromic autosomal recessive macular dystrophy. Ophthalmology In press Roosing, S.*, Lamers, I.J.*, de Vrieze, E.*, van den Born, L.I.*, Lambertus, S., Arts, H.H., Study group, Peters, T.A., Hoyng, C.B., Kremer, H., Hetterschijt, L., Letteboer, S.J., van Wijk, E.#, Roepman, R.#, den Hollander, A.I.#, Cremers, F.P.#, 2014. Disruption of the basal body protein POC1B results in autosomal recessive cone-rod dystrophy. Am J Hum Genet In press Roosing, S., Thiadens A.A., Hoyng, C.B., Klaver, C.C., den Hollander, A., Cremers, F.P., 2014. Causes and Consequences of Inherited Cone Disorders. Prog Retin Eye Res. In press Roosing, S., Collin, R.W., den Hollander, A.I., Cremers, F.P.#, Siemiatkowska, A.M.#, 2014. Prenylation defects in inherited retinal dystrophies. J Med Genet. 2014 51:143-151 Roosing, S., Rohrschneider, K., Beryozkin, A., Sharon, D., Weisschuh, N., Staller, J., Kohl, S., Zelinger, L., Peters, T.A., Neveling, K., Strom, T.M., European Retinal Disease Consortium, van den Born, L.I., Hoyng, C.B., Klaver, C.C., Roepman, R., Wissinger, B., Banin, E., Cremers, F.P., den Hollander, A.I., 2013. Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy. Am J Hum Genet 93, 110-117. Roosing, S., van den Born, L.I., Hoyng, C.B., Thiadens, A.A.H.J., de Baere, E., Collin, R.W.J., Koenekoop, R.K., Leroy, B.P., van Moll-Ramirez, N., Venselaar, H., Riemslag, F.C., Cremers, F.P.M., Klaver, C.C.W., den Hollander, A.I., 2013. Maternal uniparental isodisomy of chromosome 6 reveals a TULP1 mutation as a novel cause of cone dysfunction. Ophthalmology. 2013 Jun;120(6):1239-46 Kohl, S., Coppieters, F., Meire, F., Schaich, S., Roosing, S., Brennenstuhl, C., Bolz, S., van Genderen, M.M., Riemslag, F.C., European Retinal Disease Consortium, Lukowski, R., den Hollander, A.I., Cremers, F.P., De Baere, E., Hoyng, C.B., Wissinger, B., 2012. A nonsense mutation in PDE6H causes autosomal-recessive incomplete achromatopsia. Am J Hum Genet 91, 527-532. Thiadens, A.A., Phan, T.M., Zekveld-Vroon, R.C., Leroy, B.P., van den Born, L.I., Hoyng, C.B., Klaver, C.C., Roosing, S., Pott, J.W., van Schooneveld, M.J., van Moll-Ramirez, N., van Genderen, M.M., Boon, C.J., den Hollander, A.I., Bergen, A.A., De Baere, E., Cremers, F.P., Lotery, A.J., 2012. Clinical course, genetic etiology, and visual outcome in cone and cone-rod dystrophy. Ophthalmology 119, 819-826. Coene, K.L., Mans, D.A., Boldt, K., Gloeckner, C.J., van Reeuwijk, J., Bolat, E., Roosing, S., Letteboer, S.J., Peters, T.A., Cremers, F.P., Ueffing, M., Roepman, R., 2011. The ciliopathyassociated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase. Hum Mol Genet 20, 3592-3605. Azam, M., Collin, R.W., Shah, S.T.A., Shah, A.A., Khan, M.I., Hussain, A., Sadeque, A., Strom, T.M., Thiadens, A.A., Roosing, S., den Hollander, A.I., Cremers, F.P., Qamar, R., 2010. Novel CNGA3 and CNGB3 mutations in two Pakistani families with achromatopsia. Mol Vis 16, 774-781.

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Thiadens, A.A., Roosing, S., Collin, R.W., van Moll-Ramirez, N., van Lith-Verhoeven, J.J., van Schooneveld, M.J., den Hollander, A.I., van den Born, L.I., Hoyng, C.B., Cremers, F.P., Klaver, C.C., 2010. Comprehensive analysis of the achromatopsia genes CNGA3 and CNGB3 in progressive cone dystrophy. Ophthalmology 117, 825-830. Thiadens, A.A., Somervuo, V., van den Born, L.I., Roosing, S., van Schooneveld, M.J., Kuijpers, R.W., van Moll-Ramirez, N., Cremers, F.P., Hoyng, C.B., Klaver, C.C., 2010. Progressive loss of cones in achromatopsia: an imaging study using spectral-domain optical coherence tomography. Invest Ophthalmol Vis Sci 51, 5952-5957. Littink, K.W., Koenekoop, R.K., van den Born, L.I., Collin, R.W., Moruz, L., Veltman, J.A., Roosing, S., Zonneveld, M.N., Omar, A., Darvish, M., Lopez, I., Kroes, H.Y., van Genderen, M.M., Hoyng, C.B., Rohrschneider, K., van Schooneveld, M.J., Cremers, F.P., den Hollander, A.I., 2010. Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations. Invest Ophthalmol Vis Sci 51, 5943-5951. Littink, K.W., van Genderen, M.M., Collin, R.W.J., Roosing, S., de Brouwer, A.P., Riemslag, F.C., Venselaar, H., Thiadens, A.A.H.J., Hoyng, C.B., Rohrschneider, K., den Hollander, A.I., Cremers, F.P., van den Born, L.I., 2009. A novel homozygous nonsense mutation in CABP4 causes congenital cone-rod synaptic disorder. Invest Ophthalmol Vis Sci 50, 2344-2350. Thiadens, A.A., den Hollander, A.I., Roosing, S., Nabuurs, S.B., Zekveld-Vroon, R.C., Collin, R.W., De Baere E., Koenekoop, R.K., van Schooneveld, M.J., Strom, T.M., van Lith-Verhoeven, J.J., Lotery, A.J., van Moll-Ramirez, N., Leroy, B.P., van den Born, L.I., Hoyng, C.B., Cremers, F.P., Klaver, C.C., 2009. Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders. Am J Hum Genet 85, 240-247. Thiadens, A.A., Slingerland, N.W., Roosing, S., van Schooneveld, M.J., van Lith-Verhoeven, J.J., van Moll-Ramirez, N., van den Born, L.I., Hoyng, C.B., Cremers, F.P., Klaver, C.C., 2009. Genetic etiology and clinical consequences of complete and incomplete achromatopsia. Ophthalmology 116, 1984-1989 e1981. Frank, V., den Hollander, A.I., Bruchle, N.O., Zonneveld, M.N., Nurnberg, G., Becker, C., Du Bois, G., Kendziorra, H., Roosing, S., Senderek, J., Nurnberg, P., Cremers, F.P.M., Zerres, K., Bergmann, C., 2008. Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome. Hum Mutat 29, 45-52. Rodenburg, W., Keijer, J., Kramer, E., Roosing, S., Vink, C., Katan, M.B., van der Meer, R., Bovee-Oudenhoven, I.M., 2007. Salmonella induces prominent gene expression in the rat colon. BMC Microbiol 7, 84. * Authors contributed equally # Last authors contributed equally

273 List of publications

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Acknowledgements

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Geen doel is te ver, als je plezier hebt in wat je doet. Ik heb m’n doel gehaald, en met plezier!!! Dit had ik nooit kunnen doen zonder de steun van ontzettend veel mensen. Ik wil dan ook van deze gelegenheid gebruiken maken om al deze mensen in het zonnetje te zetten. Frans en Anneke, bedankt dat jullie mij destijds als research analist hebben aangenomen. Zonder twijfel heb ik de juiste keuze gemaakt. Wat voelde ik me snel op mijn plek binnen de groep. Daarnaast ook voor het gestelde vertrouwen in mij om de uitdaging aan te gaan om te gaan promoveren, een keuze waar ik zonder spijt op terugkijk. Beste Anneke, bedankt voor je begeleiding in de afgelopen jaren. Het eerste jaar vanuit Amerika en werkbesprekingen via Skype op een diaprojector waren een hele belevenis. Jouw enorm wetenschappelijk talent zorgt voor een overvolle agenda, toch konden we steeds voldoende tijd vinden om onze besprekingen te houden. Bedankt voor al je waardevolle input en zorgvuldige aanpassingen aan mijn manuscripten en altijd eerlijke en oprechte mening. Beste Frans, wat ben ik jou veel dank verschuldigd. Heel wat uurtjes hebben we samen doorgebracht op jouw kamer om samen de resultaten te analyseren, presentaties te perfectioneren, en manuscripten te bespreken. Jij hebt mijn onzekerheid over promoveren als analist langzaam weten weg te poetsen en mij gebracht tot waar ik nu ben. Teamspirit is één van jouw vaandels, welke samen met je begeleiding, kennis, precisie en enthousiasme voor dit werk enorm inspirerend is en maakt werken met jou echt een plezier. Beste Caroline en Carel, naast Frans en Anneke had ik dit onderzoek ook niet kunnen uitvoeren zonder jullie input van het klinische perspectief. De geweldige hoeveelheid patiëntengegevens en jullie aandeel bij het beantwoorden van mijn klinische vraagstukken waren zeer belangrijk. Beste Ingeborgh, ook al behoorde je niet tot mijn directe begeleiders, toch heb ik een groot deel van mijn klinische kennis aan jou te danken. Bedankt voor de vele telefonische leermomenten en voor je verbluffende oog voor detail bij het verwerken van manuscripten. Ik ben ervan overtuigd dat het CEP290 artikel ‘hoge ogen’ gaat gooien! Hans en Lucy, bedankt dat jullie zoveel extra vervelende onderzoeken wilden ondergaan voor ons artikel. Het is voor mij erg bijzonder g eweest om de mensen achter de DNA-monsters te leren kennen. Lieve Marijke, vanaf de eerste dag hebben we samen gewerkt aan het kegelproject. Naast alle meetings en besprekingen hebben we ontelbare pauzes gehad en persoonlijke gesprekken gevoerd. Ik ben je dan ook enorm dankbaar voor de geweldige resultaten en bevindingen die we samen hebben gedaan en deze mooie tijd samen op het lab. Alberta, samen met Caroline heb je een enorme hoeveelheid aan patiëntengegevens en DNA verzameld voor het project. Voor mij was het als beginnend analist heel spannend om met jou te mogen samenwerken en wat was het ontzettend leuk om met jou al die ‘helse karweien’ van experimenten te doen. Inmiddels al een hele tijd ver weg in Tanzania en gelukkig nog steeds in contact over het project maar ook voor de gezelligheid. Karin, wat is de tijd voorbij gevlogen sinds ik jouw paranimf mocht zijn! Bedankt voor alle geweldige reizen, ervaringen, epjespost, telefoongesprekken en gezelligheid die we hebben mogen delen. Geen ander zoals jij kan zich inleven in ‘de andere kant’, hou dat vast!! Rob, bedankt voor al die jaren. Jij bent een grote steun en altijd mijn grote vraagbaak. Bedankt ook voor alle lol en geweldige feestjes die we hebben meegemaakt.

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Sylvia, al sinds ik bij de afdeling werk ben je mijn ‘hulplijn’ voor allerlei ‘kwesties’. Bedankt voor al je adviezen en enorme gezelligheid deze jaren. Vanaf nu, hoop ik, op alleen nog maar gezelligheid!! A big thank you for all (ex-)members of the group of Blindness Genetics, Blindness Therapy, and Multifactorial Eye Diseases in the first lab. Kostas, Alejo, Bjorn, Marijke, Ellen, Frederieke, Arjen, Lonneke, Galuh, Alex, Rob, Anna, Imran, Maleeha, Julio, Laura L.d.M., Maleeha Maria, Danielle, Lonneke H., Silvia, Dyah, Shazia, Mahesh, Constantin, Maartje, Humaira, Eiko, Krysta. Please remain the dynamic and great group as you are. Riccardo, I am sure you’ll do great in your PhD project, it was a pleasure supervising you during your first months! Sylvia, Lisette, Ka Man, Ideke, Margo, Stef, Machteld, Erwin, Jeroen, Dorus, Ralph, Erik, Hannie en Theo heel erg bedankt voor het beantwoorden van mijn vele eiwit-vragen, helpen bij mijn RAB28-experimenten en voor alle onmisbare gezelligheid op het lab. Anne Kusters, als stagiaire heb je in een korte tijd een start met het ABCA4 project kunnen maken. Merve Mütlu continued this project during her summer internship. Although this procedure seemed not robust enough, it led to the new PhD project on the topic of ABCA4. Thank you girls for your help and enthusiasm with this project. I am really grateful for all the work-related or holiday trips I have been able to make in the last years with colleagues, to colleagues, and with colleagues to colleagues or colleagues family. Visiting Riga with Karin (iets met knoflook...), going to the European cup final the NetherlandsRussia in Basel with Karin & Daphne, a great visit with Karin and Alejo to Madrid with Almudena & Jorge as our host (Thanks for having us! Alejo, did you already improve your ‘bowling’ skills?), visit the Ardennen with Marloes, Petra, Konny and Eveline (Mama Mia Karaoke? Nooit meer haha!). Then there was the unforgettable experience (AVENTURAAA) of visiting Peru and Alejo’s family (Thanks for having us!) with Karin, Peer, Marisol, Christian and the amazing tour guide Alejo. The great girls weekend in Drenthe with Evelyn, Laura T., Minh, Anna C. Celia, Bonnie, Merel, and Monique. Furthermore, I really enjoyed the amazing trips with Galuh, Bjorn, Laura S. and Alex to touristic Barcelona (BCN 1.0) and the non-touristic Barcelona (BCN 2.0). Thanks Alex and Laura S. for being our guide and muchas gracias Nati for having us! Hope to see you soon for NY 1.0!! Dear Kentar, Hennie and the kids, I feel privileged to have been able to visit you in Semarang for the Genetics teaching. Adith, thanks for being our perfect tour guide (with or without cheesy music). Almudena, Maleeha, Kentar, Esin, Saima (and family), Marta de Castro MirOOOO, thank you for your visit’s to the lab, to all of you I treasure great memories. Carmel, James, and Layal from Leeds, let’s put it this way... my ARVO experiences would definitely have been different without you. De afdeling zou niet draaien zonder de geweldige dames van het secretariaat, Ineke, Miranda, Doménique en Brenda en natuurlijk projectmanager Dennis. Zij verdienen dan ook een grote ‘bedankt!’ voor het beantwoorden van mijn vele vragen (hállóóóó). Natuurlijk Nick voor de kwaliteit (-analyse van de sfeer en het bier). Bart, Berna en Liz van het secretariaat van de afdeling Oogheelkunde, en Riet Bernaerts van het ErasmusMC. Bedankt! Het is echt onmogelijk om iedereen apart te bedanken, en in 7 jaar leer je zoveel mensen kennen, daarom ook een ontzettende thank-you aan collega’s van de rare genomics disorder-, doofheid-, neuro-, multifactorieel-, onco-, en vliegen-groep, de bioinformatici, en de mensen van diagnostiek met wie ik heb samengewerkt.

277 Acknowledgements

Dear Susanne Kohl, thank you for the great advises, small talk and lunches at conferences. You are truly the nicest ‘competitor’ and deserve the best! Big hug! Ronald, vooral de laatste maanden heb ik heel veel aan onze gesprekken gehad en mede dankzij jou heb ik nu mijn post-doc bij Prof. Joe Gleeson. Bedankt hiervoor en ik hoop en weet zeker dat we nog veel zullen gaan samenwerken! Lies, Kornelia, en Dorien, bedankt voor alle hulp met exomes en input tijdens besprekingen vanuit diagnostisch oogpunt. Dearest P2-people, I wouldn’t know how I would have finished my thesis without all the necessary coffee, lunch, fruit or no-reason breaks and sharing good and bad moments with Bonnie, Minh, Evelyn, Celia, Anna C., Laura T., Galuh, Anna S., Merel, Korinna, Alex, Tom, Zafar and Human. Evelyn, m’n PhD-retreat-roomie!! Bedankt voor alle gezellige etentjes en drankjes die we samen hebben gehad en dat ik ook nog eens jouw ceremoniemeesteres mocht zijn!! Marloes, wat heb ik genoten van al onze hap en/of stapavondjes. Altijd heerlijk om met je bij te kletsen! Minh, Minnetjeeee, P. Sherman 42 Wallaby Way Sidney, I’m sure you’ll get there one day! Thanks for being my sweet P2-neighbour;) And let me know if you are interested in American peanut butter! ;) The Spanish guapa’s Anna C. and Celia, your birthday’s are legendary! Bonnie, thanks voor (niet-)serieuze gesprekken en het co-ceremononiemeesteressen! Jonas, Galuh en Alex, het was heel leuk om jullie Nederlandse ‘les’ te geven. Jullie krijgen de taal al heel goed onder de knie! Peer, Simon & Martijn, jullie ook bedankt voor al de gezellige events (al dan niet met een biertje in de hand....). Bedankt! Saskia, Mieke, Willie, en Marlie, voor het helpen bij al mijn celkweek/patiënt/DNA gerelateerd kwesties en Ramon en Nathalie voor de klinische analyse van patiënten. Remco, Simon en Anna S. bij het helpen van mijn ICT-issues. Katie, voor altijd schone werkruimte en een fris bureau en natuurlijk de gezellige kletspraat. Leden van de sequentiefaciliteit, voor het runnen van ontelbare aantallen sequenties. Betsie, voor het aanleveren van schoon glaswerk, MQ en tips. DUC2012 (Tom, Michael, Simone, Zafar & Wesley) voor het organiseren van een zeer geslaagd Romeins dagje uit voor de afdeling. Blindness genetics dagje uit (o.a. skieën, fietssteppen in Nijmegen), 4-day Marches spontaneous Wednesday (which is now more ‘a rule’), Molgen day outs, zero-euro day out, 40-jarig jubileum (wat een week en WAT een feest!), kerstborrels, of which the majority ended (or let’s say: continued) in the Fuik or Malle Babbe, the countless number of Friday after work drinks in the Aesculaaf followed by pizza at Mr. Jacks or just all the spontaneous sushi dinners at Yukimi. Also I have shared many Queens days, Ascension day and many orange soccer events on the ‘Kingssquare’, great memories!! Alex & Galuh, as Burbujitos you will be my paranimphs and standing next to me on this important day for the simple reason that you were always ‘standing’ next to me during the good and bad parts during my PhD-project. Galuhtje, you are the sweetest, always helpful and always give your true opinion (with for sure the most gravity issues...).Thanks for being you! Alex, thanks for being a great friend. The hours and hours of talking on (non)-scientific matters meant a lot to me! Bjorn, the adopted Burbujito, thanks for the unconditional advises and numerous occasions of great fun througout the years! Burbujitos, thanks for all the nasi goreng’s, Alex-pasta’s, Panadero-apple pies, rummikub-evenings, movie days and ‘regular’ evenings, we had great fun!

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Lieve lieve dinnetjes, jullie zijn de familie die je zelf uitkiest, bedankt dat jullie er altijd voor me zijn en altijd goede adviezen hebben. Daarnaast natuurlijk voor alle ge-wel-di-ge memories aan al onze momenten samen! De afstand tussen ons viertjes was geen probleem en ik ben ervan overtuigd dat dit met de nieuwe afstand niet anders zal zijn!! Lieve Daphne, al dikke vriendinnen sinds de studie in Eindhoven en sindsdien hebben we samen al heel wat van de wereld gezien en natuurlijk heel wat lief en leed gedeeld. Lieve Martine, samen in de klas gezeten en elkaar pas echt leren kennen in ‘ons Nijmegen’. Bedankt voor de vele avondjes chillen en kletsen!! Lieve Claudia, dikke vriendinnen sinds je in hetzelfde huis kwam wonen (jouw bank of mijn bank?). Love you, girls!!! Lieve Esther M., ook zonder jou zou mijn tijd in Nijmegen een stuk minder leuk zijn geweest. Bedankt voor alle zorgeloze avondjes en wat ben ik blij dat je op je plek bent in Den Bosch. Lieve lieve Oma, het is zover, ik ben klaar met ‘studeren’. Bedankt voor alle lieve steun en natuurlijk gewoon bedankt omdat je mijn lieve oma bent!! Lex, Rob & Jan en de Tillers, bedankt voor de interesse voor mijn onderzoek. Lieve Esther en Ruud, bedankt voor alle geweldige steun in deze jaren, op werkgebied, maar natuurlijk tot ver daarbuiten. Niets kan op tegen gezelligheid met jullie. Es, hier is het resultaat van ook jouw werk, bedankt voor al je avonduurtjes wat geresulteerd heeft in deze super mooie lay-out. Lieve papa en mama, zonder jullie zou ik nu niet zijn waar ik nu ben. Bedankt dat ik alle kansen in m’n leven heb kunnen grijpen met alle steun van jullie. Zonder jullie zou dit boekje er nooit gekomen zijn. Hou van jullie! Tot ziens in New York!! Susanne

Nikky, miauw. Flos†, Remi & Rox, woef!

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Name PhD student: Susanne Roosing Department: Human Genetics Research School: Radboud Institute for Molecular Life Sciences

TRAINING ACTIVITIES Courses & Workshops Graduate course 8T03 Cell Growth and Differentiation, MMD course Poster presentation workshop Academic writing Seminars & lectures NCMLS Seminar series NCMLS Technical forums NCMLS Mini-symposia PhD-defences/Orations (Inter)national Symposia & congresses Medical genetics course, Bologna, Italy# Course in eye genetics, Bologna, Italy# Retina Nederland patient day* NCMLS symposium New Frontiers PhD-retreat (2010#, 2011#, 2012#, 2013*), Wageningen, The Netherlands Achromatopsia patient day* ARVO-Ned symposium# ARVO-conference (2011, Miami#; 2012,Miami#; 2013,Seattle*)USA ARVO pre-meeting (2012, Miami; 2013, Seattle)USA Conference: RabGTPases in health and disease, Cork, Ireland Dutch Opthalmology PhD-students meeting (2012, 2013*), Nijmegen, The Netherlands IGMD science day*, Nijmegen, The Netherlands Vision camp, Leibertingen, Germany* Asia-ARVO conference, New Delhi, India*

Other -

Literature discussion series*** Theme discussion series*** NCMLS committee Training and supervision Review of scientific paper

TEACHING ACTIVITIES Lecturing Teaching Genetics in retinal dystrophies, Semarang, Indonesia (15*) f) Other Orientation program PhD-students Supervising student internship projects (8 months, 8 weeks) Teaching workgroup students: From patient to genetic defect TOTAL e)

PhD period: 01-01-2010 â&#x20AC;&#x201C; 31-01-2014 Promotor: Prof. Dr. F.P.M. Cremers, Prof. Dr. C.C.W. Klaver, Prof. Dr. C.B. Hoyng Co-promotor: Dr. A.I. den Hollander Year(s) ECTS

2010 2010 2011 2011

2 5.5 0.25 3

2010-2013 2010-2013 2013 2010-2013

1.2 1.6 0.5 0.8

2010 2010 2010 2010-2012 2010-2013

1.5 1.5 0.5 3 5.25

2011 2011 2011-2013

0.75 0.75 4.75

2012-2013 2012

0.5 0.75

2012-2013

1.5

2013 2013 2013

0.75 1.25 1.5

2010-2013 2010-2013 2012-2013 2013

0.2

2013

7.5

2011 2011-2012 2011-2013

0.5 3.3 3 53.6

# = oral presentation; * = poster presentation

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About the author

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Susanne Roosing was born on July 3rd 1984, in Geldrop, the Netherlands. After finishing high school in 2002, she started her studies in Biology and Medical Laboratory Research at the Fontys Hogeschool in Eindhoven, the Netherlands. During this period, she performed a 5-month internship at the Department of Pulmonology at the University of Maastricht, the Netherlands, and a second and final internship of 10 months at the Department Food Safety at the Rikilt Institute in Wageningen, the Netherlands, where she also worked for 3 months after finishing her degree studies in 2006. In February 2007, she initiated her work as a research technician in the Blindness Genetics group headed by Prof. Dr. Frans Cremers, at the Radboud University Medical Center in Nijmegen, the Netherlands. She worked under the supervision of Dr. Anneke den Hollander on the identification of novel genes as a cause of cone dystrophy. In 2010, Susanne Roosing received the opportunity to start as a PhD student in the same group, working on the identification of novel causes for cone and cone-rod dystrophy with drs. Anneke den Hollander, Frans Cremers, Caroline Klaver and Carel Hoyng as (co)supervisors. In 2013, she was awarded with the NCMLS travel grant to attend the annual meeting of the Association for Research in Vision and Ophthalmology (Seattle, USA). In the same year, she was an invited speaker at Vision Camp in Leibertingen, Germany, and at ARVO-ASIA in New Delhi, India. After finishing her thesis manuscript at the end of January 2014, she became a postdoctoral fellow at the Department of Neuroscience, under supervision of Prof. Dr. Joe Gleeson at the University of California, San Diego, USA, and the Rockefeller University in New York, USA.

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