Phenotype | Supplement to Issue 24 I Trinity Term 2016

PHENOTYPE

Genetics Supplement to Issue 24 | Trinity Term 2016

www.phenotype.org.uk

Genetics and the control of cell division in the worm C. elegans Sophie Gilbert from Prof. Woolard’s group

Genetics Complexity of Mendelian diseases Cell division and ageing Meiotic recombination

OXFORD UNIVERSITY BIOCHEMICAL SOCIETY

Supplement to Issue 24 | Trinity 2016

CONTENTS genetics 3

C. elegans: a valuable genetic model Sophie Gilbert

4

The death of genetic determinism: a post-mendelian critique Sara Althari

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Mixing meiosis in mice and men Dr George Busby

EDITORIAL TEAM EDITOR-IN-CHIEF Rebecca Hancock Cardiovascular Medicinal Chemistry FEATURES EDITORS Heather Booth DPAG Dr Alexander Feuerborn Pathology Michael Song DPAG REGULARS EDITORS Dr Vasilika Economopoulos Oncology Chloe Ming-Han Tsai Paediatrics Lauren Chessum MRC Harwell SCIENCE & SOCIETY EDITORS Mariangela Panniello Pathology Iona Easthope Alumna SUPPLEMENT EDITORS Dr Vito Katis Pathology Ines Barreiros DPAG DESIGN & PRODUCTION Rebecca Hancock Cardiovascular Medicinal Chemistry Dr Vito Katis Pathology SPONSORSHIP Caitlin Clunie-O’Connor Cardiovascular Medicinal Chemistry

Cover Image: Dividing seam cells in C. elegans. Fluorescently tagged proteins were used to investigate the timing and plane of cell division in seam cells, as well as the choice between symmetric and asymmetric cell division. Markers for the cell membrane (pleckstrin homology domain-GFP) and DNA (histone H2B-GFP) were used to monitor seam cell divisions. See article by Sophie Gilbert (p3) for more details.

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COPY EDITORS Natalie Ng Dr Burcu Anil Kirmizitas Dr Fiona McMurray Dr Matt Kelly Antonia Langfelder Dr Tom Vizard Oriol Pavon Arocas Heather Booth Dr Rebecca Perrett Kevin Ray Beatrice Tyrrell Dr Monika Krecsmarik Tonia Thomas Dr Bryony Graham Dr Felicity Alcock Dr Jennifer Badger Dr Suvi Honkanen Angela Hoslin Manisha Jalan Terence Tang Beth Watts Caroline Woffindale Anne Tuberfield Lauren Chessum Amelie Joffrin Dr Cristina Marculescu Ashleigh Wilcox Emma Bickford Dr Laura McCulloch Jessica Hardy Gaia Donati Matthew Cooper

C. elegans: a valuable genetic model

I

n the 60’s, Sydney Brenner selected Caenorhabditis elegans, a free-living microscopic nematode worm, as a tool to study behaviour. He theorised that developing a map of the whole neuronal network of this simple animal would enable the discovery of the genes that control its behaviour (1). Although his goal of mapping the entire neuronal connectome of C. elegans would only be achieved 30 years later, the early work by Brenner and his colleagues propelled this organism to centre stage as a classic genetic model. In 2002, C. elegans was the first multicellular organism to have its genome completely sequenced, cementing its position at the forefront of genetic research.

by Sophie Gilbert

Professor Alison Woollard started her lab in Oxford focusing on the development of the C. elegans male tail. This sex-specific structure is the result of a complex series of divisions involving stemlike cells (Figure 1). Control of stem cell divisions has particular relevance to human disease due to their prevalent misregulation in cancer: too many divisions result in tumour formation, yet too few divisions result in a deficiency of specialised cells.

Gratifyingly, Alison found that mab-2, a gene which when deleted results in a loss of cells in the male tail, encodes the sole worm homologue of the mammalian Runx genes (2). Runx genes are deregulated in Acute Myeloid Leukaemia (AML) (3), a human genetic disease resulting in the overproliferation of blood stem cells. Thus, the study of Runx function in C. elegans may help us better understand the causes of this malignant disorder.

A genome-wide RNA interference (RNAi) screen, involving the systematic knockdown of every gene in the C. elegans genome, has since revealed many new players in the control of these stemlike cell divisions. Crucially, genetic screens can be performed without prior knowledge of likely candidate genes and offer the potential to discover new, unpredicted interactions. From this screen, it was found that knockdown of a gene called ceh-20 causes the population of stem-like cells in the head of the worm to undergo uncontrolled expansion (hyperplasia). Since expression of Runx is known to be required to promote proliferation of the stem-like cells (as Runx knockdown results in too few cells), it was speculated that knockdown of ceh-20 may cause an over-expression of Runx. Subsequent experiments confirmed this hypothesis, revealing CEH-20 as an upstream regulator of Runx that acts controlling its transcription, thus regulating the balance between proliferation and differentiation of stem-like cells in C. elegans (4). These studies demonstrate the power of genetics in revealing novel signaling networks between molecules, cells, tissues and organisms.

Recently, Alison’s research has branched into the genetics of ageing. Of all the recent discoveries in this rapidly developing field, perhaps the most astounding is that lifespan can be determined by only a handful of genes: mutations in a single gene are capable of causing a marked reduction (or increase) in lifespan. One such C. elegans gene is wrn-1, named after the human Werner Syndrome, which affects one in 100,000 people. People suffering from this disease experience a premature onset of old age, and often die in their late 40s.

Figure 1: Together with Lynne Cox of the Department of Biochemistry, Alison and her colleagues have shown Top: Stem-like cells in C. elegans, visualised that the lifespan of worms with a deletion in wrn-1 with a GFP reporter. is significantly compromised. Furthermore, a screen Bottom: During for genes that interact with wrn-1 has yielded a very post-embryonic surprising discovery: knocking out a second gene, development, the p53, which would normally have a detrimental impact worm undergoes upon the lifespan of the animal, curiously results in a four larval moults, significant increase in the lifespan of double mutant L1 - L4, during which the stem-like cells animals. The bringing together of two detrimental undergo a series mutations to produce a net beneficial result has been of divisions. Genes termed ‘synthetic viability’ and represents a novel controlling these concept in the field of ageing research.

References 1. Brenner S (1974) The genetics of Caenorhabditis elegans.

Genetics 77(1):71-94.

2. Nimmo R, et al. (2005) mab-2 encodes RNT-1, a C. elegans Runx homologue essential for controlling cell proliferation in a stem cell-like developmental lineage. Development 132(22):5043-54.

3. Erickson P, et al. (1992) Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt. Blood 80(7):1825-31.

divisions, whether they are asymmetric, providing cells to growing tissues (yellow squares), or symmetric (at the start of L2) to self-proliferate, have conserved roles in mammalian stem cell divisions.

4. Hughes S, et al. (2013) CEH-20/Pbx and UNC62/Meis function upstream of rnt-1/Runx to regulate asymmetric divisions of the C. elegans stem-like seam cells. Biol Open 2(7):718-27.

Sophie Gilbert is a DPhil student in Prof Alison Woollard’s group in the Department of Biochemistry.

Trinity 2016 | Phenotype Gen. | 3

The death of genetic determinism:

a post-Mendelian critique

by Sara Althari

S

ince the completion of the Human Genome Project, and with the advent of next generation sequencing technologies, the genetics and genomics communities have made tremendous accomplishments in both deciphering the molecular code and using this information to discover novel biology and therapeutic strategies. Indeed, the genomics revolution is paving the way towards a reality in which concepts such as individualised medicine and genome editing are becoming increasingly more technically, analytically, and financially tractable.

Herein, I discuss some of these paradigm-shifting findings, particularly in the context of monogenic diabetes and naturally occurring human knockouts.

Figure 1.

Juxtaposition of classic and contemporary views of variant-phenotype relationships. Solid arrows represent the discrete pre-genomic view of the division between healthy (benign), common complex (common polygenic disease risk), and Mendelian (rare monogenic disorder) disease states. Dashed arrows represent the spectrum-based reality of variant manifestation, which takes into account instances such as incomplete penetrance of bona fide pathogenic alleles in healthy individuals and monogenic disorders falsely attributed to a neutral allele in a known disease risk gene.

Determining the allelic status at a given genetic locus is no longer considered a formidable task. Instead, the main challenge in this era of big data is making reliable and accurate interpretations of the phenotypic consequences and clinical significance of identified variants. This so-called ‘interpretive’ gap in our knowledge was considered closed at least in the context of Mendelian diseases, or single-gene disorders characterised by the inheritance of rare, highly penetrant variants that produce poorly functioning protein.

However, the ever-growing amount of data from resequencing large case-control cohorts unselected for phenotypic extremes, isolated founder populations, and populations enriched for autozygosity, whereby individuals carry identical alleles by virtue of consanguinity, continue to challenge the notion that Mendelian disease variants are simpler to interpret (Figure 1). Such endeavours have not only identified presumed monogenic disease-causing alleles in healthy individuals, but also led to the discovery of biallelic loss-of-function (LOF) events (i.e. two presumably dysfunctional alleles of a gene present in the genome) that confer protection against certain diseases (Figure 1).

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Until recently, assignments of variant pathogenicity and estimations of allelic penetrance have been based predominantly on studying individuals who represent the polar ends of a phenotypic distribution. Whilst such study designs have been absolutely indispensable to the identification of disease-driving alleles, they have led to exaggerated effect-size estimations by failing to capture the genomic complexity across the phenotypic distribution. Avoiding pre-selection on the basis of extreme phenotypes and following up genetic data with thorough experimental and statistical validation allows for comprehensive and contextual understandings of variant behaviour that help to mitigate the upward bias (1).

Following these guidelines, Flannick and colleagues explored the spectrum of lowfrequency variants in the genes implicated in a rare monogenic form of diabetes known as Maturity-Onset Diabetes of the Young (MODY), characterised by Mendelian autosomal dominant inheritance (2). Using rigorously phenotyped, randomly ascertained population-based cohorts, unselected on the basis of classic MODY criteria, and targeted sequencing of known MODY genes (GCK, HNF1A, HNF4A, PDX1, INS, NEUROD1, HNF1B), the investigators found MODY-causal variants, previously reported as pathogenic with evidence of familial co-segregation and dysfunction, that were incompletely penetrant

in carriers that had normal blood glucose levels (i.e., normoglycemic) (2).

A study conducted by the SIGMA consortium revealed the association of a low frequency missense variant in HNF1A (p.E508K) – a known MODY gene and a master transcriptional regulator in the liver and pancreatic beta cells – with a five-fold increase in type 2 diabetes (T2D) risk among carriers (OR, 5.48; 95% CI, 2.83-10.61; p= 4.4 x 107) (3). The variant was identified as part of a systematic whole exome screen of a large Mexican and US Latino T2D case-control cohort and taken through a comprehensive functional characterisation pipeline (3). The unbiased interrogation led to the discovery of an incompletely penetrant, population-specific, moderately damaging allele in a gene typically linked to rare monogenic disease and yet manifested in a common complex disease risk elevation (T2D).

Equally valuable have been studies of populations that are genetically homogenous by virtue of genetic drift and/or enrichment for autozygosity. In-depth assessment of the genetic architecture of isolated/inbred populations has led to the discovery of rare human knockouts, i.e., individuals who carry homozygous LOF alleles of genes often thought to be indispensable, who exhibit normal or even disease-protective phenotypes. Some of the poster children of this work include LPA, PRMD9 and PCSK9. LPA (lipoprotein A gene) was identified in a biallelic LOF state in a Finnish cohort and found to be associated with a reduced risk of cardiovascular disease (4). In a very recent study, Narasimhan and colleagues sequenced the exomes of over 3,000 British-Pakistani individuals with a high rate of paternal relatedness (5). This effort revealed homozygous predicted LOF expression of nearly 800 genes. Interestingly, the redundant role of PRMD9 (a gene which influences meiotic recombination) in humans became apparent when phased whole-genome sequencing was performed on a healthy PRMD9 knockout mother from this cohort, and her child, which revealed localisation of double strand break sites away from typically dense PRMD9-dependant recombination hot spots in controls (5). Finally, perhaps the most celebrated human knockout story is that of the PCSK9 gene (responsible for recycling LDL receptors): individuals with biallelic LOF of PCSK9 showed markedly reduced LDL cholesterol levels (6). The development of a novel class of cholesterol

lowering agents which mimic PCSK9 LOF shortly followed this discovery (7).

Indeed, the efforts described in this article have challenged longstanding notions of heritability and allele manifestation. In so doing, they have posed new and interesting biological and translational questions, which the genetics community is now more than equipped to tackle. It is an exciting time to be a geneticist!

References 1. MacArthur DG, et al. (2014) Guidelines for investigating causality of sequence variants in human disease. Nature 508(7497):469-476.

2. Flannick J, et al. (2013) Assessing the phenotypic effects in the general population of rare variants in genes for a dominant Mendelian form of diabetes. Nat Genet 45(11):1380-1385.

3. Consortium STD, et al. (2014) Association of a lowfrequency variant in HNF1A with type 2 diabetes in a Latino population. J Am Med Assoc 311(22):2305-2314.

4. Lim ET, et al. (2014) Distribution and medical impact of loss-of-function variants in the Finnish founder population. PLoS Genet 10(7):e1004494.

5. Narasimhan VM, et al. (2016) Health and population effects of rare gene knockouts in adult humans with related parents. Science.

6. Lange LA, et al. (2014) Whole-exome sequencing identifies rare and low-frequency coding variants associated with LDL cholesterol. Am J Hum Genet 94(2):233-245.

7. Blom DJ, et al. (2014) A 52-week placebo-controlled trial of evolocumab in hyperlipidemia. N Engl J Med 370(19):1809-1819.

Sara Althari is a DPhil student in Prof Mark McCarthy’s group in the Wellcome Trust Centre for Human Genetics.

Trinity 2016 | Phenotype Gen. | 5

Mixing meiosis in mice and men G

enetic diversity induces the subtle phenotypic differences between individuals of the same species, which natural selection exploits to send valuable genes into the next generation.

Whilst many of us know that genetic variation results from mutation events, far fewer are aware of the additional, yet equally important role that meiosis has in setting up genetic diversity.

As a human, you have 23 pairs of chromosomes, having received one member of each pair from each of your parents. You started life as a fertilised egg, formed by the fusion of two gametes; an egg cell from your mother and a sperm cell from your father. Gametes therefore must have half the full genetic complement of normal cells so that the number of chromosomes does not double every time someone has a child. Meiosis is the process that occurs in all sexually reproducing eukaryotes (animals, plants, and fungi) to split germline cells into gametes (Figure 1).

During the first step of meiosis, each of the 23 pairs of parental chromosomes come together and duplicate, such that there are now two homologous copies of each chromosome.

After this copying step, the homologous chromosomes pair up and recombine. In the two chromosomes, both strands of DNA break at the same point, leading to an exchange of genetic material, which generates mixed chromosomes with parts from each parent (Figure 1). The 46 pairs then segregate into two separate cells (23 pairs in each), which then divide again, leading to the formation of four daughter cells (gametes) from the original cell, each with 23 chromosomes.

by Dr George Busby

generating novel diversity. Similar to mutation, an understanding of recombination is crucial to understand many aspects of genetics.

When we perform studies that aim to find associations between different genes and disease, we have a better chance of finding mutations underlying disease susceptibility by genotyping many thousands of people. This can now be done cheaply using assays that look at known human genetic variants, or genetic markers. If you have enough markers, which usually do not cause the disease themselves, there will potentially be one close enough to the disease-causing mutation that no recombination will have happened between the mutation and the markers. Markers can therefore tag causal disease mutations.

Other applications where the understanding of recombination is crucial include inferring natural selection and the understanding of human ancestry. If it has been selected, a beneficial stretch of DNA will resist being split up by recombination, leading to the observation of long stretches of similar genetic sequence in lots of people (1). When two populations from different parts of the world cometogether and reproduce, recombination can fuse together bits of chromosomes chromosomes with different Meiosis generates genetic novelty in two ways. histories. Human ancestry can therefore be First, recombination between homologous traced by understanding the distribution of chromosomes leads to mosaic daughter genetic material from different groups along chromosomes, which are made up of DNA chromosomes in a population (2). sequences that were originally on different Whilst recombination in male individuals can maternal and paternal chromosomes. On be observed in the lab by comparing sperm average, there is one recombination event cell DNA sequences with somatic cell DNA per chromosome per generation in humans. sequences (3), this approach is expensive and Second, during segregation, chromosomes laborious, and provides a become further mixed up, relatively small amount meaning that the final set Human ancestry of data. An alternative of chromosomes in gametes approach is to look at can be traced by originates from both genetic sequence data, either parents. Because genes that understanding from families or from large were formerly on different numbers of people, and the distribution parental chromosomes can use statistical inference to now be found on the same of genetic find where recombination chromosome, recombination is happening. The general material...along shuffles existing genes idea of these statistical into new combinations, chromosomes

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A germline cell, like most cells in your body, has two copies of each chromosome, one inherited from each parent.

The initial stage of meiosis involves the duplication of each chromosome to form homologous pairs.

breaks which initiate recombination (Figure 2).

Because the zinc-finger can only bind to specific sequences of DNA, called motifs, PRDM9 causes recombination to be punctate along chromosomes, giving rise to the recombination hotspots which had previously been observed (6).

So far, PRDM9 appears to be relevant only to Next, RECOMBINATION occurs, allowing homologous pairs to join up and swap DNA recombination in mammals. to form mosaic chromosomes.

The zinc-finger array is, however, highly polymorphic both within and between species, meaning that the actual sequence motif that it binds evolves, perhaps quite rapidly. Strangely, this suggests that hotspots move After two rounds of cell over time. To investigate division four gametes are produced, each the evolution of hotspots with a single copy of further, researchers in each chromosome.

Oxford looked at the role of PRDM9 in mice (7). The hybrid male offspring of two different mouse subspecies is infertile. The researchers first showed that this was because PRDM9 had evolved in separate directions in the different subspecies. The differential evolution of the approaches is to look at how mutations are PRDM9 zinc-finger domain distributed along chromosomes in many people caused the chromosomes to break in different within a population. Certain combinations places during genetically engineered new of mutations along chromosomes can only be parents from the two recombination, leading seen if, and only if, recombination has occurred to broken homologous chromosomes failing between particular mutations. Statistical to reconnect (Figure 2). They then genetically analyses of recombination needs a large amount engineered new parents from the two mouse of sequence data, which has only really been subspecies to have human PRDM9 alleles. possible over the last decade or so.

This resulted in viable offspring, confirming both that the reason for hybrid sterility is Recently, members of the Department of because of breakpoint mismatch in the unStatistics and Wellcome Trust Centre for engineered parents, and also that introducing Human Genetics have made key advances in a PRDM9 that binds human-like motifs leads our understanding of this important process. to symmetry in chromosome break points One fundamental discovery made by these and thus viable recombination. This finding researchers, in research published at the same raises the tantalizing possibility that PRDM9 time as work from Chicago and Montpellier and the Jackson Laboratory, is that the PRDM9 evolution has a key role in speciation, so that populations that have been isolated from protein plays a key role in recombination (4, 5). each other and whose PRDM9 alleles will PRDM9 has several functional domains, one have evolved in separate directions generate of which is a DNA-binding zinc-finger array infertile offspring. This work also highlights the which allows it to bind to particular sequences continuing role that Oxford researchers have of DNA bases, causing the double stranded

Figure 1.

An overview of meiosis.

Trinity 2016 | Phenotype Gen. | 7

PRDM9 CONTROLS RECOMBINATION

PRDM9

PRDM9 has a zinc finger array that binds to a specific DNA sequence motif initiating a double stranded break. This allows recombination to occur symmetrically on the two homologous chromosomes, one blue, which originated from the individual's father, and one red from their mother.

zinc-finger array

paternal chromosome PRDM9 binding motif

maternal chromosome

Each chromosome has two strands, both of which are broken to form double stranded breaks (DSBs) at the same place on both parental chromosomes. + RECOMBINATION

SEQUENCE EVOLUTION CHANGES BINDING MOTIF

Because the DSBs occur in the same place, recombination can both join homologous chromosomes together and mix contiguous lengths of chromosome together, resulting in mosaic daughter chromosomes.

MOUSE SUB-SPECIES HYBRIDS ARE INFERTILE A Mus mus. domesticus x Mus mus. musculus hybrid has chromosomes with different ancestries. In this example, independent evolution of the PRDM9 binding motif in the M. m. domesticus lineage stops the M. m. musculus PRDM9 from recognising the binding site, causing a DSB to occur on only one chromosome, causing problems with meiosis and infertility.

MOUSE PRDM9

Mus musculus domesticus chromosome Mus musculus musculus chromosome

HUMANISED PRDM9 Mus musculus domesticus chromosome Mus musculus musculus chromosome

HUMANISING PRDM9 RESTORES FERTILITY When the Prdm9 gene is engineered to have a human zinc-finger array (humanised) the resulting PRDM9 molecule matches binding motifs on both chromosomes, which are different from mouse PRDM9 motifs, and which have not been evolving independently.

Symmetrical DSBs can form and recombination can now occur and fertility is restored.

Figure 2.

The role of PRDM9 in recombination.

played in understanding the important process of recombination.

of Meiotic Recombination Hotspots in Humans and Mice. Science 327(5967):836–840.

5. Parvanov ED, et al. (2010) PRDM9 controls activation of mammalian recombination hotspots. Science 327(5967):835.

References

1. Sabeti PC, et al. (2007) Genome-wide detection and characterization of positive selection in human populations. Nature 449(7164):913–918.

6. Jeffreys AJ, et al. (2005) Human recombination hot spots hidden in regions of strong marker association.

Nat Genet 37(6):601–606.

2. Hellenthal G, et al. (2014) A genetic atlas of human admixture history. Science 343(6172):747–751.

7. Davies B, et al. (2016) Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice. Nature 530(7589):171–176.

3. Jeffreys AJ & Rita N. (2002) Reciprocal crossover asymmetry and meiotic drive in a human recombination hot spot. Nat Genet 31(3):267–271.

4. Baudat F, et al. (2009) PRDM9 Is a Major Determinant

Dr George Busby is a postdoctoral researcher in Professor Dominic Kwiatkowski’s group at the Wellcome Trust Centre for Human Genetics.

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