Genetic modifiers in DMD
Kevin Flanigan, MD
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Outline The DMD gene and the dystrophin protein
New insights into BMD from “DMD” mutations •Altered protein translation initiation •Nonsense-associated splicing New insights into other genetic modifiers
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Dystrophinopathies: Clinical diagnosis Duchenne muscular dystrophy (DMD): Onset age 3-5 Pelvic girdle weakness Tight heel cords CK 50-100X normal Loss of ambulation by age 12 (range 7-12) Death by age 20 (historically)
Becker muscular dystrophy (BMD): Classic definition: loss of ambulation > age 12 Alternatively: “intermediate muscular dystrophy� for loss of ambulation ages 12 through15 BMD for loss of ambulation >age 15
Limb-girdle syndromes in adulthood Muscle aches (myalgias) Isolated cardiomyopathy
Dystrophin Mutations
Dystrophin gene (Xp21.1) is huge:
2.4 million nucleotides 79 exons and 8 promoters
Large deletions (≥ 1 exon) account for ~65% of DMD/BMD patients
~5% have duplications ~15% of boys have nonsense mutations Remainder are frameshifting insertions/deletions, splice site mutations, missense mutations
X Roberts, Genome Biology, 2001
Dystrophin mutations: Duchenne vs Becker
Size of deletion does not correlate well with phenotype Best correlation is whether the deletion is “in-frame” or “out-of-frame” In-frame deletions are more likely to result in translation of a protein with partial function (i.e.,
out-of-frame deletions are DMD ~90% of the time)
Roberts, Genome Biology, 2001
N
B
D
N Dystrophin (427 kd)
GAPDH
Antisense oligomer
van Deutekom et al, N Engl J Med. 2007 Dec 27;357(26):2677-86
One type of “modifier”: specific mutations at the DMD gene locus itself BMD from “DMD” (nonsense) mutations: Potential molecular mechanisms of phenotypic amelioration
Altered translational initiation (in exon 1 point mutations)
Nonsense mutations and exon skipping
p.Trp3X BMD Pedigrees 12/6/2006
12/6/2006
Family 1
Proband = 62 yr. old male, mild BMD, still ambulatory Utah
Family 2
DC 145
42970
43048
43406
43046
43047
43045
43044
43194
43279
43373
43339
43341
2
3
43293
43305
43111
Proband = 3.5 yr. old male, incidental to elevated CK levels, Younger brother CK = 5080 iu/L Michigan 43043
Concordant resequencing haplotypes across the DMD gene
43640
• Family 3: patient 43800; Kansas City,Missouri
– presented at age 7 years with bilateral calf pain and elevated serum CK level (8,000-24,000 iu/L). Muscle biopsy showed degenerating and regenerating fibers and reduced N-terminal, rod, and C-terminal dystrophin antibody staining.
• Family 4: patient 43676; also from Kansas City
– incidentally found at age 4 years to have an elevated serum CK level (4558 iu/L and 14,559 iu/L on separate occasions).
• Family 5: patient 43831; Milwaukee, Wisconsin
– presented in childhood with myalgias but no weakness; an elevated serum CK led to a muscle biopsy with decreased amino-terminal and rod domain dystrophin antibody staining.
• Family 6: patient 43889; Philadelphia, PA
– presented at age 13 years for evaluation of “hyperCKemia” found incidentally during an evaluation of short stature. His serum CK ranged from 5877 iu/L when active to 712 iu/L when more sedentary. Maternal grandfather (73 yrs) also Trp3X.
Evidence for functional rescue of 5’ mutations from a DMD founder allele mutation (p.Trp3X) • Associated with childhood hyperCKemia, with or without myoglobinuria • Compatible with no significant weakness at age 70 • No effect on reproductive fitness
DMD control
Mandra1 ex.66-79 (C-term)
Dys3 ex.10-12 (N-term)
Manex7B ex.7-8 (N-term)
Trp3X
WT
WT
Gurvich et al, 2009; Hum Mutat 30:633-40
60kDa
FS
W3X_AUG2&3
W3X_AUG3
W3X_AUG2
W3X
WT_AUG1
WT 74kDa
Alternate initiation from at least two exon 6 ATG sites contributes to the amelioration of the disease phenotype, and may be a general mechanism of rescue for early (exon 1-5) mutations. Hypothesized that initiation is driven by an internal ribosome entry site (IRES) in exon 5. Gurvich OL Human Mutation 2009
What is an IRES?
Normal conditions
Stress conditions
Internal Ribosome Entry Sites (IRES) are cis-acting RNA sequences able to mediate internal entry of the 40S ribosomal subunit under cell stress conditions
The highly functional N-terminal truncated isoform is lacking the first half (CH1) of the ABD1 domain
Absence of the first half of the dystrophin actin binding domain may be consistent with very mild disease
Ervasti, JM, J. Biol. Chem. 2003;278:13591-13594
BMD from “DMD” (nonsense) mutations: Potential molecular mechanisms of phenotypic amelioration
Altered translational initiation (in exon 1 point mutations)
Nonsense mutations and exon skipping
Reading Frame Rule in DMD/BMD
45%-55% of BMD patients have out-offrame mutations
Flanigan KM, et al. Hum Mutat. 2009;30(12):1657-1666.
Nonsense Mutations Do Not Always Predict DMD • Mutations predicted as nonsense mutations may instead affect exon splice regulatory signals1,2 – This results in exclusion of exons – The remaining mRNA may be in-frame 1. Disset A, et al. Hum Mol Genet. 2006;15(6);999-1013. 2. Flanigan KM, et al. Hum Mutat. 2011;32(3):299-308.
Disset A, et al. Hum Mol Genet. 2006;15(6);999-1013. ©Disset A, et al. 2006. Published by Oxford University Press. All rights reserved.
Distribution of BMD versus DMD nonsense mutations In-frame exons (39) shaded Out-of-frame (40) unshaded p.Trp3X
p = 0.004
DMD I/BMD
176 26
BMD with nonsense mutations occurs preferentially in some exons more than others i.e, Those with: The weakest aggregate splice site signals of all DMD exons Weaker competing 3’-splice site strengths than those exons that only have DMD mutations
Lower exon splice enhancer densities than those exons that only have DMD mutations
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Exon splice regulatory elements alterations occur within an exon definition context described in A. Disset et al., Human Molecular Genetics 2006
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Nonsense-induced exon skipping
c.4240C>T p.1414Gly>X
Conclusions (1) 1. Altered translational initiation may result in milder phenotypes in patients with nonsense mutations in exons 1-5. 2. Nonsense mutations in the central rod domain may result in BMD in a subset of in-frame exons
3. Genotype alone cannot always predict phenotype, and phenotypic predictions should be made with caution in an in-frame flanking context ………………..……………………………………………………………………………………………………………………………………..
Outline The DMD gene and the dystrophin protein
New insights into BMD from “DMD” mutations •Altered protein translation initiation •Nonsense-associated splicing New insights into other genetic modifiers
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• Cytokine involved in immune cell migration and survival • Implicated in fibrosis through the TGF-beta pathway
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Pegoraro et al, 2011
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Sgcg LGMD 2C g
mdx DMD
Mapping modifiers of muscular dystrophy E. McNally, Univ of Chicago
Membrane leak
Fibrosis
Evans Blue Dye (EBD)
Hydroxyproline (HOP)
Using mice to map genetic modifiers Parental strains:
SgcgD2 X (severe)
Sgcg129 (mild)
F1 generation:
SgcgD2/129
F2 generation
SgcgD2/129
•270 F2 animals were analyzed.
LOD score
Genomewide scan for modifiers of membrane leak Locus on Chromosome 7 =DMOD1
SNPs/Chromosomes
LOD score
Genomewide scan for modifiers of fibrosis Locus on Chromosome 7 = DMOD1
SNPs/Chromosomes
Membrane permeability and fibrosis are both modified by a region of chr 7
10.0
20.0
30.0
Mb
40.0
Chromosome 7
Membrane Permeability 30.88
19.88
Fibrosis 25.24
34.88
50.0
Latent TGFb binding protein 4 (LTBP4) is within a memberthe of modifier the Fibrillin superfamily region
An insertion/deletion in LTBP4 modifies muscular dystrophy exon12
mild severe
exon12
Heydemann et al. 2009
LTBPs regulate TGFb availability
proteolysis
SMAD-P
LTBP
Large latent complex
TGFb
TGFb receptor
Gene expression
LTBP +TGFb
SECRETING CELL
extracellular matrix
RECEIVING CELL
Variability of the LTBP4 proline rich region across species
Ceco, Heydemann
Single nucleotide polymorphisms in human LTBP4
Linkage disequilibrium across LTBP4
United Dystrophinopathy Project
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Does LTBP4 influence DMD phenotype? • Use loss of ambulation as a dichotomous outcome • To take an unbiased approach, we included all subjects who had a recorded loss of ambulation before age 20
• N = 254 subjects – 244 (96%) were catalogued as DMD – 8 = IMD (lost ambulation between age 12 and 15) – 2 = BMD (lost ambulation after age 15 but before age 20)
The LTBP4 “IAAM” haplotype predicts prolonged ambulation in DMD
Prolonged ambulation with LTBP4 “IAAM” is not explained by DMD mutation
Steroid use with LTBP4 “IAAM” predicts prolonged ambulation in DMD
Steroid use with LTBP4 “IAAM” predicts prolonged ambulation in DMD
Haplotype analysis of nonsynonymous LTBP4 ambulatory loss for DMD patients. variants associated with age of ambulatory loss
TABLE 1. Haplotype analysis of nonsynonymous LTBP4 variants associated with age of
Haplotype VTTT IAAM
b
All (n=254) a Global P = 0.002 c freq score p-val 0.53 -1.51 0.1 -4 0.31 3.43 6 x 10
Steroid treated (n=137) a Global P = 0.013 c freq score p-val 0.52 -1.04 0.3 0.32 2.92 0.004
Steroid naive (n=102) a Global P = 0.12 c freq score p-val 0.52 -1.12 0.3 0.29 1.91 0.06
a
The haplo.stats package was used to test for association between haplotypes and age of ambulatory loss as a quantitative trait using a recessive model. b These haplotypes consist of SNPS: rs2303729, rs1131620, rs10880 respectively. c p value from the 2, df=1, distribution of the haplotype-specific score.
Mean age of ambulatory loss for “IAAM” versus other haplotypes: • In steroid-treated patients: 12.5 ± 3.3 years vs. 10.7 ± 2.1 years • In steroid-naïve patients: 11.2 ± 2.7 vs. 9.8 ± 2.0 years
LTBP4 “IAAM” fibroblasts have reduced TGFb signaling
Conclusions (2) • The LTBP4 IAAM haplotype is associated with decreased TGFb signaling. • LTBP4 genotype effect is: • seen independent of the primary mutation (truncating or not) • seen in both glucocorticoid treated and naïve DMD subjects • the magnitude of effect suggests that glucocorticoid treated subjects homozygous for the protective allele have the most improved ambulation. • Stratification for LTBP4 genotype should be considered for clinical trials. ………………..……………………………………………………………………………………………………………………………………..
Genetic Modifiers of DMD 1R01NS085238-01A1; May 2014 • Update and analyze phenotypic data within the UDP cohort. – Flanigan Lab – Igor Dvorchik/Biostatistics
• Map modifier traits by high-density single nucleotide polymorphism (SNP) arrays. – Robert Weiss, PhD, University of Utah – Veronica Vieland, PhD, Nationwide Children’s Hospital
• Capture rare variants by exome sequencing of individuals with extreme outlier phenotypes. – Stan Nelson, MD, PhD, UCLA
• Validate newly identified putative genetic modifiers. – Other DMD cohorts: CINRG (E. Hoffman, Children’s National/C. McDonald, UC Davis); Dutch cohort (P. Spitali, Leiden) – Other mouse-identified candidates: Elizabeth McNally, Univ of Chicago
Acknowledgments
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Flanigan Lab – Adeline Vulin-Chaffiol, PhD – Nicolas Wein, PhD – Tabatha Simmons – Andrea Rutherford – Jack Kaminoh – Andrew Findlay NCH – Louise Rodino-Klapac, PhD – Kristin Heller, PhD – NCH Transgenic Core
• •
Veronica Vieland, PhD Igor Dvorchik, PhD
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Ohio State University – Paul Janssen, PhD – Dan Schoenberg, PhD – Baskar Bakthavachalu, PhD University of Utah – Michael Howard, PhD – Robert Weiss, PhD Steve Wilton, PhD Alessandra Ferlini, MD Maria Sofia Falzarano, PhD Francesca Gualandi, PhD Sonia Messina, MD Giuseppe Vita, MD Chiara Passarellit, PhD Cristina Szigyarto, PhD Jim Ervasti, PhD
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Acknowledgements University of Chicago Elizabeth McNally, MD, PhD Ermelinda Ceco Kay-Marie Lamar
Nationwide Children’s Hospital/OSU Jerry Mendell, MD Jack Kaminoh Wendy King Washington University in St. Louis Alan Pestronk, MD Julaine Florence Anne Connolly University of Iowa Katherine Mathews
University of Utah Children’s Hospital of Philadelphia Robert Weiss, PhD Richard Finkel Diane Dunn University of Minnesota Michael Howard John Day Kathy Swoboda University of California-Davis Eduard Gappmaier Craig McDonald Jay Maiti ………………..……………………………………………………………………………………………………………………………………..