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Investigating Genetic Mechanisms Underlying Clinical Heterogeneity in Neuromuscular Diseases Open Access

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Neuromuscular disorders show broad variation in clinical symptoms, and much of this variability can be explained by genetic and allelic heterogeneity, presence of genetic modifiers, and/or mosaicism. The goal of this dissertation research was to provide experimental evidence for different genetic mechanisms that result in clinical variability in neuromuscular diseases. In one study, we decipher genetic and allelic heterogeneity in a cohort of neuromuscular disease patients using next-generation sequencing. In a second study, we characterize a case of revertant mosaicism in a patient with an unusual phenotype of Duchenne muscular dystrophy. Finally, we examine the effect of genetic modifiers, i.e. genes or polymorphisms in other genes that modify disease. This last study screened candidate genetic modifiers within the transforming growth factor-β (TGFβ) pathway as a means of explaining variation in symptoms in a cohort of Duchenne muscular dystrophy (DMD) patients.In our first study, we tested a highly parallel targeted sequencing approach using a gene panel of 45 myopathy-related genes in a cohort of 95 patients with undiagnosed muscle diseases. We were able to detect likely pathogenic mutations in approximately 35% of the patients. We have highlighted two examples of novel mutations found in the COL12A1 gene by targeted sequencing and the DYNC1H1 gene using exome sequencing methods. We have also reported an unusual case of a reversion mutation leading to somatic mosaicism in a patient with DMD. To further study clinical variation, we analyzed the known genetic modifiers of DMD i.e. polymorphisms in SPP1 and LTBP4, that alter age at loss of ambulation in DMD patients. We sought to determine if these genetic modifiers also showed associations with timed function and quantitative strength outcome measures. We also screened polymorphisms in additional members of the transforming growth factor-β (TGFβ) pathway (IBSP and TGFBR2). The sensitivity of eight outcome measures (timed functions tests, strength) was measured by comparing 6 and 12 month changes in a natural history cohort (CINRG DNHS, n=40) to a prednisone treated clinical trial (n=29). Our study showed that timed function tests were sensitive outcome measures for younger DMD patients who have not yet lost ambulation. Genotype association studies with IBSP rs2616262, a polymorphism previously shown to be associated with circulating SPP1 protein levels in population-based studies, showed significant association with age at loss of ambulation (p=0.04). Our findings support the use of next-generation sequencing studies as the standard approach for the diagnosis of inherited myopathies. Discovery of novel mutations in the COL12A1 and DYNC1H1 genes led to an extension of the phenotypes associated with them. We reported a novel genetic mechanism i.e. revertant mosaicism seen in a patient with an unusual phenotype of DMD. Our genetic modifier studies provide evidence for the use of timed function tests as sensitive outcome measures of gross motor skills in younger DMD boys. They also provide further support for polymorphisms in the TGFβ pathway acting as genetic modifiers of DMD. In conclusion, we used alternative molecular methods to discover the molecular basis for phenotypic heterogeneity in a cohort of neuromuscular disease patients.

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