Targeted long-read sequencing enables comprehensive analysis of the genetic and epigenetic landscape of inherited myopathies

Inherited myopathies are a group of disorders with diverse and complex genetic aetiologies. The causative genetic variants vary widely in type, size and sequence context, encompassing small sequence variants and large structural variants in protein-coding genes, mitochondrial variants, repeat expansions, and more complex events, such as the chromosome 4 D4Z4 macrosatellite contraction and hypomethylation that causes facioscapulohumeral muscular dystrophy (FSHD). This poses a challenge for analysis with next generation sequencing and other molecular methods. This is further compounded by phenotypic variability between patients and phenotypic overlap of different forms of genetic myopathy. To accelerate myopathy research and improve diagnosis we have developed a targeted long-read sequencing assay and an integrated bioinformatics analysis framework that captures the full suite of genes, variants and epigenetic signatures currently implicated in all forms of inherited myopathy. Applying this to a cohort of 53 myopathy patients with and without previous genetic diagnoses, we demonstrate the analytical validity of our approach, as well as its improved accuracy and resolution compared to existing methods - especially for FSHD. Our LRS assay identified an array of new information about the genetic and epigenetic landscape of inherited myopathies and provided new diagnoses for 29% of patients who had remained undiagnosed following clinical genetic testing. In our cohort, FSHD and oculopharyngodistal myopathies were the most common new diagnoses that were missed or mis-diagnosed by standard clinical genetic testing. Our new method constitutes a single streamlined assay for comprehensive genetic and epigenetic characterisation of inherited myopathies.