P.10.21 Next-generation sequencing meets genetic diagnostics: Development of a comprehensive workflow for neuromuscular disorders

Abstract

Muscle disease shows extensive genetic and allelic heterogeneity, with many genes causing related phenotypes. Some of the gene loci causing muscular dystrophy are the largest in the human genome (e.g. titin, dystrophin) making molecular diagnostics particularly challenging and expensive. Next generation sequencing promises single-test approach to diagnostics. A commonly utilized nextgen approach is whole exome sequence (WES), where hybrid capture of exons enables parallel sequencing of most exons in a single sequencing run. However, WES has an exonic drop-out rate of about 10% – 1 in 10 dystrophin, nebulin, titin or other gene exons would remain ‘non-sequenced’ with the WES approach. Thus, a negative result on WES does not necessarily rule out common genetic causes of muscle disease. An alternative approach is targeted sequencing panels. Here, each exon of specific candidate myopathy genes is individually amplified by microbubble PCR (RainDance), and this highly enriched DNA is then subjected to nextgen sequencing. Here, we develop and test a targeted muscle disease gene panel for 45 of the most common myopathy genes (1851 amplimers). We report our experience with this panel in 89 myopathy patients. We found greater consistency in depth of coverage (average 1000-fold coverage), less drop-out (2% instead of 10% in exomes), and genotype/phenotype concordance with clinician-determined differential diagnosis. Overall, targeted re-sequencing panels allow for more definitive testing of known muscular dystrophy genes. We give some specific case examples, with mutation in titin (TTN), ryanodine receptor (RYR1), and nebulin (NEB) genes. Both WES and targeted sequencing panels permit more centralized testing at greatly reduced cost and more rapid turn-around compared to single gene testing. The routine implementation of nextgen sequencing approaches should streamline molecular diagnostics, genetic counseling, and patient navigation for clinical trials.