D.O.4 Next generation sequencing for genetic diagnosis and gene identification in myopathies

Abstract Myopathies are rare diseases with a high impact on patients, families and the health care system. Despite tremendous efforts, about half of patients do not have a molecular diagnosis. This is mainly due to genetic heterogeneity, the fact that very large genes known to be mutated in myopathies are difficult to screen, and the presence of yet unidentified genes. We provide the proof-of-principle that next generation sequencing (NGS) can be used for molecular diagnosis, to screen large genes, and to identify novel genes. For molecular diagnosis, we used a custom capture library to enrich the coding sequence and intron–exon boundaries of 267 genes known to be mutated in neuromuscular diseases. We could detect all known mutations in previously characterized patients, including homozygous, heterozygous, exonic, intronic, point, small indel mutations and a large deletion. The cost to sequence these 267 genes is lower than to test one gene by the conventional Sanger method. We then tested several patients without molecular diagnosis and could find mutations in several of them including mutations in TTN, the largest human gene. We also used exome sequencing in different myopathy cohorts and identified disease-causing mutations in RYR1 and NEB genes, large genes not screened on routine if RNA is not available. Phenotypes of patients with RYR1 mutations were very heterogeneous, supporting that NGS broadens genotype–phenotype correlations and represents an unbiased approach to investigate mutation/gene frequency in myopathies. Next we used exome and genome sequencing to identify disease-causing genes in specific myopathies for which no causative genes were previously known. We found mutations either in genes previously linked to other myopathies or in novel genes. Examples will be presented. Next generation sequencing will accelerate mutation discovery for the benefit of patient diagnosis and a better understanding of muscle function under normal and pathological conditions.