A Reliable Targeted Next-Generation Sequencing Strategy for Diagnosis of Myopathies and Muscular Dystrophies, Especially for the Giant Titin and Nebulin Genes

Myopathies and muscular dystrophies (M-MDs) are genetically heterogeneous diseases, with >100 identified genes, including the giant and complex titin ( TTN ) and nebulin ( NEB ) genes. Next-generation sequencing technology revolutionized M-MD diagnosis and revealed high frequency of TTN and NEB variants. We developed a next-generation sequencing diagnostic strategy targeted to the coding sequences of 135 M-MD genes. Comparison of two targeted capture technologies (SeqCap EZ Choice library capture kit and Nextera Rapid Capture Custom Enrichment kit) and of two whole-exome sequencing kits (SureSelect V5 and TruSeq RapidExome capture) revealed best coverage with the SeqCap EZ Choice protocol. A marked decrease in coverage was observed with the other kits, affecting mostly the first exons of genes and the repeated regions of TTN and NEB . Bioinformatics analysis strategy was fine-tuned to achieve optimal detection of variants, including small insertions/deletions (INDELs) and copy number variants (CNVs). Analysis of a cohort of 128 patients allowed the detection of 52 substitutions, 13 INDELs (including a trinucleotide repeat expansion), and 3 CNVs. Two INDELs were localized in the repeated regions of NEB , suggesting that these mutations may be frequent but underestimated. A large deletion was also identified in TTN that is, to our knowledge, the first published CNV in this gene.