D.P.2 Next generation sequencing after selected DNA capture as a tool for molecular diagnosis of neuromuscular disorders

Abstract

Currently in most laboratories, molecular investigations of neuromuscular disorders (NMDs) are based on a differential molecular diagnosis by a complex and time-consuming gene by gene approach guided by a clinical approach. As a consequence, it is estimated that 35–50% of patients remain devoid of a precise molecular diagnosis. On one hand, many causal genes for NMDs are still to be identified but, on the other hand, it becomes evident that the current approaches are not sufficiently powerful and accurate to perform exhaustive screenings even in known genes, and that around 50% of patients without a definite molecular diagnosis carry unidentified mutations in known genes. In the recent years, technological tools have been developed towards performing massive molecular analyses using DNA sequence capture (SC) and next generation sequencing (NGS). In the context of the European project “NMD-Chip” (FP7 Health call), we have taken advantage of these technologies to develop specific custom SC DNA arrays to explore via NGS, known and candidate genes for NMDs. A custom in-solution DNA SC library has been designed. It includes 820 genes: 50 are known to be involved in common muscular dystrophies or myopathies (Duchenne/Becker, limb girdle, congenital), the others are candidate genes for those disorders, selected from experimental and published data. To validate this DNA chip, we have selected 20 patient DNAs: five with one known mutation, including point mutations and large rearrangements (1 COL6A2, 1 COL6A1, 1 DOK7, 1 DES, 1 TNXB) and 15 with no molecular diagnosis. We also explored the DNA of three of these patients via Exome sequencing to further compare and validate our custom SC design. The analysis of our data will help to demonstrate the proof of concept of NGS as a robust and accurate approach towards exploring multiple genes in NMD patients.