Next generation sequencing for molecular diagnosis of neuromuscular diseases

Abstract

Inherited neuromuscular disorders (NMD) are chronic genetic diseases posing a significant burden on patients and the health care system. Despite tremendous research and clinical efforts, the molecular causes remain unknown for nearly half of the patients, due to genetic heterogeneity and conventional molecular diagnosis based on a gene-by-gene approach. We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis. We designed a capture library to target the coding and splice site sequences of all known NMD genes and used NGS and DNA multiplexing to retrieve the pathogenic mutations in patients with heterogeneous NMD with or without known mutations. We retrieved all known mutations, including point mutations and small indels, intronic and exonic mutations, and a large deletion in a patient with Duchenne muscular dystrophy, validating the sensitivity and reproducibility of this strategy on a heterogeneous subset of NMD with different genetic inheritance. Most pathogenic mutations were ranked on top in our blind bioinformatic pipeline. Following the same strategy, we characterized probable TTN, RYR1 and COL6A3 mutations in several patients without previous molecular diagnosis. The cost was less than conventional testing for a single large gene. With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations. An earlier genetic diagnosis should provide improved disease management and higher quality genetic counseling, and ease access to therapy or inclusion into therapeutic trials.

Fig. 1

Bioinformatic filtering and ranking

Fig. 2

Detection of copy number and mapping of a deletion in patient C with DMD. a, b Gender determination: comparison of sequence reads mapping to the X chromosome between two female DNAs in (a) and a female (black) and a male (red) in (b). In b a deletion of several exons is detected on the X chromosome for the male (squared). c Next generation sequencing data showing the detection of a 27 exons deletion in patient C with DMD (middle panel) compared to two other DNAs (top and bottom panels). Random off-target reads allow a more precise mapping of the deletion breakpoints. Off-target reads varied between two different experiments. d CGH-array results showing the 5′ and 3′ breakpoints map between 32,538,435 and 32,538,443 and between 32,187,417 and 32,187,427, respectively

Fig. 3

Detection of different types of mutations from patients with previously known and unknown molecular diagnosis. Compound heterozygous exonic point mutation (a) and heterozygous indel mutation (b) in the SETX gene in patient H with ataxia. c Homozygous exonic point mutation in the BIN1 gene in patient B with centronuclear myopathy. d Intronic mutation in the MTM1 gene in patient G with myotubular myopathy. e, f Novel compound heterozygous mutations detected in patient P with muscular dystrophy and arthrogryposis in the RYR1 gene by next generation sequencing and confirmed by Sanger sequencing. Displayed with the integrative genomics viewer IGV [25]. The normal nucleotide and protein sequences are depicted at the bottom