Copy Number Variants Account for a Tiny Fraction of Undiagnosed Myopathic Patients

Abstract

Next-generation sequencing (NGS) technologies have led to an increase in the diagnosis of heterogeneous genetic conditions. However, over 50% of patients with a genetically inherited disease are still without a diagnosis. In these cases, different hypotheses are usually postulated, including variants in novel genes or elusive mutations. Although the impact of copy number variants (CNVs) in neuromuscular disorders has been largely ignored to date, missed CNVs are predicted to have a major role in disease causation as some very large genes, such as the dystrophin gene, have prone-to-deletion regions. Since muscle tissues express several large disease genes, the presence of elusive CNVs needs to be comprehensively assessed following an accurate and systematic approach. In this multicenter cohort study, we analyzed 234 undiagnosed myopathy patients using a custom array comparative genomic hybridization (CGH) that covers all muscle disease genes at high resolution. Twenty-two patients (9.4%) showed non-polymorphic CNVs. In 12 patients (5.1%), the identified CNVs were considered responsible for the observed phenotype. An additional ten patients (4.3%) presented candidate CNVs not yet proven to be causative. Our study indicates that deletions and duplications may account for 5⁻9% of genetically unsolved patients. This strongly suggests that other mechanisms of disease are yet to be discovered.

Keywords: copy number variants; next-generation sequencing; skeletal muscle disorders; variants of uncertain significance.

Figure 1

Graphic view of 12 causative copy number variants (CNVs). Gene representation and array CGH panels showing: (a) 4 DMD deletions (patients I–IV); (b) 2 deletions and 2 duplications in LAMA2 (patients V–VIII); (c) 3 deletions involving sarcoglycan genes (patients IX–XI); (d) SPAST gene deletion in patient XII. Green bars indicate deletions and red bars indicate duplications. The results of genomic quantification by real-time PCR are schematically represented (bottom of c). The number of each coding exon along gene is surmounted by a square corresponding to the number of detected copies.

Figure 2

Molecular characterization of LAMA2 variants in patients V and VIII. Single nucleotide variants in LAMA2 occurred in compound heterozygosity with a duplication. (a) Integrative Genomics Viewer (IGV) [27] visualization and Sanger confirmation of c.5374G>T (patient V); (b) Characterization of exon 21–55 duplication on RNA (patient V). (c) The splice variant c.6429+3A>C characterized on muscle transcript, leads to skipping of exons 44–45 (patient VIII); (d) Fine mapping of the tandem duplication on muscle RNA transcript (patient VIII).