P172 Exploring the diagnostic ability of RNA-seq to identify disease-causing variants in muscular dystrophy

• Published in: Neuromuscular disorders : NMD 2023-10, Vol.33, p.S108-S108
• Main Authors: Gaynor, A., Hale, M., Lek, M., Provenzano, M., Bates, K., Johnson, N.
• Format: Article
• Language: English
• ISSN: 0960-8966; 1873-2364
• DOI: 10.1016/j.nmd.2023.07.172

The muscular dystrophies are caused by genetic mutations that result in progressive weakness in individuals. It is now typical practice to begin genetic diagnostics with a next-generation sequencing (NGS) panel. However, cohort studies have shown that these panels do not provide a confirmatory diagnosis in most of these individuals. Furthermore, these panels can be populated by variants of unknown significance (VUS). One alternative approach is to use transcriptomic analysis of muscle to improve genetic diagnosis. Here we propose to perform RNA-seq on a cohort of patients with previously negative/unclear NGS testing. Patients in a muscular dystrophy clinic were enrolled if they had previous negative genetic testing as defined by no pathogenic variants or a single VUS in a recessive gene. A muscle biopsy was obtained. Extracted RNA from diseased and healthy tissue underwent paired-end sequencing on an Illumina platform. After alignment to GRCh38, samples were passed through the DROP pipeline consisting of packages OUTRIDER (differential expression) and FRASER (differential splicing). Results were filtered against a comprehensive list of genes related to neuromuscular disease, as well as VUS identified from previous genetic testing. Candidate variants emerging from analysis were modeled in vitro using a range of functional assays to provide evidence of variant pathogenesis. To date, 14 patients have been sequenced. In one instance, aberrant splicing was observed in a patient sample housing a homozygous VUS in SGCA. The variant was modeled in a minigene assay, revealing disruption of the canonical donor splice site predicted to lead to a non-functional protein product. RNA-seq provided evidence to support pathogenicity for a homozygous VUS in SGCA. Current undiagnosed patients being evaluated by RNA-seq include a triad (parent and two siblings) with an oculopharyngeal muscular dystrophy phenotype.