Closing the Gap - Detection of 5q-Spinal Muscular Atrophy by Short-Read Next-Generation Sequencing and Unexpected Results in a Diagnostic Patient Cohort

The importance of early diagnosis of 5q-Spinal muscular atrophy (5q-SMA) has heightened as early intervention can significantly improve clinical outcomes. In 96% of cases, 5q-SMA is caused by a homozygous deletion of SMN1. Around 4 % of patients carry a SMN1deletion and a single-nucleotide variant (SNV) on the other allele. Traditionally, diagnosis is based on multiplex ligation probe amplification (MLPA) to detect homozygous or heterozygous exon 7 deletions in SMN1. Due to high homologies within the SMN1/SMN2locus, sequence analysis to identify SNVs of the SMN1gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods. The objective was to overcome the limitations in high-throughput srNGS with the aim of providing SMA patients with a fast and reliable diagnosis to enable their timely therapy. A bioinformatics workflow to detect homozygous SMN1deletions and SMN1SNVs on srNGS analysis was applied to diagnostic whole exome and panel testing for suggested neuromuscular disorders (1684 patients) and to fetal samples in prenatal diagnostics (260 patients). SNVs were detected by aligning sequencing reads from SMN1and SMN2to an SMN1reference sequence. Homozygous SMN1deletions were identified by filtering sequence reads for the ,, gene-determining variant“ (GDV). 10 patients were diagnosed with 5q-SMA based on (i) SMN1deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1(2 patients). Applying our workflow in srNGS-based panel and whole exome sequencing (WES) is crucial in a clinical laboratory, as otherwise patients with an atypical clinical presentation initially not suspected to suffer from SMA remain undiagnosed.