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

Abstract

Background: 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 SMN1 deletion 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/SMN2 locus, sequence analysis to identify SNVs of the SMN1 gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods.

Objective: 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.

Methods: A bioinformatics workflow to detect homozygous SMN1 deletions and SMN1 SNVs 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 SMN1 and SMN2 to an SMN1 reference sequence. Homozygous SMN1 deletions were identified by filtering sequence reads for the ,, gene-determining variant" (GDV).

Results: 10 patients were diagnosed with 5q-SMA based on (i) SMN1 deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1 deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1 (2 patients).

Conclusions: 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.

Keywords: 5q-SMA; bioninformatics; clinical genetics; dark genes; neuromuscular disorders; spinal muscular atrophy.

Fig. 1

Schematic representation of the SMN1/2 locus on chromosome 5q. SMN1 and SMN2 genes differ in the “Gene-determining variant” (GDV) NM_000344.4:c.840C>T p.(Phe280=) (rs1164325688) in exon 7, depicted as C (blue) in SMN1 and T (red) in SMN2. By masking of SMN2 in the reference genome, all SMN1/2 reads are mapped to SMN1. SMN1 can be analyzed for SNVs by the caller. (A) Example of a normal control: The T (GDV of SMN2) is called in a VAF of <0.9 indicating absence of a homozygous deletion of exon 7 of SMN1. (B) Example of a patient with a homozygous SMN1 deletion: There are no reads with the C (SMN1). Thus, the variant caller indicates T (GDV of SMN2) with a VAF of >0.9 (“homozygous”). (C) Example of a patient with a pathogenic SNV in SMN1 (indicated as red star) and a benign variant in SMN2 (indicated as green star). After applying the SMN2-masked pipeline, SNVs in SMN1 as well SMN2 reads are called. It has to be considered that the assumed diploid sequence for the variant calling is not anymore true and is at least doubled or even more tripled resulting in variant allele frequencies (VAF) ≠ 0.5 for heterozygous variants. The VAF depends on the SMN1 and SMN2 copy number of the individual. A heterozygous SNV in oneSMN1copy may have a low VAF (e.g. of 0.2 in case of two SMN1 and three SMN2 copies).

Fig. 2

Example of a heterozygous pathogenic variant in SMN1, NM_000344.4:c.815A>G p.(Tyr272Cys) in the IGV (Integrative Genomics Viewer (27)).