The next generation of population-based spinal muscular atrophy carrier screening: comprehensive pan-ethnic SMN1 copy-number and sequence variant analysis by massively parallel sequencing

Purpose: To investigate pan-ethnic SMN1 copy-number and sequence variation by hybridization-based target enrichment coupled with massively parallel sequencing or next-generation sequencing (NGS). Methods: NGS reads aligned to SMN1 and SMN2 exon 7 were quantified to determine the total combined copy number of SMN1 and SMN2. The ratio of SMN1 to SMN2 was calculated based on a single-nucleotide difference that distinguishes the two genes. SMN1 copy-number results were compared between the NGS and quantitative polymerase chain reaction and/or multiplex ligation-dependent probe amplification. The NGS data set was also queried for the g.27134T>G single-nucleotide polymorphism (SNP) and other SMN1 sequence pathogenic variants. Results: The sensitivity of the test to detect spinal muscular atrophy (SMA) carriers with one copy of SMN1 was 100% (95% confidence interval (CI): 95.9-100%; n = 90) and specificity was 99.6% (95% CI: 99.4-99.7%; n = 6,648). Detection of the g.27134T>G SNP by NGS was 100% concordant with an restriction fragment-length polymorphism method (n = 493). Ten single-nucleotide variants in SMN1 were detectable by NGS and confirmed by gene-specific amplicon-based sequencing. This comprehensive approach yielded SMA carrier detection rates of 90.3-95.0% in five ethnic groups studied. Conclusion: We have developed a novel, comprehensive SMN1 copy-number and sequence variant analysis method by NGS that demonstrated improved SMA carrier detection rates across the entire population examined.