Evaluating the performance of four assays for carrier screening of spinal muscular atrophy

Background and aims: Spinal muscular atrophy (SMA) is an autosomal recessive inherited neuromuscular condition caused by biallelic mutations in the survival of motor neuron 1 (SMN1) gene. A homozygous deletion of the SMN1 gene accounts for approximately 95-98% of SMA patients. A highly homologous gene survival motor neuron 2 (SMN2) can partially compensate for SMN1 deletion, and its copy number is associated with disease severity. Population-based carrier screening by simultaneous quantification of SMN1 and SMN2 copy numbers is the best method to prevent SMA.Materials and methods: In this study, a total of 516 samples were re-tested for the SMN1 copy number by using quantitative polymerase chain reaction (qPCR), multiplex ligation probe amplification (MLPA), droplet digital PCR (ddPCR), high-resolution melting (HRM) analysis, and PCR-based capillary electrophoresis (PCR/CE) simultaneously. Then, the performance of these methods was compared by using MLPA results as the reference. Results: The results of qPCR, ddPCR, HRM, and PCR/CE in detecting heterozygous deletion of SMN1 exon 7 and the results of ddPCR, HRM, and PCR/CE in detecting =2 copies of SMN1 exon7 are totally consistent with those of MLPA. The sensitivity and specificity of qPCR for detection of 2 copies of SMN1 exon 7 were 99.7% and 98.8%, respectively. The sensitivity and specificity of qPCR for detection of >2 copies of SMN1 exon 7 were 96.3% and 99.8%, respectively. Compared with the MLPA results, the sensitivity and specificity of qPCR and HRM for detection of heterozygous deletion of SMN1 exon 8 were 100% and 100%, respectively. They were 99.4% and 100%, respectively for detection of 2 copies, and 100% and 100%, respectively for detection of >2 copies. The results of PCR/CE in detecting SMN1 exon 8 were consistent with those of MLPA.Conclusion: All these four methods show excellent performance in detecting heterozygous deletion of SMN1 exon 7. All PCR/CE results are totally concordant with those of MLPA. As the most cost-effective method, qPCR also shows high sensitivity and specificity in detecting SMN1. Taken together, our study provides useful information to select appropriate methods for SMA carrier screening.

Automated summary

In this study, different methods including qPCR, ddPCR, HRM, and PCR/CE were compared for detecting SMN1 gene copy number in spinal muscular atrophy (SMA) patients. All four methods showed excellent performance in detecting heterozygous deletion of SMN1 exon 7. Among these methods, qPCR was found to be the most cost-effective. This study provides useful information for SMA carrier screening.