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.