Copy number assessment of SMN1 based on real-time PCR with high-resolution melting: fast and highly reliable testing

Spinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by the absence of both copies of the survival motor neuron 1 (SMN1) gene. Multiple regions recommended population-wide SMA screening to quantify the copy number of SMN1. SMN1 diagnostic assays for the simplified procedure, high sensitivity, and throughput continue to be needed.Real-time PCR with high-resolution melting for the quantifying of the SMN1 gene exon 7 copies and exon 8 copies were established and confirmed by multiplex ligation-dependent probe amplification (MLPA). The diagnosis of 2563 individuals, including SMA patients, suspected cases, and the general population, was tested by real-time PCR. The results were compared with the gold standard test MLPA.In this study, the homozygous and heterozygous deletions were detected by real-time PCR with a high-resolution melting method with an incidence of 10.18% and 2.26%, respectively. In addition, the R-value distribution (P > 0.05) among 8 replicates and the coefficient of variation (CV < 0.003) suggested that the real-time PCR screening test had high reproducibility. High concordance was obtained between real-time PCR with high-resolution melting and MLPA.The real-time PCR based on high-resolution melting provides a sensitive and high-throughput approach to large-scale SMA carrier screening with low cost and labor.