Copy Number Assessment of SMN1 Based on Real-Time PCR With High-Resolution Melting: Fast and Highly Reliable Testing

Background: Spinal muscular atrophy (SMA) is a common neuromuscular disorder, caused by absence of both copies of the survival motor neuron 1 (SMN1) gene. Population-wide SMA screening to quantify copy number of SMN1 is recommended by multiple regions. SMN1 diagnostic assay with simplified procedure, high sensitivity and throughput is still needed.Methods: Real-Time PCR with High-Resolution Melting for the quantification of the SMN1 gene exon 7 copies and SMN1 gene exon 8 copies was established and confirmed by multiplex ligation-dependent probe amplification (MLPA). The diagnosis of 2563 individuals including SMA patients, suspected cases and the general population were analyzed by the real-time PCR. The results were compared with the gold standard test MPLA. Results: In this study, the homozygous deletions, heterozygous deletions were identified by Real-Time PCR with High-Resolution Melting method with an incidence of 10.18% and 2.42%, respectively. In addition, the R value distribution (P>0.05) among the 8 replicates and the coefficient of variation (CV<0.003) suggested that the qPCR screening test had high reproducibility. High concordance was obtained between Real-Time PCR with High-Resolution Melting and MPLA. Conclusions: The qPCR based on High-Resolution Melting provides a sensitive and high-throughput approach to large-scale SMA carrier screening with low cost and labor.