Comprehensive Analysis of Spinal Muscular Atrophy: SMN1 Copy Number, Intragenic Mutation, and 2 + 0 Carrier Analysis by Third-Generation Sequencing.

Population-wide carrier screening for spinal muscular atrophy (SMA) is recommended by the American College of Medical Genetics and Genomics. However, the methods used currently mainly focus on SMN1 copy number and fail to identify carriers with pathogenic intragenic mutations and silent (2 + 0) carriers. We developed a method termed comprehensive analysis of SMA (CASMA) based on long-range PCR and third-generation sequencing of full-length and downstream regions of SMN1/2. The sensitivity and specificity of CASMA to detect SMA carriers with one copy of SMN1 were 100% (n = 101) and 99.2% (n = 236), respectively. CASMA confirmed three SMN1 intragenic mutations and pinpointed an inframe mutation c.661_666del to SMN2, which was misreported to SMN1 by allele-specific long-range nested PCR plus Sanger sequencing. CASMA also correctly predicted 8 of 16 samples (50%) with SMN1 duplication alleles. CASMA was expected to increase the detection rate of SMA carriers from 91% to 98% and decrease the residual risk ratio from 1:415 to 1:1868 after negative results of two SMN1 copies in the Chinese population. CASMA presents a comprehensive approach for identifying SMN1 and SMN2 copy number, intragenic mutations, and potential silent carriers that significantly reduces the residual risk ratio in SMA carrier screening and has great clinical utility.