Amplification refractory mutation system polymerase chain reaction-capillary electrophoresis's applicability for newborn screening in dried blood spots of spinal muscular atrophy.

内容摘要

Presymptomatic treatment for infants diagnosed with spinal muscular atrophy (SMA) through newborn screening (NBS) can dramatically improve the prognosis. This study aims to investigate the feasibility of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) in SMA NBS. This technology combines multiplex fluorescence and automated capillary electrophoresis, achieving high sensitivity and multiplex detection through the size differences of fluorescently amplified fragments. The copy numbers (CNs) of survival motor neuron 1 (SMN1) and SMN2 exons 7 and 8 can be simultaneously determined in a single tube using specific primers designed based on differential bases. Two endogenous control genes were simultaneously assayed within the single-tube reaction system, while concordance of total CNs between exons 7 and 8 was comparatively assessed to ensure experimental veracity. Heterozygous with SMN1 1-copy were screened for 19 minor variants, and long-range amplification and exon sequencing were conducted to identify compound heterozygous SMA newborns. Among 20,193 dried blood spots (DBS) from eastern China, we identified 2 cases of SMN1 homozygous deletions and 380 individuals with heterozygous deletion yielding a carrier frequency of 1 in 53. Three minor SMN1 variations were found in 7 cases with SMN1 1-copy. The average detection time per sample was approximately 2.5 h, with median diagnostic time points occurring on the 7th postnatal day for SMN1/SMN2 CNs and the 11th day for minor variants. ARMS-PCR-CE is a comprehensive, accurate and rapid method for SMA DBS screening for SMN1/SMN2 CNs variations and minor variants detection.