Comparative study of third-generation sequencing-based CASMA-trio and STR linkage analysis for identifying SMN1 2 + 0 carriers.

Spinal Muscular Atrophy (SMA, MIM#253300) is an autosomal recessive neuromuscular disorder caused by defects in the Survival Motor Neuron (SMN) gene. The SMN1 gene, recognized as the primary pathogenic gene for SMA, exhibits a high degree of sequence homology with SMN2 gene. Individuals with the SMN1 2 + 0 genotype represent a unique type of SMA carrier, characterized by two SMN1 copies on one chromosome and zero copies on the other. Accurate identification of this type of carrier is crucial for genetic counseling in families. This study included 28 samples from five SMA families, each with an affected patient carrying a homozygous deletion of the SMN1 gene and a parent suspected to be a SMN1 2 + 0 carrier. Comprehensive Analysis of SMA (CASMA), based on third-generation sequencing technology, was used to detect the SMN1 and SMN2 copy numbers in the samples, and SMN1 2 + 0 carriers were identified through SMN1 haplotypes in parent-child trios (CASMA-trio). The results were compared with those obtained using Multiplex Ligation-dependent Probe Amplification (MLPA) combined with Short Tandem Repeat (STR) linkage analysis. The SMN1 and SMN2 copy numbers detected by MLPA and CASMA were concordant across 25 peripheral blood samples, whereas CASMA failed to accurately determine the copy numbers in the remaining 3 amniotic fluid samples. CASMA-trio identified 5 members from 4 families as SMN1 2 + 0 carriers, which were consistent with the results from STR linkage analysis. However, the two methods yielded inconsistent results for the proband's father in one family. These findings suggest that CASMA has the potential to detect SMN1 and SMN2 copy numbers. Compared to STR linkage analysis, CASMA-trio only requires a parent-child trio to analyze SMN1 2 + 0 carriers, demonstrating a broader application prospect. Implementing CASMA-trio can facilitate comprehensive screening for SMA carriers.