Sequencing Technology in Molecular Diagnosis of Spinal Muscular Atrophy Caused by SMN1 Deletion / 天津医药

ABSTRACT

Objective To investigate the feasibility of DNA sequencing analysis in molecular diagnosis for spinal muscular atrophy (SMA). Methods Two pairs of primers were utilized to amplify the region including 5 different bases in SMA-causative gene SMN1 and its homologue copy SMN2 by polymerase chain reaction (PCR). The first primer amplified a fragment 501 bp long spanning from SMN intron 6 to intron 7 targeting four different bases (g.31957, 32006, 32154 and 32269). The second primer reversely amplified a 189 bp long fragment within SMN exon 8 including one base-pair differ-ence (g.32734). PCR procedure was followed by Sanger sequencing technique to identify the 5 different bases. SMA patients caused by SMN1 homozygous deletion were distinguished from carriers or normal controls by absence of SMN1 specific bas-es in sequence chromatograms. This assay was performed in 7 SMA suspected patients and their parents. The specimens were also detected by PCR- restriction fragment length polymorphism (RFLP) method. Results It was found that 6 of 7 SMA suspected patients showed only SMN2 specific bases at the 5 different base positions among the region from intron 6 to exon 8, which meant the patient displaying only SMN2-specific nucleotide a, T, g, g and A at g.31957, 32006, 32154, 32269 and 32734, while their parents (carriers) showed a/g, T/C, g/a, g/a and A/G at the same sites. SMN1 gene was deleted in the patient, and the deletion region was inferred from intron 6 to exon 8. Because carriers had both SMN1 and SMN2 genes, they can be discriminated from the SMN1 deleted patient. One of 7 patients yield an unique sequence chromatogram of a, T, g, g and A/G, indicating that exon 8 of SMN1 was not deleted in this patient. Conclusion DNA sequencing analysis is an alter-native simple method for detecting SMA caused by homozygous deletion of SMN1. We recommend to replace the widely used PCR-RFLP method with DNA sequencing assay.