Abstract:

[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 theregion including5different bases in SMA-causative geneSMN1and its homologue copySMN2bypolymerase chain reaction (PCR). The first primer amplified a fragment 501bp long spanning fromSMNintron 6to intron 7targeting four different bases (g.31957, 32006, 32154and 32269). The second primer reversely amplified a 189bp long fragment withinSMNexon8including one base-pair differ? ence (g.32734). PCR procedure was followed by Sanger sequencing technique to identify the 5different bases. SMA patients caused bySMN1homozygous deletion were distinguished from carriers or normal controls by absence ofSMN1specific bas? es in sequence chromatograms. This assay was performed in7SMA suspected patients and their parents. The specimens were also detected by PCR- restriction fragment length polymorphism (RFLP) method.Results It was found that 6of7 SMA suspected patients showed onlySMN2specific bases at the5different base positionsamong the region from intron6to exon8, which meant the patient displaying only SMN2-specific nucleotide a, T, g, g and A at g.31957, 32006, 32154, 32269 and32734, while their parents (carriers) showed a/g, T/C, g/a, g/a and A/G at the same sites. SMN1gene was deleted in the patient, and the deletion region was inferred from intron6to exon 8. Because carriers had bothSMN1andSMN2genes, they can be discriminated from theSMN1deleted patient. One of7patients yield an unique sequence chromatogram of a, T, g, g and A/G, indicating thatexon8ofSMN1was not deleted in this patient.Conclusion DNA sequencing analysis is an alter? native simple method for detecting SMA caused by homozygous deletion ofSMN1. We recommend to replace the widely used PCR-RFLP method with DNA sequencing assay.

Key words: spinal muscular atrophies of childhood, sequence analysis, DNA, genetic testing