[Limitation of PCR-RFLP method for the detection of genetic mutations in spinal muscular atrophy].

Objective To explore the applicability and limitation of PCR-restriction fragment length polymorphism (PCR-RFLP) method for genetic diagnosis of spinal muscular atrophy (SMA).Methods PCR-RFLP was applied to detect potential deletion in exons 7 and 8 of SMN1 gene in 935 suspected cases with SMA.Multiplex ligation-dependent probe amplification(MLPA) was carried out to analyze dosage alteration of SMN1 gene in 339 of such cases. To confirm the accuracy of PCR-RFLP method for homozygous and heterozygous deletions detection,the consistency of PCR-RFLP and MLPA results were assessed with a Pearson Chi-square test.Results Homozygous deletion of exon 7 of SMN1 was detected in 590 suspected cases.The rate of diagnosis was therefore 63.1％ (590/935).For the 339 suspected cases,PCR-RFLP and MLPA respectively identified 194 and 196 homozygous deletions in the exon 7 of SMN1 gene,suggesting a good consistency (98.9％)(x2 =0.2,P=0.88).However,only 4 of 339 cases was found to carry a heterozygous deletion of SMN1 exon 7 by PCR-RFLP,in contrast with 17 detected by MLPA.The consistency only reached 23.5％,for which statistical significance was detected (x2 =8.29,P＜0.01).Conclusion Although PCR-RFLP is a simple,specific and efficient method for SMA diagnosis,it has obvious limitation for the diagnosis of 5％-10％ SMA patients who have carried a compound heterozygous mutation. Key words: Spinal muscular atrophy;  SMN1 gene;  PCR-restriction fragment length polymorphism;  Multiplex ligation-dependent probe amplification