儿童进行性脊肌萎缩症的分子诊断(附10例报告)

目的 对10例儿童进行性脊肌萎缩症(SMA)患者进行分子诊断.方法 提取10例SMA患者和其19例家系成员(患者的父母)以及20例健康正常人对照的基因组DNA.常规PCR扩增SMN基因第7外显子(E7),PCR产物经Dra I酶切后,行琼脂糖凝胶电泳.同时行特异性PCR扩增SMN1和SMN2的E7.结果 常规PCR中,所有10例SMA患者的SMN1 PCR产物(189bp)全部被Dra I切割,所有39例对照组成员(包括19例家系成员和20例健康正常人对照)的SMN1 PCR产物仅有部分可被Dra I切割.在位点特异性PCR中,所有10例SMA患者只有SMN2的E7扩增,所有39例对照组成员既有SMN1、也有SMN2的E7扩增产物.结论 所有10例SMA患者可见SMN1纯合性缺失.所有39例对照组成员未见SMN1纯合性缺失.本研究采用PCR-RFLP和位点特异性PCR相结合,相互佐证,形成一套特有的分子诊断方法,确保了诊断的准确性.

Molecular diagnosis of puerile spinal muscular atrophy (with report of 10 cases)

Objective To make molecular diagnosis for puerile spinal muscular atrophy(SMA).Methods Genomic DNA was extracted directly from the blood of both the case group(10 children with SMA) and the control group(including 19 parents of SMA patients and 20 healthy individuals).Two methods,polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and allele-specific PCR,were used to analyze exon 7 of SMN gene from genomic DNA,and consequent electrophoresis of PCR products on agarose gel was performed.Results Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed.In conventional PCR-RFLP,part of the PCR products(189bp) from genomic DNA of all 39 members in the control group remained intact after digestion with Dra I,while the PCR products from genomic DNA of all 10 SMA children in the case group was completely digested by Dra I.In allele-specific PCR,exon 7 of both SMN1 and SMN2 could be seen when genomic DNA of all 39 members in the control group was used,while only SMN2's exon 7 could be seen when genomic DNA of all 10 SMA children in the case group was used.Conclusion Homozygous deletion of SMN1 was present in all 10 SMA children in the case group,while homozygous deletion of SMN1 was not detected in all 39 members in the control group.The combination of PCR-RFLP and allele-specific PCR,both their results can be references for each other,offers efficient and accurate methodology for molecular diagnosis of SMA.