聚合酶链反应-单链构象多态性和限制性酶切分析法对脊髓性肌萎缩症的基因诊断

Diagnosis of SMA by PCR-SSCP and PCR followed by restriction enzyme digestion

目的对脊髓性肌萎缩症(SMA)的早期诊断提供基因学特征和可靠的辅助检测手段。方法用PCR-单链构象多态性(SSCP)和PCR-限制性酶切分析法对首都儿科研究所附属儿童医院神经科门诊的30个SMA家系和50名入托查体正常儿童运动神经元存活基因(SMN)的第7和8外显子进行缺失检测。结果SMN基因外显子7和8缺失检测结果:在30例SMA患儿中,22例(73.3%,22/30)同时缺失SMN1基因外显子7和外显子8,4例(13.3%,4/30)显示单纯SMN1基因外显子7纯合缺失,4例(13.3%,4/30)SMN1基因外显子7或8均未见缺失,未见单纯SMN1基因外显子8纯合缺失。1例SMAⅠ型患儿父亲为SMN1基因外显子7和8的纯合缺失。1名正常儿童有SMN2基因外显子7和8的纯合缺失。经过PCR-限制性酶切法检测不伴有缺失的2例SMAⅢ型患儿及其家系SMN1基因外显子8SSCP电泳图中出现了异常条带。结论PCR-限制性酶切和PCR-SSCP分析法对SMN1基因外显子7和8缺失进行检测是诊断SMA的有效辅助手段,两者联合应用可以相互验证、互为补充;SMN1基因外显子7或8的缺失检测对SMA进行基因诊断是一种简便、特异的诊断方法,并且由于其为一种无创性检查,易被家长接受,是SMA临床症状前诊断、鉴别诊断和临床确诊的重要辅助手段。

Objective To provide reliable methods for the gene diagnosis of spinal muscular atrophy （SMA） patients. Methods Exon 7 and 8 of SMN gene were tested by PCR-RFLP and PCR-SSCP in 30 patients who were clinically diagnosed as SMA and in their parents. 50 normal children were tested as control. Results In PCR-RFLP,Two segments （188 bp, 164 bp ） in normal children and one segment （164 bp） in SMA patients when exon 7 was detected; And two segments （188 bp, 125 bp） in normal children and one segment （ 125 bp） who were found when exon 8 was detected. In PCR-SSCP the bands denoted SMN1 exon7 and exon8 missed in SMA patients. SMN1 gene exon7 and 8 are both missed in 22 of 30 patients, only exon 7 missed in 4 patients. No missing of exon7 and/or exon8 was found in the left 4 patients. Nobody was showed exon8 missing only. 16 SMA I patients（94. 1% ） and 10 SMA Ⅲ patients（100% ） were found SMN1 gene missing. Abnormal bands were found in two of the three SMAIII patients who did not lose SMN1 gene exon 7or 8 . One SMA patient＇s father missed SMN1 gene exon 7or 8 homozygously. No one lost SMN1 gene exon 7or 8 in 50 normal children, but one child missed SMN2 gene exon7 and 8. We found that PCR-RFLP and PCR-SSCP had the same results in testing deletion of SMN1 exon7 and exon 8. And in two SMA patients without deletion of SMN1 gene exon 7or 8 by PCR-RFLP,we found abnormal bands in their parents by PCR-SSCP. Conclusions The test of missing of SMN1 exon 7 and 8 is maybe helpful to early diagnosis, prenatal diagnosis, differentiate diagnosis and exact diagnosis. Both PCR-RFLP and PCR-SSCP are useful assistant ways in diagnosis of SMA more exactly. Gene diagnosis of SMA by testing absence of SMN EV and E8 is convenient,and can be accepted by the parents.