甲基化特异性PCR方法诊断Prader-Willi综合征

Diagnosis of Prader-Willi syndrome by methylationspecific PCR

目的Prader-Willi综合征(PWS)是多系统异常的复杂临床综合征,仅根据临床症状很难诊断,国外已建立快速、高效、特异性、敏感性均佳的甲基化特异性PCR(MS-PCR)方法用于临床诊断,但我国还未有系统的对照研究。该研究目的便是建立PWS的MS-PCR诊断方法,并对临床疑似患者进行筛查。方法将44例受试者,分为正常对照组(16例)、临床确诊患者组(7例)及临床疑似患者组(21例)。采用盐析法提取基因组DNA;应用CpGemoneTMFast Modification试剂盒行亚硫酸盐修饰;以正常人为阴性对照,未修饰的基因组DNA为系统对照,以M(母源)、P(父源)两对引物同时对修饰产物行PCR;扩增产物以琼脂糖凝胶电泳分离。结果①16例正常对照的PCR结果同时显示M,P两条带,7例临床确诊的PWS患者均只显示一条M带;②经MS-PCR筛查的21例临床疑似患者,2例确诊为PWS,其他19例排除PWS。结论该研究成功建立MS-PCR,并对疑似患者进行了筛查确诊。MS-PCR为特异高效的PWS确诊方法且方便易行。

Objective Prader-Willi syndrome （PWS） is a complex, muhisystem disorder, which is difficult to be diagnosed based on clinical symptoms and the purpose of this study is to establish methylation-specific PCR （MS-PCR） assay for the diagnosis of PWS, and evaluate its use in clinical cases. MS-PCR assay has been developed abroad for 10 years, and it is efficient, fast, specific and sensitive but it has not yet been used in clinical diagnosis in our country. Methods Forty-four subjects were assigned to 3 groups： normal controls （n = 16）, typical PWS patients （ n = 7） and suspected PWS patients （n = 21 ）. Genome DNA was extracted by salt fractionation method and treated with CpGemone^TM Fast Modification Kit. Using unmodified genome DNA as system control, the modified DNA was amplified by PCR with two primer pairs （ M and P） , and separated by agarose gel electrophoresis. Results All normal controls showed both 174 bp （M） and 100 bp （P） products, while all of the seven typical PWS patients demonstrated only 174 bp （M） product. In the 21 suspected patients, two cases were confirmed with PWS by MS-PCR, while others were excluded from PWS. Conclusions MS-PCR appears to be a specific, efficient and convenient assay for the diagnosis of PWS.