粘多糖贮积症Ⅱ型患者艾杜糖-2-硫酸酯酶基因常见突变的检测

The detection of the frequent mutations of iduronate-2-sulphatase gene in mucopolysaccharidosis type Ⅱ patients in Chinese

目的 探讨并建立粘多糖贮积症 型 (mucopolysaccharidosis ,MPS )患者艾杜糖 - 2 -硫酸酯酶 (iduronate- 2 - sulphatase,IDS)基因常见突变的检测方法。方法 应用聚合酶链反应 -单链构象多态性(polymerase chain reaction- single strand conformation polymorphism,PCR- SSCP)对 IDS基因突变热点外显子 3、8和 9进行点突变检测 ;应用 DNA测序对 PCR- SSCP检出的突变进行序列分析 ;应用聚合酶链反应 -限制性片段长度多态性 (polymerase chain reaction- restriction fragment length polymorphism,PCR-RFL P)对 DNA测序的结果进行检测。结果 经 PCR- SSCP发现该患者的 IDS基因外显子 9有明显异常泳动的条带 ;DNA测序发现患儿的外显子 9发生点突变 (C16 72 T) ,从而导致患者艾杜糖 - 2 -硫酸酯酶蛋白发生氨基酸替换 (R46 8W) ;PCR- RFL P电泳检测结果显示粘多糖贮积症 型患者仅出现 5 5 4bp 1条带 ,而患儿父母出现 2 5 7bp和 2 97bp 2条带 ,进一步验证了序列分析的结果。结论 PCR- SSCP分析、DNA序列分析和 PCR- RFL P分析是诊断 MPS 的有效方法 ,三者联合使用可以相互验证、互为补充 ,提高基因诊断的准确率和成功率。

Objective To investigate and establish the gene diagnosis methods for the frequent mutations of iduronate-2-sulphatase(IDS) gene in mucopolysaccharidosis typeⅡ patients. Methods polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) analysis was applied to detect the mutations of exons 3,8 and 9 which were hot spots in the iduronate-2-sulfatase gene; DNA sequencing was applied to analyze the mutations which had been detected by PCR-SSCP; PCR-restriction fragment length polymorphism (PCR-RFLP) was applied to detect the results of DNA sequencing. Results Obvious and abnormal bands in exon 9 of the IDS gene were found by applying PCR-SSCP; the mutation(C1672T) of exon 9 was found in the patient through DNA sequencing, which led to amino acid replacement(R468W); the PCR-restriction enzyme digestion showed that only one band(554 bp) appeared in the patient, but there were two bands (257 bp and 297 bp) in his parents, and it verified the results of sequencing analysis. Conclusion PCR-SSCP analysis, DNA sequencing analysis and PCR-restriction enzyme digestion are effective methods for MPSⅡ diagnosis. Combined applications of these methods can verify and complement each other and improve the accuracy of diagnosis.