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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 a method for detecting common mutations of iduronate - 2 - sulphatase (IDS) gene in mucopolysaccharidosis (MPS) patients. Methods Mutations of exon 3, 8 and 9 of IDS mutation were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) - sequence analysis of the mutations detected by SSCP. The DNA sequencing results were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results There was a significantly abnormal band of IDS gene exon 9 in this patient detected by PCR-SSCP. DNA sequencing revealed a mutation of C16 72 T in exon 9 of the patient, 2-sulfatase protein (R46 8W). PCR-RFLP electrophoresis showed that only 5 5 4bp 1 bands were found in the mucopolysaccharidosis patients, while those of the parents showed 257 bp and 2 97 bp 2 Bands further validated the results of the sequence analysis. Conclusion PCR-SSCP analysis, DNA sequence analysis and PCR-RFLP analysis are effective methods to diagnose MPS. The combination of these two methods can be mutually validated and complement each other to improve the accuracy and success rate of gene diagnosis.