应用变性高效液相色谱技术检测parkin基因突变

目的应用变性高效液相色谱技术(denaturing high performance liquid chromatography,DHPLC)检测早发性帕金森综合征(early-onset parkinsonism,EOP)致病基因parkin突变。方法散发EOP患者82例,提取外周血细胞DNA,通过PCR扩增parkin基因的12个外显子,应用DHPLC进行变异筛查,峰形异常者进行DNA测序以明确序列变异的种类和位置。结果以100名健康者为正常对照,在82例EOP患者中检测出3种突变,均为点突变,内含子区突变包括IVS1-39G→T和IVS9 18C→T;编码区突变为T1422C,导致所编码的441位氨基酸由半胱氨酸变为精氨酸(Cys441Arg)。结论在散发EOP患者中发现3个杂合型点突变,探讨应用DHPLC在EOP患者中开展parkin基因诊断的可行性。

Mutation detection of parkin gene by denaturing high performance liquid chromatography

OBJECTIVE: To detect parkin gene mutation of early-onset parkinsonism (EOP) by denaturing high performance liquid chromatography (DHPLC). METHODS: The blood cell genomic DNA of 82 EOP patients was isolated. Exons of parkin gene were amplified by PCR. The PCR products were detected by DHPLC. The sample with abnormal peak shape was sequenced. RESULTS: Three point mutations were identified in 82 EOP patients compared with 100 healthy controls. Mutations in intron include IVS1-39 G --> T and IVS9 +18 C --> T. The T1422C mutation was in coding region and resulted in 441 Cys --> Arg. CONCLUSION: Three heterozygous mutations are found in sporadic EOP patients and genetic diagnosis of parkin gene by DHPLC is applicable in EOP patients.