帕金森病相关的线粒体多态性基因型分析

Detection System for Identifying Parkinson Disease-Associated Mitochondrial DNA Polymorphisms

目的建立基于荧光杂交探针和熔解曲线分析方法,检测线粒体单核苷酸多态性的快速而可靠的技术,为与帕金森病相关的环境和遗传因素相互作用的人群流行病学研究提供技术支持。方法从全血中提取线粒体DNA,设计特异性引物和荧光素标记探针,在LightCycler仪器上进行实时PCR和熔解曲线分析,根据检测探针上特异等位基因的荧光探针的熔点温度(Tm值)给样本的10个单核苷酸多态性分型,测序验证熔解曲线分析结果。结果在线粒体DNA的1719、4580、7028、8251、9055、10398、12308、13368、13708和16391位点上的野生型向突变型变化,引起检测探针的Tm值依次降低6.51、8.29、3.26、7.82、4.79、2.84、2.73、9.04、8.53和9.52℃。熔解曲线分析获得的150个基因型分析结果与测序结果100/一致。结论荧光杂交探针技术能快速和有效地检测线粒体DNA多态性。这种方法将能够用于大规模的环境流行病学研究,并有助于研究帕金森病的发病机制,识别遗传易感生物标志和保护易感人群。

Objective To establish a system for rapidly detecting single nucleotide polymorphisms （SNPs） in mitochondrial DNA （mtDNA） using hybridization probes and melting temperature （Tm） curve analysis. This technique is suitable for populationbased studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson＇s disease （PD）. Methods mtDNA was extracted from the blood. Rapid polymerase chain reaction （PCR） and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler. Genotyping of 10 SNPs was based on the analysis of allele-specific Tm of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products. Results Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The changes at nucleotide positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, 9.52℃ decline in Tm of the detection probes respectively. Genotyping of all detected genes was verified by 100% correspondence with the results of sequencing. Conclusion A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes has ben developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.