WD患者ATP7B基因exon8 DNA突变检测方法的研究

Study on the methods of analysing the mutation in exon 8 of Wilson′s disease ATP7B gene

目的对肝豆状核变性(又称Wilson病,WD)ATP7B基因的突变热点外显子8进行PCR扩增产物Msp I酶切和电泳分析及DNA直接双向测序,进而对实验中的方法进行优化研究。方法对102例患者和20例健康人提取基因组DNA,PCR扩增ATP7B基因第8号外显子,扩增产物进行Msp I酶切反应,并进行DNA直接双向测序;讨论分析改进PCR扩增、Msp I酶切的方法学,并对测序结果与临床表型做相关性研究。结果102例WD患者,用反复多次改进的实验方法研究分析后,发现35例存在Msp I酶切结果异常并经测序证实,8号外显子Arg778Leu纯合突变占所有WD病人的34.31%,其中1例伴Leu770Leu多态性同义突变;对照组未检出突变。结论改进实验方法后发现PCR扩增良好,适宜测序;WD突变热点8号外显子中Arg778Leu为主要突变形式,PCR-Msp I酶切反应可作为WD病人ATP7B8号外显子Arg778Leu突变的筛选方法,直接双向测序是确定8号外显子突变位点的可靠方法之一。

Objective： Mspl digesting and sequencing the hot mutation point in exon 8 of hepatolenticular degeneration （Wilson＇ s disease, WD） ATP7B gene. Study on the methods in the experiment. Methods： Extracting the genomic DNA of 102 WD patients and 20 normal controls, and amplifying exon 8 of ATP7B gene by polymerase chain reaction （PCR）. Analyzeing the amplification by digestion of Mspl, and then sequencing the DNA of all the patients and normal controls. After that, discussing and analyzing the amelioration of the process in PCR amplification, Mspl digestion, and studying the correlation between the mutation of exon 8 and clinical manifestation. Results： Digested by Mspl through amelioration, 35 cases were abnormal after the experiment method was improved time and time again. Sequence results showed that 34. 31% of the cases had homozygous Arg778Leu mutation in exon 8, among which 1 was found to have Leu770Leu polymorphism simultaneously, while no mutation in exon 8 was foud in the control group. Conclusions ： After careful experimental amelioration, Arg778Leu was thought to be the mutation hot point of ATP7B gene, and PCR - Mspl digestion and sequcncing was reliable for molecular diagnosis in patients with Wilson＇s disesse.