应用变性高效液相色谱技术进行肝豆状核变性的基因突变筛查及产前诊断

Mutation screening and prenatal diagnosis of Wilson＇ s disease by denature high performance liquid chromatography

目的探讨变性高效液相色谱（denature high performance liquid chromatography，DHPLC）技术在肝豆状核变性（wilson’s disease，WD）的突变筛查及产前诊断中的临床应用。方法以6个WD家系中的患者及其父母的DNA为模板，采用PCR技术扩增ATP7B基因的21个外显子及5’非翻译区，PCR产物经DHPLC技术进行突变筛查，对峰型有改变者进行测序验证。在确定了先证者突变类型的基础上，采用相同方法对其中4个家系（1个双胎和3个单胎）进行产前诊断。结果6例患者中检测出5种已知的致病突变及8种多态类型。患者的父母均为相应突变类型的携带者。产前诊断结果显示，两例妊娠为异常胎儿，其中1例双胎为Arg778Leu／IVS4—1G〉C双重杂合子，1例单胎为Ser975Tyr／Pro992Leu双重杂合子，这两对妊娠夫妇选择了终止妊娠。另两例妊娠中，1例为Ser975Tyr杂合子，1例完全正常，他们选择了继续妊娠，出生了表型正常儿。结论DHPLC在Wilson病的突变检测和产前诊断中有良好的应用前景。

Objecive To study the clinical application of denature high performance liquid etu＇omatography （DHPLC） technique on mutation screening and prenatal diagnosis for Wilson＇ s disease （WD）. Methods Genomic DNA of the probands with Wilson＇ s disease and their parents from 6 families was subjected to polymerase chain reaction （PCR） for the 21 exons and the 5＇ untranslated region of ATP7B gene. Mutation screening of the PCR products was performed by DHPLC. The abnormal peaks were confirmed by further sequencing analysis. Based on the successful gene diagnosis for the patients, prenatal diagnosis was performed in 4 families, including 1 twin and 3 singletons. Results Five disease-causing mutations and 8 polyrnorphisms were fotmd in the 6 probands by DHPLC and sequencing. The parents were carriers with the same mutation as their affected children. Prenatal diagnosis showed that two pregnancies were abnormal, including a twin pregnancy with compound heterozygote for Arg778Leu and IVS4-1G 〉 C mutation, and a single pregnancy with a compound heterozygote for Ser975Tyr and Pro992Leu mutations. These two pregnancies were terminated after genetic counseling. Another two pregnancies included a singleton carrier with Ser975Tyr mutation and a normat genotype fetus, respectively. These two pregnancies were continued and the babies were healthy. Conclusion DHPLC is a powerful tool in prenatal diagnosis as well as in postnatal diagnosis.