Functional genetics in patients with long QT syndrome

长QT综合征患者的功能遗传学

基本信息

批准号：
09670714
负责人：
HORIE Minoru
金额：
$ 2.05万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670714/
关键词：
long QT syndrome myocardial ion channels channelopathy gene expression system PCR SSCP

项目摘要

Patients with familial and secondary long QT syndrome have been enrolled for the study to identify the responsible gene mutations, by using polymerase chain reaction/single-strand conformation polymorphism (PC R/SSCP) analyses. To screen for mutaions in KCNQ1 (formerly KvLQT1), hERG, KCNE1 and SCN5A, we constructed specific primer pairs to amplify the short cDNAs that are thought to encode the segment of functional importance. The PCR products were heat-denatured with formamide and applied to a 12% polyacrylamide gel. The staining was achieved by SYBR Green II.If aberant spots were detected, the PCR product was subcloned into pGEM-T vector for the subsequent SSCP and DNA sequencing. Among 48 families with long QT syndrome we examined so far we found three different mutations in KVLQT1 and three in hERG.In a different sporadic long QT syndrome patient, we also found a mutation in SCN5A.By employing a site-directed mutagenesis technique, several naturally-occurring mutations were constructed and were transfected with COS7 cells by the LipofectAMlNE method. Whole-cell patch-clamp experiments were conducted 48-60 hours after transfection. Using this type of structure-function analyses, we seek to elucidate the correlation between mutations in different ion channels and the clinical features of the disease.

通过使用聚合酶链反应/单链构象多态性（PC R/SSCP）分析，已将家族性和继发性QT综合征的患者纳入了该研究，以鉴定负责任的基因突变。为了筛选在KCNQ1（以前是KVLQT1），HERG，KCNE1和SCN5A中的突变，我们构建了特定的引物对，以扩大被认为可以编码功能重要性段的简短CDNA。将PCR产物用甲酰胺进行热增生，并应用于12％的聚丙烯酰胺凝胶。通过SYBR绿色II实现染色。如果检测到了ABERANT斑点，则将PCR产物亚克隆到PGEM-T载体中，以进行随后的SSCP和DNA测序。在48家患有长QT综合征的家庭中，我们检查了到目前为止，我们在KVLQT1中发现了三个不同的突变，在HERG中发现了三个不同的突变。在一个不同的零星长QT综合征患者中，我们还发现SCN5A中的突变。通过使用定位的诱变技术，通过几种自然探测突变进行了构建，并通过cos7构建了lip。转染后48-60小时进行了全细胞斑块钳实验。使用这种类型的结构功能分析，我们试图阐明不同离子通道中的突变与疾病的临床特征之间的相关性。