Functional genetics in patients with long QT syndrome

家族性和继发性长QT综合征患者已被纳入研究，以确定负责的基因突变，通过使用聚合酶链反应/单链构象多态性（PCR/SSCP）分析。为了筛选KCNQ 1（以前的KvLQT 1）、hERG、KCNE 1和SCN 5A中的突变，我们构建了特异性引物对来扩增被认为编码功能重要片段的短cDNA。PCR产物用甲酰胺热变性，并施加到12%聚丙烯酰胺凝胶上。用SYBR绿色Ⅱ染色，如有异常斑点，将PCR产物亚克隆到pGEM-T载体上，进行SSCP和DNA测序。在48个长QT综合征家系中，我们发现了3个KVLQT 1和3个hERG突变，并在一个散发性长QT综合征患者中发现了SCN 5A突变。转染后48-60小时进行全细胞膜片钳实验。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的突变和疾病的临床特征之间的相关性。

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.