Functional genetics in patients with long QT syndrome

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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.