A common human SCN5A polymorphism modifies expression of an arrhythmia causing mutation

Abstract

Ye, Bin, Carmen R. Valdivia, Michael J. Ackerman, and Jonathan C. Makielski. A common human SCN5A polymorphism modifies expression of an arrhythmia causing mutation. Physiol Genomics 12: 187-193, 2003. First published November 26, 2002 10.1152 10. /physiolgenomics.00117. 2002 encodes the ␣-subunit of the ion channel that carries Na current in human heart. From a human cardiac cDNA library we recloned SCN5A. The new clone hH1b differed from existing clones hH1 in four and from hH1a in three positions. The common polymorphism H558R was uniquely present in hH1b. Voltage clamp study showed minor but potentially important kinetic differences between hH1b and the other clones. More dramatically, when the LQT3 mutation M1766L was introduced into the different clones, Na current was markedly reduced in the hH1 and hH1a backgrounds, whereas in hH1b the Na current was not reduced. Immunocytochemistry experiments showed a trafficking defect for M1766L Na channels in hH1 and hH1a but not in hH1b. The double-mutation M1766L/H558R in the hH1a background restored normal trafficking and current including persistent late current, suggesting the disease phenotype was the result of a "double hit" that included the common polymorphism, H558R. These results show that the choice of background clone must be carefully considered in mutagenesis studies. This also represents an example of intragenic complementation, the first for such a large protein. ion channels; trafficking defect; polymorphisms; LQT3; intragenic complementation THE HUMAN cardiac Na channel gene SCN5A encodes the ion channel hNa v 1.5 (9), which carries inward Na current (I Na ) in heart. Mutations in SCN5A cause arrhythmia syndromes including congenital long QT syndrome LQT3 and Brugada syndrome (2, 12). Two distinct full-length hNa v 1.5 clones isolated from human cardiac cDNA libraries, hH1 (8) and hH1a (10), have been used previously to characterize human I Na for wild-type channels and for channels containing putative arrhythmia-causing mutations. The sequences for hH1 and hH1a reportedly differ in 9 of the 2,016 amino acids (8, 10) and have never been studied under identical conditions. We hypothesized that these differences might affect function, either of wild-type channels, or when arrhythmia mutations were inserted into the different backgrounds. For this study, RT-PCR was used to obtain a third complete clone (designated hH1b) for hNa v 1.5. hH1b contained a known common polymorphism H558R whereby histidine (H) at position 558 is replaced by an arginine (R) (11, 17). We also studied an LQT3 mutation M1766L that was previously reported to have both decreased current expression and at the same time enhanced late current (relative to peak current) when expressed in the hH1a background (14). In the present study, I Na expression for the M1766L mutation expressed in the three background clones depended upon the polymorphism at position 558. Thus SCN5A serves as its own modifier gene. These results represent a novel form of "intragenic complementation" where a m...

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