Abstract 1532: Rare Genetic Variant in the hH1 cDNA Clone, R1027Q, Dramatically Alters the Cellular Electrophysiological Phenotype Ascribed to the Common Polymorphism, H558R, and a Brugada Syndrome Mutation, R1512W: Implications for Functional Studies
Background: Previous studies in the SCN5A-encoded Na channel showed that amino acid substitutions at one position affected function of a Na channel containing a pathogenic mutation elsewhere. The first and most commonly used SCN5A cDNA clone (hH1) is the minor alternatively spliced transcript with an extra glutamine at position 1077 (Q1077), and also differs from the Na channel found in nearly all humans by virtue of a rare amino acid substitution (R1027Q). We hypothesized that R1027Q in previous studies using hH1 may have increased expression levels of the Brugada syndrome (BrS)-associated mutation (R1512W) and the common polymorphism (H558R).
Methods: We generated mutations in the alternatively-spliced SCN5A transcript containing Q1077 (as in the hH1 clone) as the wild type (WT) channel to generate channels with the following amino acid substitutions in addition to Q1077ins: H558R, H558R/R1027Q, R1512W, and R1027Q/R1512W. These channels were expressed in HEK-293 cells and current densities measured by whole cell voltage clamp.
Results: The current density for H558R was reduced dramatically (−7±2 pA/pF) whereas H558R/R1027Q exhibited normal current density (−297±56 pA/pF) comparable to WT (−310±52 pA/pF). The BrS mutation, R1512W-hH1, was characterized originally as having only subtle inactivation defects but normal current density. However, when we expressed R1512W in the patient’s true WT background (i.e. without R1027Q), the current density was attenuated significantly (−79±19 pA/pF) whereas the double mutant (R1027Q/R1512W) which replicated R1512W-hH1 showed nearly normal density (−180±21 pA/pF, −253±35 pA/pF for hH1, n=10–25 cells).
Conclusions: R1027Q, the rare genetic variant present in the hH1 cDNA clone, rescues expression of Na current for channels with H558R and the BrS1-associated mutation, R1512W. Using the hH1 clone which contains R1027Q as background (as is the case for nearly all previous studies of SCN5A mutations) may yield erroneous conclusions regarding the functional impact assigned to putative pathogenic mutations. It may be critical to study mutations in either true WT clones or in the patient’s own genetic background.
This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).