Tan, Bi‐Hua

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).

Background— Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8 -encoded Kir6.1 (K ATP ) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8 -deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white; age, 2.9±1.9 months). KCNJ8 mutations were coexpressed heterologously with SUR2A in COS-1 cells and characterized using whole-cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared with WT, the pinacidil-activated K ATP current was decreased 45% to 68% for Kir6.1-E332del and 40% to 57% for V346I between −20 mV and 40 mV. Conclusions— Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency.

The common polymorphism SCN5A-S1103Y (∼13% allelic frequency in African Americans) is a risk factor for arrhythmia, sudden unexplained death (SUD), and sudden infant death syndrome. Prompted by a case of autopsy-negative SUD in a 23-year-old African American man who collapsed while playing football, we hypothesized that S1103Y interacted with other SCN5A variants to pathologically modify sodium current ( I Na ). Mutational analysis of arrhythmia-associated genes in the victim revealed the variants SCN5A-R680H and SCN5A-S1103Y. These variants were made both separately and in the same cDNA construct of the alternative splice variant backgrounds (SCN5A-Q1077del and Q1077) and expressed in HEK293 cells. In the most abundant SCN5A-Q1077del, late I Na for S1103Y alone was not significantly different from wild type (WT). However, late I Na for R680H, R680H+S1103Y (coexpressed), and R680H/S1103Y (on the same cDNA) was increased 2.1-, 3.4-, and 3.6-fold, respectively, compared with WT. Intracellular acidosis (pH 6.7) increased late I Na for S1103Y, R680H, R680H+S1103Y, and R680H/S1103Y by 2.2-, 2.4-, 5.0-, and 5.5-fold, respectively, compared with WT at pH 6.7. Expression in the less abundant SCN5A-Q1077 showed no increased late I Na . This is the initial report of a functional interaction for the common polymorphism S1103Y with another mutation in the major transcript Q1077del of SCN5A. The “double hit” and environmental factor of acidosis may have converged to cause arrhythmic sudden death in this case.

Background— Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in α1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current ( I Na ) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1 ’s open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (≈3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction–based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I Na was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I Na and 2.3- to 2.7-fold increase in late I Na compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current. Conclusions— Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as α1-syntrophin from similarly rare but innocuous ones.