Elucidation of ALG10B as a Novel Long-QT Syndrome-Susceptibility Gene.

BACKGROUND: Long-QT syndrome (LQTS) is characterized by QT prolongation and increased risk for syncope, seizures, and sudden cardiac death. The majority of LQTS stems from pathogenic mutations in KCNQ1, KCNH2, or SCN5A. However, ≈10% of patients with LQTS remain genetically elusive. We utilized genome sequencing to identify a novel LQTS genetic substrate in a multigenerational genotype-negative LQTS pedigree. METHODS: Genome sequencing was performed on 5 affected family members. Only rare nonsynonymous variants present in all affected family members were considered. The candidate variant was characterized functionally in patient-derived induced pluripotent stem cell and gene-edited, variant corrected, isogenic control induced pluripotent stem cell-derived cardiomyocytes. RESULTS: A missense variant (p.G6S) was identified in ALG10B-encoded α-1,2-glucosyltransferase B protein. ALG10B (alpha-1,2-glucosyltransferase B protein) is a known interacting protein of KCNH2-encoded Kv11.1 (HERG [human Ether-à-go-go-related gene]). Compared with isogenic control, ALG10B-p.G6S induced pluripotent stem cell-derived cardiomyocytes showed (1) decreased protein expression of ALG10B (p.G6S, 0.7±0.18, n=8 versus control, 1.25±0.16, n=9; P<0.05), (2) significant retention of HERG in the endoplasmic reticulum (P<0.0005), and (3) a significantly prolonged action potential duration confirmed by both patch clamp (p.G6S, 531.1±38.3 ms, n=15 versus control, 324.1±21.8 ms, n=13; P<0.001) and multielectrode assay (P<0.0001). Lumacaftor-a compound known to rescue HERG trafficking-shortened the pathologically prolonged action potential duration of ALG10B-p.G6S induced pluripotent stem cell-derived cardiomyocytes by 10.6% (n=31 electrodes; P<0.001). CONCLUSIONS: Here, we demonstrate that ALG10B-p.G6S downregulates ALG10B, resulting in defective HERG trafficking and action potential duration prolongation. Therefore, ALG10B is a novel LQTS-susceptibility gene underlying the LQTS phenotype observed in a multigenerational pedigree. ALG10B mutation analysis may be warranted, especially in genotype-negative patients with an LQT2-like phenotype.

Long QT Syndrome KCNH2 Variant Induces hERG1a/1b Subunit Imbalance in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

[Figure: see text].

A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants.

PURPOSE: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. METHODS: We report an E. coli-based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. RESULTS: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. CONCLUSION: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

BACKGROUND: Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. METHODS AND RESULTS: Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. CONCLUSIONS: The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Mouse ERG K(+) channel clones reveal differences in protein trafficking and function.

BACKGROUND: The mouse ether-a-go-go-related gene 1a (mERG1a, mKCNH2) encodes mERG K(+) channels in mouse cardiomyocytes. The mERG channels and their human analogue, hERG channels, conduct IKr. Mutations in hERG channels reduce IKr to cause congenital long-QT syndrome type 2, mostly by decreasing surface membrane expression of trafficking-deficient channels. Three cDNA sequences were originally reported for mERG channels that differ by 1 to 4 amino acid residues (mERG-London, mERG-Waterston, and mERG-Nie). We characterized these mERG channels to test the postulation that they would differ in their protein trafficking and biophysical function, based on previous findings in long-QT syndrome type 2. METHODS AND RESULTS: The 3 mERG and hERG channels were expressed in HEK293 cells and neonatal mouse cardiomyocytes and were studied using Western blot and whole-cell patch clamp. We then compared our findings with the recent sequencing results in the Welcome Trust Sanger Institute Mouse Genomes Project (WTSIMGP). CONCLUSIONS: First, the mERG-London channel with amino acid substitutions in regions of highly ordered structure is trafficking deficient and undergoes temperature-dependent and pharmacological correction of its trafficking deficiency. Second, the voltage dependence of channel gating would be different for the 3 mERG channels. Third, compared with the WTSIMGP data set, the mERG-Nie clone is likely to represent the wild-type mouse sequence and physiology. Fourth, the WTSIMGP analysis suggests that substrain-specific sequence differences in mERG are a common finding in mice. These findings with mERG channels support previous findings with hERG channel structure-function analyses in long-QT syndrome type 2, in which sequence changes in regions of highly ordered structure are likely to result in abnormal protein trafficking.

Large-scale mutational analysis of Kv11.1 reveals molecular insights into type 2 long QT syndrome.

It has been suggested that deficient protein trafficking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafficking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that deficient protein trafficking is the dominant mechanism for all domains except for the distal carboxy-terminus. Also, most pore mutations--in contrast to intracellular domain mutations--were found to have severe dominant-negative effects when co-expressed with wild-type subunits. Finally, pharmacological correction of the trafficking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild-type subunits to form heteromeric channels.

Calcium transients closely reflect prolonged action potentials in iPSC models of inherited cardiac arrhythmia.

Long-QT syndrome mutations can cause syncope and sudden death by prolonging the cardiac action potential (AP). Ion channels affected by mutations are various, and the influences of cellular calcium cycling on LQTS cardiac events are unknown. To better understand LQTS arrhythmias, we performed current-clamp and intracellular calcium ([Ca(2+)]i) measurements on cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPS-CM). In myocytes carrying an LQT2 mutation (HERG-A422T), APs and [Ca(2+)]i transients were prolonged in parallel. APs were abbreviated by nifedipine exposure and further lengthened upon releasing intracellularly stored Ca(2+). Validating this model, control iPS-CM treated with HERG-blocking drugs recapitulated the LQT2 phenotype. In LQT3 iPS-CM, expressing NaV1.5-N406K, APs and [Ca(2+)]i transients were markedly prolonged. AP prolongation was sensitive to tetrodotoxin and to inhibiting Na(+)-Ca(2+) exchange. These results suggest that LQTS mutations act partly on cytosolic Ca(2+) cycling, potentially providing a basis for functionally targeted interventions regardless of the specific mutation site.

Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum.

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K(+) current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

Effect of microculture on cell metabolism and biochemistry: do cells get stressed in microchannels?

Microfluidics is emerging as a promising platform for cell culture, enabling increased microenvironment control and potential for integrated analysis compared to conventional macroculture systems such as well plates and Petri dishes. To advance the use of microfluidic devices for cell culture, it is necessary to better understand how miniaturization affects cell behavior. In particular, microfluidic devices have significantly higher surface-area-to-volume ratios than conventional platforms, resulting in lower volumes of media per cell, which can lead to cell stress. We investigated cell stress under a variety of culture conditions using three cell lines: parental HEK (human embryonic kidney) cells and transfected HEK cells that stably express wild-type (WT) and mutant (G601S) human ether-a-go-go related gene (hERG) potassium channel protein. These three cell lines provide a unique model system through which to study cell-type-specific responses in microculture because mutant hERG is known to be sensitive to environmental conditions, making its expression a particularly sensitive readout through which to compare macro- and microculture. While expression of WT-hERG was similar in microchannel and well culture, the expression of mutant G601S-hERG was reduced in microchannels. Expression of the endoplasmic reticulum (ER) stress marker immunoglobulin binding protein (BiP) was upregulated in all three cell lines in microculture. Using BiP expression, glucose consumption, and lactate accumulation as readouts we developed methods for reducing ER stress including properly increasing the frequency of media replacement, reducing cell seeding density, and adjusting the serum concentration and buffering capacity of culture medium. Indeed, increasing the buffering capacity of culture medium or frequency of media replacement partially restored the expression of the G601S-hERG in microculture. This work illuminates how biochemical properties of cells differ in macro- and microculture and suggests strategies that can be used to modify cell culture protocols for future studies involving miniaturized culture platforms.

Mechanism of loss of Kv11.1 K+ current in mutant T421M-Kv11.1-expressing rat ventricular myocytes: interaction of trafficking and gating.

BACKGROUND: Type 2 long QT syndrome involves mutations in the human ether a-go-go-related gene (hERG or KCNH2). T421M, an S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking, and pharmacological mechanisms in adult rat ventricular myocytes. METHODS AND RESULTS: Isolated adult rat ventricular myocytes were infected with wild-type (WT)-Kv11.1- and T421M-Kv11.1-expressing adenovirus and analyzed with the use of patch clamp, Western blot, and confocal imaging techniques. Expression of WT-Kv11.1 or T421M-Kv11.1 produced peak tail current (I(Kv11.1)) of 8.78±1.18 and 1.91±0.22 pA/pF, respectively. Loss of mutant I(Kv11.1) resulted from (1) a partially trafficking-deficient channel protein with reduced cell surface expression and (2) altered channel gating with a positive shift in the voltage dependence of activation and altered kinetics of activation and deactivation. Coexpression of WT+T421M-Kv11.1 resulted in heterotetrameric channels that remained partially trafficking deficient with only a minimal increase in peak I(Kv11.1) density, whereas the voltage dependence of channel gating became WT-like. In the adult rat ventricular myocyte model, both WT-Kv11.1 and T421M-Kv11.1 channels responded to ß-adrenergic stimulation by increasing I(Kv11.1). CONCLUSIONS: The T421M-Kv11.1 mutation caused a loss of I(Kv11.1) through interactions of abnormal protein trafficking and channel gating. Furthermore, for coexpressed WT+T421M-Kv11.1 channels, different dominant-negative interactions govern protein trafficking and ion channel gating, and these are likely to be reflected in the clinical phenotype. Our results also show that WT and mutant Kv11.1 channels responded to ß-adrenergic stimulation.

Trafficking-deficient hERG K⁺ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER.

The human ether-a-go-go related gene (hERG) encodes the voltage-gated K(+) channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an "immature" N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.

Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels.

L-type Ca(2+) channels (LTCCs) play a critical role in Ca(2+)-dependent signaling processes in a variety of cell types. The number of functional LTCCs at the plasma membrane strongly influences the strength and duration of Ca(2+) signals. Recent studies demonstrated that endosomal trafficking provides a mechanism for dynamic changes in LTCC surface membrane density. The purpose of the current study was to determine whether the small GTPase Rab11b, a known regulator of endosomal recycling, impacts plasmalemmal expression of Ca(v)1.2 LTCCs. Disruption of endogenous Rab11b function with a dominant negative Rab11b S25N mutant led to a significant 64% increase in peak L-type Ba(2+) current (I(Ba,L)) in human embryonic kidney (HEK)293 cells. Short-hairpin RNA (shRNA)-mediated knockdown of Rab11b also significantly increased peak I(Ba,L) by 66% compared when with cells transfected with control shRNA, whereas knockdown of Rab11a did not impact I(Ba,L). Rab11b S25N led to a 1.7-fold increase in plasma membrane density of hemagglutinin epitope-tagged Ca(v)1.2 expressed in HEK293 cells. Cell surface biotinylation experiments demonstrated that Rab11b S25N does not significantly impact anterograde trafficking of LTCCs to the surface membrane but rather slows degradation of plasmalemmal Ca(v)1.2 channels. We further demonstrated Rab11b expression in ventricular myocardium and showed that Rab11b S25N significantly increases peak I(Ba,L) by 98% in neonatal mouse cardiac myocytes. These findings reveal a novel role for Rab11b in limiting, rather than promoting, the plasma membrane expression of Ca(v)1.2 LTCCs in contrast to its effects on other ion channels including human ether-a-go-go-related gene (hERG) K(+) channels and cystic fibrosis transmembrane conductance regulator. This suggests Rab11b differentially regulates the trafficking of distinct cargo and extends our understanding of how endosomal transport impacts the functional expression of LTCCs.

Microfluidic cell culture and its application in high-throughput drug screening: cardiotoxicity assay for hERG channels.

Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval and hence risk for life-threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, the authors demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. They validate their microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP), and polydimethylsiloxane (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG) and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. The authors demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. The authors further show that experimental procedures can be streamlined by using direct in-channel immunofluorescence staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with 5 different drugs suggests that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules.

Novel chemical suppressors of long QT syndrome identified by an in vivo functional screen.

BACKGROUND: Genetic long QT (LQT) syndrome is a life-threatening disorder caused by mutations that result in prolongation of cardiac repolarization. Recent work has demonstrated that a zebrafish model of LQT syndrome faithfully recapitulates several features of human disease, including prolongation of ventricular action potential duration, spontaneous early afterdepolarizations, and 2:1 atrioventricular block in early stages of development. Because of their transparency, small size, and absorption of small molecules from their environment, zebrafish are amenable to high-throughput chemical screens. We describe a small-molecule screen using the zebrafish KCNH2 mutant breakdance to identify compounds that can rescue the LQT type 2 phenotype. METHODS AND RESULTS: Zebrafish breakdance embryos were exposed to test compounds at 48 hours of development and scored for rescue of 2:1 atrioventricular block at 72 hours in a 96-well format. Only compounds that suppressed the LQT phenotype in 3 of 3 fish were considered hits. Screen compounds were obtained from commercially available small-molecule libraries (Prestwick and Chembridge). Initial hits were confirmed with dose-response testing and time-course studies. Optical mapping with the voltage-sensitive dye di-4 ANEPPS was performed to measure compound effects on cardiac action potential durations. Screening of 1200 small molecules resulted in the identification of flurandrenolide and 2-methoxy-N-(4-methylphenyl) benzamide (2-MMB) as compounds that reproducibly suppressed the LQT phenotype. Optical mapping confirmed that treatment with each compound caused shortening of ventricular action potential durations. Structure activity studies and steroid receptor knockdown suggest that flurandrenolide functions via the glucocorticoid signaling pathway. CONCLUSIONS: Using a zebrafish model of LQT type 2 syndrome in a high-throughput chemical screen, we have identified 2 compounds, flurandrenolide and the novel compound 2-MMB, as small molecules that rescue the zebrafish LQT type 2 syndrome by shortening the ventricular action potential duration. We provide evidence that flurandrenolide functions via the glucocorticoid receptor-mediated pathway. These 2 molecules and future discoveries from this screen should yield novel tools for the study of cardiac electrophysiology and may lead to novel therapeutics for human LQT patients.

Properties of WT and mutant hERG K(+) channels expressed in neonatal mouse cardiomyocytes.

Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming alpha-subunits that coassemble to form rapidly activating delayed rectifier K(+) current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The DeltaY475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue.

Rescue of mutated cardiac ion channels in inherited arrhythmia syndromes.

Inherited arrhythmia syndromes comprise an increasingly complex group of diseases involving mutations in multiple genes encoding ion channels, ion channel accessory subunits and channel interacting proteins, and various regulatory elements. These mutations serve to disrupt normal electrophysiology in the heart, leading to increased arrhythmogenic risk and death. These diseases have added impact as they often affect young people, sometimes without warning. Although originally thought to alter ion channel function, it is now increasingly recognized that mutations may alter ion channel protein and messenger RNA processing, to reduce the number of channels reaching the surface membrane. For many of these mutations, it is also known that several interventions may restore protein processing of mutant channels to increase their surface membrane expression toward normal. In this article, we reviewed inherited arrhythmia syndromes, focusing on long QT syndrome type 2, and discuss the complex biology of ion channel trafficking and pharmacological rescue of disease-causing mutant channels. Pharmacological rescue of misprocessed mutant channel proteins, or their transcripts providing appropriate small molecule drugs can be developed, has the potential for novel clinical therapies in some patients with inherited arrhythmia syndromes.

Alpha1-syntrophin mutations identified in sudden infant death syndrome cause an increase in late cardiac sodium current.

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 alpha1-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 (approximately 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 alpha1-syntrophin from similarly rare but innocuous ones.