Dao W. Wang

Prevalence of Long-QT Syndrome Gene Variants in Sudden Infant Death Syndrome

Background— The hypothesis that some cases of sudden infant death syndrome (SIDS) could be caused by long-QT syndrome (LQTS) has been supported by molecular studies. However, there are inadequate data regarding the true prevalence of mutations in arrhythmia-susceptibility genes among SIDS cases. Given the importance and potential implications of these observations, we performed a study to more accurately quantify the contribution to SIDS of LQTS gene mutations and rare variants. Methods and Results— Molecular screening of 7 genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2, CAV3) associated with LQTS was performed with denaturing high-performance liquid chromatography and nucleotide sequencing of genomic DNA from 201 cases diagnosed as SIDS according to the Nordic Criteria, and from 182 infant and adult controls. All SIDS and control cases originated from the same regions in Norway. Genetic analysis was blinded to diagnosis. Mutations and rare variants were found in 26 of 201 cases (12.9%). On the basis of their functional effect, however, we considered 8 mutations and 7 rare variants found in 19 of 201 cases as likely contributors to sudden death (9.5%; 95% CI, 5.8 to 14.4%). Conclusions— We demonstrated that 9.5% of cases diagnosed as SIDS carry functionally significant genetic variants in LQTS genes. The present study demonstrates that sudden arrhythmic death is an important contributor to SIDS. As these variants likely modify ventricular repolarization and QT interval duration, our results support the debated concept that an ECG would probably identify most infants at risk for sudden death due to LQTS either in infancy or later on in life.

Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A)

Sick sinus syndrome (SSS) describes an arrhythmia phenotype attributed to sinus node dysfunction and diagnosed by electrocardiographic demonstration of sinus bradycardia or sinus arrest. Although frequently associated with underlying heart disease and seen most often in the elderly, SSS may occur in the fetus, infant, and child without apparent cause. In this setting, SSS is presumed to be congenital. Based on prior associations with disorders of cardiac rhythm and conduction, we screened the alpha subunit of the cardiac sodium channel (SCN5A) as a candidate gene in ten pediatric patients from seven families who were diagnosed with congenital SSS during the first decade of life. Probands from three kindreds exhibited compound heterozygosity for six distinct SCN5A alleles, including two mutations previously associated with dominant disorders of cardiac excitability. Biophysical characterization of the mutants using heterologously expressed recombinant human heart sodium channels demonstrate loss of function or significant impairments in channel gating (inactivation) that predict reduced myocardial excitability. Our findings reveal a molecular basis for some forms of congenital SSS and define a recessive disorder of a human heart voltage-gated sodium channel.

Molecular basis of an inherited epilepsy

Epilepsy is a common neurological condition that reflects neuronal hyperexcitability arising from largely unknown cellular and molecular mechanisms. In generalized epilepsy with febrile seizures plus, an autosomal dominant epilepsy syndrome, mutations in three genes coding for voltage-gated sodium channel alpha or beta1 subunits (SCN1A, SCN2A, SCN1B) and one GABA receptor subunit gene (GABRG2) have been identified. Here, we characterize the functional effects of three mutations in the human neuronal sodium channel alpha subunit SCN1A by heterologous expression with its known accessory subunits, beta1 and beta2, in cultured mammalian cells. SCN1A mutations alter channel inactivation, resulting in persistent inward sodium current. This gain-of-function abnormality will likely enhance excitability of neuronal membranes by causing prolonged membrane depolarization, a plausible underlying biophysical mechanism responsible for this inherited human epilepsy.

Cardiac Sodium Channel Dysfunction in Sudden Infant Death Syndrome

Background— Mutations in genes responsible for the congenital long-QT syndrome, especially SCN5A, have been identified in some cases of sudden infant death syndrome. In a large-scale collaborative genetic screen, several SCN5A variants were identified in a Norwegian sudden infant death syndrome cohort (n=201). We present functional characterization of 7 missense variants (S216L, R680H, T1304M, F1486L, V1951L, F2004L, and P2006A) and 1 in-frame deletion allele (delAL586-587) identified by these efforts. Methods and Results— Whole-cell sodium currents were measured in tsA201 cells transiently transfected with recombinant wild-type or mutant SCN5A cDNA (hH1) coexpressed with the human &bgr;1 subunit. All variants exhibited defects in the kinetics and voltage dependence of inactivation. Five variants (S216L, T1304M, F1486L, F2004L, and P2006A) exhibited significantly increased persistent sodium currents (range, 0.5% to 1.7% of peak current) typical of SCN5A mutations associated with long-QT syndrome. These same 5 variants also displayed significant depolarizing shifts in voltage dependence of inactivation (range, 5 to 14 mV) and faster recovery from inactivation, but F1486L uniquely exhibits a depolarizing shift in the conductance-voltage relationship. Three alleles (delAL586-587, R680H, and V1951L) exhibited increased persistent current only under conditions of internal acidosis (R680H) or when expressed in the context of a common splice variant (delQ1077), indicating that they have a latent dysfunctional phenotype. Conclusions— Our present results greatly expand the spectrum of functionally characterized SCN5A variants associated with sudden infant death syndrome and provide further biophysical correlates of arrhythmia susceptibility in this syndrome.

Pharmacological targeting of long QT mutant sodium channels.

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a delay in cardiac cellular repolarization leading to cardiac arrhythmias and sudden death often in young people. One form of the disease (LQT3) involves mutations in the voltage-gated cardiac sodium channel. The potential for targeted suppression of the LQT defect was explored by heterologous expression of mutant channels in cultured human cells. Kinetic and steady state analysis revealed an enhanced apparent affinity for the predominantly charged, primary amine compound, mexiletine. The affinity of the mutant channels in the inactivated state was similar to the wild type (WT) channels (IC50 approximately 15-20 microM), but the late-opening channels were inhibited at significantly lower concentrations (IC50 = 2-3 microM) causing a preferential suppression of the late openings. The targeting of the defective behavior of the mutant channels has important implications for therapeutic intervention in this disease. The results provide insights for the selective suppression of the mutant phenotype by very low concentrations of drug and indicate that mexiletine equally suppresses the defect in all three known LQT3 mutants.

Congenital Long-QT Syndrome Caused by a Novel Mutation in a Conserved Acidic Domain of the Cardiac Na+ Channel

BACKGROUND Congenital long-QT syndrome (LQTS) is an inherited condition of abnormal cardiac excitability characterized clinically by an increased risk of ventricular tachyarrhythmias. One form, LQT3, is caused by mutations in the cardiac voltage-dependent sodium channel gene, SCN5A. Only 5 SCN5A mutations have been associated with LQTS, and more work is needed to improve correlations between SCN5A genotypes and associated clinical syndromes. METHODS AND RESULTS We researched a 3-generation white family with autosomal dominant LQTS who exhibited a wide clinical spectrum from mild bradycardia to sudden death. Molecular genetic studies revealed a single nucleotide substitution in SCN5A exon 28 that caused the substitution of Glu1784 by Lys (E1784K). The mutation occurs in a highly conserved domain within the C-terminus of the cardiac sodium channel containing multiple, negatively charged amino acids. Two-electrode voltage-clamp recordings of a recombinant E1784K mutant channel expressed in Xenopus oocytes revealed a defect in fast inactivation characterized by a small, persistent current during long membrane depolarizations. Coexpression of the mutant with the human sodium channel beta1-subunit did not affect the persistent current, even though we did observe shifts in the voltage dependence of steady-state inactivation. Neutralizing multiple, negatively charged residues in the same region of the sodium channel C-terminus did not cause a more severe functional defect. CONCLUSIONS We characterized the genetics and molecular pathophysiology of a novel SCN5A sodium channel mutation, E1784K. The functional defect exhibited by the mutant channel causes delayed myocardial repolarization, and our data on the effects of multiple charge neutralizations in this region of the C-terminus suggest that the molecular mechanism of channel dysfunction involves an allosteric rather than a direct effect on channel gating.

The transient outward current in mice lacking the potassium channel gene Kv1.4

1 The transient outward current (Ito) plays a prominent role in the repolarization phase of the cardiac action potential. Several K+ channel genes, including Kv1.4, are expressed in the heart, produce rapidly inactivating currents when heterologously expressed, and may be the molecular basis of Ito. 2 We engineered mice homozygous for a targeted disruption of the K+ channel gene Kv1.4 and compared Ito in wild‐type (Kv1.4+/+), heterozygous (Kv1.4+/‐) and homozygous ‘knockout’ (Kv1.4−/−) mice. Kv1.4 RNA was truncated in Kv1.4−/− mice and protein expression was absent. 3 Adult myocytes isolated from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice had large rapidly inactivating outward currents. The peak current densities at 60 mV (normalized by cellular capacitance, in pA pF−1; means ± s.e.m.) were 53.8 ± 5.3, 45.3 ± 2.2 and 44.4 ± 2.8 in cells from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice, respectively (P < 0.02 for Kv1.4+/+vs. Kv1.4−/−). The steady‐state values (800 ms after the voltage clamp step) were 30.9 ± 2.9, 26.9 ± 3.8 and 23.5 ± 2.2, respectively (P < 0.02 for Kv1.4+/+vs. Kv1.4−/−). The inactivating portion of the current was unchanged in the targeted mice. 4 The voltage dependence and time course of inactivation were not changed by targeted disruption of Kv1.4. The mean best‐fitting V½ (membrane potential at 50 % inactivation) values for myocytes from Kv1.4 +/+, Kv1.4+/− and Kv1.4−/− mice were ‐53.5 ± 3.7, ‐51.1 ± 2.6 and ‐54.2 ± 2.4 mV, respectively. The slope factors (k) were ‐10.1 ± 1.4, ‐8.8 ± 1.4 and ‐9.5 ± 1.2 mV, respectively. The fast time constants for development of inactivation at ‐30 mV were 27.8 ± 2.2, 26.2 ± 5.1 and 19.6 ± 2.1 ms in Kv1.4+/+, Kv1.4+/− and Kv1.4−/− myocytes, respectively. At +30 mV, they were 35.5 ± 2.6, 30.0 ± 2.1 and 28.7 ± 1.6 ms, respectively. The time constants for the rapid phase of recovery from inactivation at ‐80 mV were 32.5 ± 8.2, 23.3 ± 1.8 and 39.0 ± 3.7 ms, respectively. 5 Nearly the entire inactivating component as well as more than 60 % of the steady‐state outward current was eliminated by 1 mm 4‐aminopyridine in Kv1.4+/+, Kv1.4+/− and Kv1.4−/− myocytes. 6 Western blot analysis of heart membrane extracts showed no significant upregulation of the Kv4 subfamily of channels in the targeted mice. 7 Thus, Kv1.4 is not the molecular basis of Ito in adult murine ventricular myocytes.

Striking in Vivo Phenotype of a Disease-Associated Human SCN5A Mutation Producing Minimal Changes in Vitro

Background— The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. Methods and Results— We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of &bgr;1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a dilated cardiomyopathy phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose–dependent increases in SCN5A mRNA abundance but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, 41.0±2.9 pA/pF at −30 mV; DN/H, 19.2±3.1 pA/pF, P<0.001 vs H/H; DN/DN, 9.3±1.1 pA/pF, P<0.001 versus H/H). Conclusions— Although D1275N produces near-normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a dilated cardiomyopathy phenotype by reducing cardiac sodium current.

Divergent Biophysical Defects Caused by Mutant Sodium Channels in Dilated Cardiomyopathy With Arrhythmia

Mutations in SCN5A encoding the principal Na+ channel &agr;-subunit expressed in human heart (NaV1.5) have recently been linked to an inherited form of dilated cardiomyopathy with atrial and ventricular arrhythmia. We compared the biophysical properties of 2 novel NaV1.5 mutations associated with this syndrome (D2/S4 – R814W; D4/S3 – D1595H) with the wild-type (WT) channel using heterologous expression in cultured tsA201 cells and whole-cell patch-clamp recording. Expression levels were similar among WT and mutant channels, and neither mutation affected persistent sodium current. R814W channels exhibited prominent and novel defects in the kinetics and voltage dependence of activation characterized by slower rise times and a hyperpolarized conductance-voltage relationship resulting in an increased “window current.” This mutant also displayed enhanced slow inactivation and greater use-dependent reduction in peak current at fast pulsing frequencies. By contrast, D1595H channels exhibited impaired fast inactivation characterized by slower entry into the inactivated state and a hyperpolarized steady-state inactivation curve. Our findings illustrate the divergent biophysical defects caused by 2 different SCN5A mutations associated with familial dilated cardiomyopathy. Retrospective review of the published clinical data suggested that cardiomyopathy was not common in the family with D1595H, but rather sinus bradycardia was the predominant clinical finding. However, for R814W, we speculate that an increased window current coupled with enhanced slow inactivation and rate-dependent loss of channel availability provided a unique substrate predisposing myocytes to disordered Na+ and Ca2+ homeostasis leading to myocardial dysfunction.

Cardiac Na+ Channel Dysfunction in Brugada Syndrome Is Aggravated by β1-Subunit

Background—Mutations in the gene encoding the human cardiac Na+ channel α-subunit (hH1) are responsible for chromosome 3–linked congenital long-QT syndrome (LQT3) and idiopathic ventricular fibrillation (IVF). An auxiliary β1-subunit, widely expressed in excitable tissues, shifts the voltage dependence of steady-state inactivation toward more negative potentials and restores normal gating kinetics of brain and skeletal muscle Na+ channels expressed in Xenopus oocytes but has little if any functional effect on the cardiac isoform. Here, we characterize the altered effects of a human β1-subunit (hβ1) on the heterologously expressed hH1 mutation (T1620M) previously associated with IVF. Methods and Results—When expressed alone in Xenopus oocytes, T1620M exhibited no persistent currents, in contrast to the LQT3 mutant channels, but the midpoint of steady-state inactivation (V1/2) was significantly shifted toward more positive potentials than for wild-type hH1. Coexpression of hβ1 did not significantly alter cu...