Long-QT mutation p.K557E-Kv7.1: dominant-negative suppression of IKs, but preserved cAMP-dependent up-regulation.

AIMS: Mutations in KCNQ1, encoding for Kv7.1, the α-subunit of the IKs channel, cause long-QT syndrome type 1, potentially predisposing patients to ventricular tachyarrhythmias and sudden cardiac death, in particular, during elevated sympathetic tone. Here, we aim at characterizing the p.Lys557Glu (K557E) Kv7.1 mutation, identified in a Dutch kindred, at baseline and during (mimicked) increased adrenergic tone. METHODS AND RESULTS: K557E carriers had moderate QTc prolongation that augmented significantly during exercise. IKs characteristics were determined after co-expressing Kv7.1-wild-type (WT) and/or K557E with minK and Yotiao in Chinese hamster ovary cells. K557E caused IKs loss of function with slowing of the activation kinetics, acceleration of deactivation kinetics, and a rightward shift of voltage-dependent activation. Together, these contributed to a dominant-negative reduction in IKs density. Confocal microscopy and western blot indicated that trafficking of K557E channels was not impaired. Stimulation of WT IKs by 3'-5'-cyclic adenosine monophosphate (cAMP) generated strong current up-regulation that was preserved for K557E in both hetero- and homozygosis. Accumulation of IKs at fast rates occurred both in WT and in K557E, but was blunted in the latter. In a computational model, K557E showed a loss of action potential shortening during ß-adrenergic stimulation, in accordance with the lack of QT shortening during exercise in patients. CONCLUSION: K557E causes IKs loss of function with reduced fast rate-dependent current accumulation. cAMP-dependent stimulation of mutant IKs is preserved, but incapable of fully compensating for the baseline current reduction, explaining the long QT intervals at baseline and the abnormal QT accommodation during exercise in affected patients.

Subepicardial phase 0 block and discontinuous transmural conduction underlie right precordial ST-segment elevation by a SCN5A loss-of-function mutation.

Two mechanisms are generally proposed to explain right precordial ST-segment elevation in Brugada syndrome: 1) right ventricular (RV) subepicardial action potential shortening and/or loss of dome causing transmural dispersion of repolarization; and 2) RV conduction delay. Here we report novel mechanistic insights into ST-segment elevation associated with a Na(+) current (I(Na)) loss-of-function mutation from studies in a Dutch kindred with the COOH-terminal SCN5A variant p.Phe2004Leu. The proband, a man, experienced syncope at age 22 yr and had coved-type ST-segment elevations in ECG leads V1 and V2 and negative T waves in V2. Peak and persistent mutant I(Na) were significantly decreased. I(Na) closed-state inactivation was increased, slow inactivation accelerated, and recovery from inactivation delayed. Computer-simulated I(Na)-dependent excitation was decremental from endo- to epicardium at cycle length 1,000 ms, not at cycle length 300 ms. Propagation was discontinuous across the midmyocardial to epicardial transition region, exhibiting a long local delay due to phase 0 block. Beyond this region, axial excitatory current was provided by phase 2 (dome) of the M-cell action potentials and depended on L-type Ca(2+) current ("phase 2 conduction"). These results explain right precordial ST-segment elevation on the basis of RV transmural gradients of membrane potentials during early repolarization caused by discontinuous conduction. The late slow-upstroke action potentials at the subepicardium produce T-wave inversion in the computed ECG waveform, in line with the clinical ECG.

Mapping a novel locus for familial atrial fibrillation on chromosome 10p11-q21.

BACKGROUND: Atrial Fibrillation (AF), the most common cardiac arrhythmia, is a significant public health problem in the United States, affecting approximately 2.2 million Americans. Recently, several chromosomal loci and genes have been found to be associated with familial AF. However, in most other AF cases, the genetic basis is still poorly understood. OBJECTIVE: The purpose of this study was to investigate the molecular basis of familial AF in a Dutch kindred group. METHODS: We analyzed a four-generation Dutch family in which AF segregated as an autosomal dominant trait. After the exclusion of linkage to 10q22-24, 6q14-16, 5p13, KCNQ1, KCNE2, KCNJ2 and some ion-channel-associated candidate genes, a genome-wide linkage scan using 398 microsatellite markers was performed. RESULTS: Two-point logarithms of odds (LOD) scores >1 at recombination fraction [theta] = 0.00 and a haplotype segregating with the disorder were demonstrated only across regions of chromosome 10. Subsequent fine mapping gave a maximum two-point LOD score of 4.1982 at D10S568 at [theta] = 0.00. Distinct recombination in several individuals narrowed the shared region among all affected individuals to 16.4 cM on the Genethon map (flanking markers: D10S578 and D10S1652), which corresponds to chromosome 10p11-q21. Thirteen candidate genes residing in this region, which could be associated with AF, were screened. No mutation has been found in their coding regions including the intron splice regions. CONCLUSION: We identify a novel locus for AF on chromosome 10p11-q21, which provides further evidence of genetic heterogeneity in this arrhythmia.

Chip-based mtDNA mutation screening enables fast and reliable genetic diagnosis of OXPHOS patients.

PURPOSE: Oxidative phosphorylation is under dual genetic control of the nuclear and the mitochondrial DNA (mtDNA). Oxidative phosphorylation disorders are clinically and genetically heterogeneous, which makes it difficult to determine the genetic defect, and symptom-based protocols which link clinical symptoms directly to a specific gene or mtDNA mutation are falling short. Moreover, approximately 25% of the pediatric patients with oxidative phosphorylation disorders is estimated to have mutations in the mtDNA and a standard screening approach for common mutations and deletions will only explain part of these cases. Therefore, we tested a new CHIP-based screening method for the mtDNA. METHODS: MitoChip (Affymetrix) resequencing was performed on three test samples and on 28 patient samples. RESULTS: Call rates were 94% on average and heteroplasmy detection levels varied from 5-50%. A genetic diagnosis can be made in almost one-quarter of the patients at a potential output of 8 complete mtDNA sequences every 4 days. Moreover, a number of potentially pathogenic unclassified variants (UV) were detected. CONCLUSIONS: The availability of long-range PCR protocols and the predominance of single nucleotide substitutions in the mtDNA make the resequencing CHIP a very fast and reliable method to screen the complete mtDNA for mutations.

Long QT syndrome caused by a large duplication in the KCNH2 (HERG) gene undetectable by current polymerase chain reaction-based exon-scanning methodologies.

BACKGROUND: The numerous mutations in the long QT syndrome (LQTS)-associated genes reported to date are point mutations or small insertions and deletions in coding regions or at splice junctions. OBJECTIVES: The purpose of this study was to determine the relative copy number of gene exons in a series of mutation-negative LQTS probands. METHODS: We used a quantitative multiplex approach because the polymerase chain reaction (PCR)-based exon-scanning methodologies routinely utilized in mutation analysis are unable to detect large genomic alterations. RESULTS: We identified the first large gene rearrangement consisting of a tandem duplication of 3.7 kb in KCNH2 responsible for LQTS in a Dutch family. This large duplication is expected to lead to nonfunctional or severely debilitated channels, thereby decreasing I(Kr). CONCLUSION: Our findings have implications for genetic testing in the approximately 30% of LQTS patients in whom conventional mutation screening fails to uncover a mutation. Analysis for large gene alterations such as the one described herein in routine genetic testing may provide a genetic diagnosis in a number of these patients.

HERG mutation predicts short QT based on channel kinetics but causes long QT by heterotetrameric trafficking deficiency.

OBJECTIVE: Mutations in the KCNH2 (hERG, human ether-à-go-go related gene) gene may cause a reduction of the delayed rectifier current I(Kr), thereby leading to the long QT syndrome (LQTS). The reduced I(Kr) delays the repolarisation of cardiac cells and renders patients vulnerable to ventricular arrhythmias and sudden death. We identified a novel mutation in a LQTS family and investigated its functional consequences using molecular and microscopic techniques. METHODS AND RESULTS: Genetic screening in the LQTS family revealed a heterozygous frameshift mutation p.Pro872fs located in the C-terminus of the KCNH2 gene. The mutation leads to a premature truncation of the C-terminus of the hERG protein. p.Pro872fs channels lack 282 amino acids at the C-terminus and possess an extra 4-amino acid tail. Both the kinetic and biochemical properties of the p.Pro872fs and p.Pro872fs/WT channels were studied in HEK293 cells and resulted in a novel proof of concept for heterozygous LQTS mutations: homotetrameric p.Pro872fs channels displayed near-normal expression, trafficking, and channel kinetics. Unexpectedly, upon co-expression of p.Pro872fs and WT channels, the repolarising power (the proportion of hERG current contributing to the action potential as the percentage of the total current available) was substantially higher during action potential clamp experiments as compared to WT channels alone. This would lead to a shorter rather than a prolonged QT interval. However, at the same time, heterotetramerisation of p.Pro872fs and WT channels also caused a dominant negative effect on trafficking by an increase in ER retention of these heterotetrameric channels, which surpassed the former gain in repolarising power. CONCLUSION: The LQTS phenotype in the studied family is caused by a mutation with novel properties. We demonstrate that a KCNH2 mutation that clinically leads to long QT syndrome causes at the cellular level both a "gain" and a "loss" of HERG channel function due to a kinetic increase in repolarising power and a decrease in trafficking efficiency of heteromultimeric channels.

Novel mutation in the Per-Arnt-Sim domain of KCNH2 causes a malignant form of long-QT syndrome.

BACKGROUND: It has been proposed that the highest risk for cardiac events in patients with long-QT syndrome subtype 2 (LQT2) is related to mutations in the pore region of the KCNH2 channel. It has also been suggested that a subpopulation of LQT2 patients may benefit from pharmacological therapy with modified KCNH2 channel-blocking drugs. METHODS AND RESULTS: In a large LQT2 family (n=33), we have identified a novel nonpore missense mutation (K28E) in the Per-Arnt-Sim (PAS) domain of the KCNH2 channel associated with a malignant phenotype: One third of the suspected gene carriers experienced a major cardiac event. Wild-type and K28E-KCNH2 channels were transiently transfected in HEK293 cells. For the mutant channel, whole-cell patch-clamp analysis showed a reduced current density, a negative shift of voltage-dependent channel availability, and an increased rate of deactivation. Western blot analysis and confocal imaging revealed a trafficking deficiency for the mutant channel that could be rescued by the K+ channel blocker E-4031. In cells containing both wild-type and mutant channels, deactivation kinetics were normal. In these cells, reduced current density was restored with E-4031. CONCLUSIONS: Our data suggest that besides pore mutations, mutations in the PAS domain may also exhibit a malignant outcome. Pharmacological restoration of current density is promising as a mutation-specific therapy for patients carrying this trafficking-defective mutant.

A novel mutation L619F in the cardiac Na+ channel SCN5A associated with long-QT syndrome (LQT3): a role for the I-II linker in inactivation gating.

Congenital long QT syndrome type 3 (LQT3) is caused by mutations in the gene SCN5A encoding the alpha-subunit of the cardiac Na(+) channel (Nav1.5). Functional studies of SCN5A mutations in the linker between domains III and IV, and more recently the C-terminus, have been shown to alter inactivation gating. Here we report a novel LQT3 mutation, L619F (LF), located in the domain I-II linker. In an infant with prolonged QTc intervals, mutational analysis identified a heterozygous missense mutation (L619F) in the domain I-II linker of the cardiac Na(+) channel. Wild-type (WT) and mutant channels were studied by whole-cell patch-clamp analysis in transiently expressed HEK cells. LF channels increase maintained Na(+) current (0.79 pA/pF for LF; 0.26 pA/pF for WT) during prolonged depolarization. We found a +5.8mV shift in steady state inactivation in LF channels compared to WT (WT, V(1/2)=-64.0 mV; LF, V(1/2)=-58.2 mV). The positive shift of inactivation, without a corresponding shift in activation, increases the overlap window current in LF relative to WT (1.09 vs. 0.58 pA/pF), as measured using a positive voltage ramp protocol (-100 to +50 mV in 2s). The increase in window current, combined with an increase in non-inactivating Na(+) current, may act to prolong the AP plateau and is consistent with the disease phenotype observed in patients. Moreover, the defective inactivation imposed by the L619F mutation implies a role for the I-II linker in the Na(+) channel inactivation process.

Variable clinical manifestation of a novel missense mutation in the alpha-tropomyosin (TPM1) gene in familial hypertrophic cardiomyopathy.

OBJECTIVES: This study was initiated to identify the disease-causing genetic defect in a family with hypertrophic cardiomyopathy (HCM) and high incidence of sudden death. BACKGROUND: Familial hypertropic cardiomyopathy (FHC) is an autosomal dominant transmitted disorder that is genetically and clinically heterogeneous. Mutations in 11 genes have been associated with the pathogenesis of the disease. METHODS: We studied a large FHC family, first by linkage analysis, to identify the gene involved, and subsequently screened the gene, encoding alpha-tropomyosin (TPM1), for mutations by using single-strand conformation polymorphism and sequencing analysis. RESULTS: Twelve family members presented clinical features of HCM, five of whom died at young age, while others had only mild clinical features. Marker analysis showed linkage for the TPM1 gene on chromosome 15q22 (maximal logarithm of the odds score is 5.16, theta = 0); subsequently, a novel missense mutation (Glu62Gln) was identified. CONCLUSIONS: The novel mutation identified in TPM1 is associated with the clinical features of cardiac hypertrophy in all but one genetically affected member of this large family. The clinical data suggest a malignant phenotype at young age with a variable clinical manifestation and penetrance at older age. The Glu62Gln mutation is the sixth TPM1 mutation identified as the cause of FHC, indicating that mutations in this gene are very rare. This is the first reported amino acid substitution at the f-position within the coiled-coil structure of the tropomyosin protein.