Javad Jabbari

Mutations in the potassium channel subunit KCNE1 are associated with early-onset familial atrial fibrillation

BackgroundAtrial fibrillation (AF) is the most common arrhythmia. The potassium current IKs is essential for cardiac repolarization. Gain-of-function mutations in KV7.1, the pore-forming α-subunit of the IKs channel, have been associated with AF. We hypothesized that early-onset lone AF is associated with mutations in the IKs channel regulatory subunit KCNE1.MethodsIn 209 unrelated early-onset lone AF patients (< 40 years) the entire coding sequence of KCNE1 was bidirectionally sequenced. We analyzed the identified KCNE1 mutants electrophysiologically in heterologous expression systems.ResultsTwo non-synonymous mutations G25V and G60D were found in KCNE1 that were not present in the control group (n = 432 alleles) and that have not previously been reported in any publicly available databases or in the exom variant server holding exom data from more than 10.000 alleles. Proband 1 (female, age 45, G25V) had onset of paroxysmal AF at the age of 39 years. Proband 2 (G60D) was diagnosed with lone AF at the age of 33 years. The patient has inherited the mutation from his mother, who also has AF. Both probands had no mutations in genes previously associated with AF. In heterologous expression systems, both mutants showed significant gain-of-function for IKs both with respect to steady-state current levels, kinetic parameters, and heart rate-dependent modulation.ConclusionsMutations in KV7.1 leading to gain-of-function of IKs current have previously been described in lone AF, yet this is the first time a mutation in the beta-subunit KCNE1 is associated with the disease. This finding further supports the hypothesis that increased potassium current enhances AF susceptibility.

New Exome Data Question the Pathogenicity of Genetic Variants Previously Associated With Catecholaminergic Polymorphic Ventricular Tachycardia

Background—Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal, rare hereditary disease with an estimated prevalence of 1:10 000. The genetic variants that cause CPVT are usually highly penetrant. To date, about 189 variants in 5 genes (RYR2, CASQ2, CALM1, TRND, and KCNJ2) have been associated with CPVT pathogenesis. Methods and Results—The Exome Sequencing Project database (ESP; n= 6503) was systematically searched for previously published missense and nonsense CPVT–associated variants reported in several comprehensive reviews and in 2 databases: The Human Gene Mutation Database and The Inherited Arrhythmias Database. We used 4 different prediction tools to assess all missense variants previously associated with CPVT and compared the prediction of protein damage between CPVT-associated variants identified in the ESP and those variants not identified in the ESP. We identified 11% of the variants previously associated with CPVT in the ESP population. In the literature, 57% of these variants were reported as novel disease-causing variants absent in the healthy control subjects. These putative CPVT variants were identified in 41 out of 6131 subjects in the ESP population, corresponding to a prevalence of CPVT of up to 1:150. Using an agreement of ≥3, in silico prediction tools showed a significantly higher frequency of damaging variants among the CPVT-associated variants not identified in the ESP database (83%) compared with those variants identified in the ESP (50%; P=0.021). Conclusions—We identified a substantial overrepresentation of CPVT-associated variants in a large exome database, suggesting that these variants are not necessarily the monogenic cause of CPVT.

Screening of KCNN3 in patients with early-onset lone atrial fibrillation.

AIMS The aim of this study was to screen KCNN3 encoding the small-conductance calcium-activated K+ channel (SK3) in lone atrial fibrillation patients. Atrial fibrillation (AF) is the most common cardiac arrhythmia. A genome-wide association study has recently associated an intronic single-nucleotide polymorphism (SNP) in KCNN3 with lone AF. METHODS AND RESULTS We sequenced the coding region and splice junctions of KCNN3 in 209 early-onset lone AF patients, screening for variations. A group of 208 healthy blood donors with normal ECGs and without cardiac symptoms were used as controls. All patients and controls were of Danish ethnicity. No mutations were found in the coding regions or splice sites of KCNN3. We found one known exonic synonymous SNP (rs1131820) in KCNN3 that was associated with AF. Both the genotype distribution and allele frequencies of SNP rs1131820 were significantly different between the AF cases and controls (PGenotype=0.047 and PAllele=0.027). Being a homozygous carrier of the major allele (GG) vs. the minor allele (AA) of rs1131820 was associated with an odds ratio of 2.85 (95% CI 1.13-7.18, P=0.026) for lone AF. CONCLUSIONS In this study of 209 young lone AF patients, we found no mutations in the exons or splice sites of KCNN3, but we found an association between the synonymous SNP rs1131820 in KCNN3 and lone AF.

Rare Variants in GJA5 Are Associated With Early-Onset Lone Atrial Fibrillation

BACKGROUND Genetic factors are believed to be important in early-onset lone atrial fibrillation (AF). The gene GJA5 encodes the gap-junction protein Cx40, which together with Cx43 is responsible for the electrical coupling of the atrial cardiomyocytes. The regulatory single nucleotide polymorphism rs10465885 in GJA5 was recently associated with early-onset lone AF (< 60 years) and was also found to be strongly associated with Cx40 messenger RNA levels. We hypothesized that this gene would have a strong effect in patients with a more selected phenotype, and that the findings regarding rs10465885 could be replicated in this group. METHODS The coding region and flanking intron sequences of GJA5 were resequenced in 342 patients with onset of lone AF before the age of 50 (mean age at onset 34 ± 9 years), and in 216 controls. The single nucleotide polymorphism rs10465885 was genotyped in 342 patients and 534 control subjects and odds ratios were calculated for different genetic models. RESULTS Genotyping of rs10465885 showed that the patients with early-onset lone AF were more likely to carry the A allele compared with controls (odds ratio = 1.30; P = 0.011). When resequencing GJA5, we identified the mutation A96S, previously associated with lone AF, which was not present in our control subjects or in any publicly available database or the National Heart, Lung, and Blood Institute Exome Variant Server (NHLBI EVS; 10,758 alleles). CONCLUSIONS We show a highly significant association between the A allele of rs10465885 and onset of lone AF before age 50. This opposes a previous study, wherein the G allele was found to be associated with AF, and makes it impossible to exclude that the associations are coincidental.

Genetic Loci on Chromosomes 4q25, 7p31, and 12p12 Are Associated With Onset of Lone Atrial Fibrillation Before the Age of 40 Years

BACKGROUND Three distinct genetic loci on chromosomes 1q21, 4q25, and 16q22 have been associated with atrial fibrillation (AF) in genome-wide association studies (GWAS). Five additional loci have been associated primarily with the PR interval and subsequently with AF. We aimed to investigate if 8 single nucleotide polymorphisms (SNPs), representing the 8 genomic loci previously linked with AF in genome-wide association studies, were associated with early-onset lone AF. METHODS We included 209 patients with early-onset lone AF, and a control group consisting of 534 individuals free of AF. The 8 SNPs were genotyped using TaqMan assays (Applied Biosystems, Foster City, CA). RESULTS Three SNPs were found to be significantly associated with early-onset lone AF: rs2200733 closest to PITX2 (odds ratio [OR], 1.62; 95% confidence interval [CI], 1.16-2.27; P = 0.004), rs3807989 near to CAV1 (OR 1.35; 95% CI, 1.06-1.72; P = 0.015), and rs11047543 near to SOX5 (OR 1.70; 95% CI, 1.18-2.44; P = 0.004). When correcting for multiple testing, rs2200733 and rs11047543 were still significantly associated with AF. CONCLUSIONS Three SNPs, rs2200733 (4q25), rs3807989 (7p31), and rs11047543 (12p12), were associated with early-onset lone AF. All 3 SNPs are positioned close to genes that in previous studies have been demonstrated to be important for cardiac morphology/development, thereby suggesting a link between these SNPs and structural heart disease. Our results however, indicate that variants in these 3 loci are associated with AF through mechanisms that do not involve major structural abnormalities in the heart.

Incidence and Risk Factors of Ventricular Fibrillation Before Primary Angioplasty in Patients With First ST-Elevation Myocardial Infarction: A Nationwide Study in Denmark

Background We aimed to investigate the incidence and risk factors for ventricular fibrillation (VF) before primary percutaneous coronary intervention (PPCI) among patients with ST‐segment elevation myocardial infarction (STEMI) in a prospective nationwide setting. Methods and Results In this case‐control study, patients presenting within the first 12 hours of first STEMI who survived to undergo angiography and subsequent PPCI were enrolled. Over 2 years, 219 cases presenting with VF before PPCI and 441 controls without preceding VF were enrolled. Of the 219 case patients, 182 (83%) had STEMI with out‐of‐hospital cardiac arrest due to VF, and 37 (17%) had cardiac arrest upon arrival to the emergency room. Medical history was collected by standardized interviews and by linkage to national electronic health records. The incidence of VF before PPCI among STEMI patients was 11.6%. Multivariable logistic regression analysis identified novel associations between atrial fibrillation and alcohol consumption with VF. Patients with a history of atrial fibrillation had a 2.80‐fold odds of experiencing VF before PPCI (95% CI 1.10 to 7.30). Compared with nondrinkers, patients who consumed 1 to 7 units, 8 to 14 units, or >15 units of alcohol per week had an odds ratio (OR) of 1.30 (95% CI, 0.80 to 2.20), 2.30 (95% CI, 1.20 to 4.20), or 3.30 (95% CI, 1.80 to 5.90), respectively, for VF. Previously reported associations for preinfarction angina (OR 0.46; 95% CI 0.32 to 0.67), age of <60 years (OR 1.75; 95% CI 1.20 to 2.60), anterior infarction (OR 2.10; 95% CI 1.40 to 3.00), preprocedural thrombolysis in myocardial infarction flow grade 0 (OR 1.65; 95% CI 1.14 to 2.40), and family history of sudden death (OR 1.60; 95% CI 1.10 to 2.40) were all associated with VF. Conclusion Several easily assessed risk factors were associated with VF occurring out‐of‐hospital or on arrival at the emergency room before PPCI in STEMI patients, thus providing potential avenues for investigation regarding improved identification and prevention of life‐threatening ventricular arrhythmias.

Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations.

Background and aim The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles. Methods Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times). Results Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples. Conclusion This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.

Very early-onset lone atrial fibrillation patients have a high prevalence of rare variants in genes previously associated with atrial fibrillation

BACKGROUND Atrial fibrillation (AF) is the most common cardiac arrhythmia. Currently, 14 genes important for ion channel function, intercellular signaling, and homeostatic control have been associated with AF. OBJECTIVE We hypothesized that rare genetic variants in genes previously associated with AF had a higher prevalence in early-onset lone AF patients than in the background population. METHODS Sequencing results of KCNQ1, KCNH2, SCN5A, KCNA5, KCND3, KCNE1, 2, 5, KCNJ2, SCN1-3B, NPPA, and GJA5 from 192 early-onset lone AF patients were compared with data from the National Heart, Lung, and Blood Institute Exome Variant Server consisting of 6503 persons from 18 different cohort studies. RESULTS Among the lone AF patients, 29 (7.6%) alleles harbored a novel or very rare variant (minor allele frequency <0.1 in the Exome Variant Server), a frequency that was significantly higher than what was found in the reference database (4.1%; with minor allele frequency <0.1; P = .0012). Previously published electrophysiological data showed that 96% (n = 23) of the rare variants that has been functionally investigated (n = 24) displayed significant functional changes. CONCLUSIONS We report a much higher prevalence of rare variants in genes associated with AF in early-onset lone AF patients than in the background population. By presenting these data, we believe that we are the first to provide quantitative evidence for the role of rare variants across AF susceptibility genes as a possible pathophysiological substrate for AF.

Common and rare variants in SCN10A modulate the risk of atrial fibrillation.

Background—Genome-wide association studies have shown that the common single nucleotide polymorphism rs6800541 located in SCN10A, encoding the voltage-gated Nav1.8 sodium channel, is associated with PR-interval prolongation and atrial fibrillation (AF). Single nucleotide polymorphism rs6800541 is in high linkage disequilibrium with the nonsynonymous variant in SCN10A, rs6795970 (V1073A, r2=0.933). We therefore sought to determine whether common and rare SCN10A variants are associated with early onset AF. Methods and Results—SCN10A was sequenced in 225 AF patients in whom there was no evidence of other cardiovascular disease or dysfunction (lone AF). In an association study of the rs6795970 single nucleotide polymorphism variant, we included 515 AF patients and 2 control cohorts of 730 individuals free of AF and 6161 randomly sampled individuals. Functional characterization of SCN10A variants was performed by whole-cell patch-clamping. In the lone AF cohort, 9 rare missense variants and 1 splice site donor variant were detected. Interestingly, AF patients were found to have higher G allele frequency of rs6795970, which encodes the alanine variant at position 1073 (described from here on as A1073, odds ratio =1.35 [1.16−1.54]; P=2.3×10−5). Both of the common variants, A1073 and P1092, induced a gain-of-channel function, whereas the rare missense variants, V94G and R1588Q, resulted in a loss-of-channel function. Conclusions—The common variant A1073 is associated with increased susceptibility to AF. Both rare and common variants have effect on the function of the channel, indicating that these variants influence susceptibility to AF. Hence, our study suggests that SCN10A variations are involved in the genesis of AF.

Common polymorphisms in KCNJ5 [corrected] are associated with early-onset lone atrial fibrillation in Caucasians.

Objectives: The aim of this study was to screen lone atrial fibrillation (AF) patients for mutations in the genes KCNJ2, KCNJ3 and KCNJ5, all encoding potassium channels. Furthermore, we wanted to replicate the prior association of two single-nucleotide polymorphisms (SNPs) in KCNJ5, C171T (rs6590357) and G810T (rs7118824), with lone AF in Han Chinese. Methods: We sequenced the coding region and splice site of KCNJ2, KCNJ3 and KCNJ5 in 187 early-onset lone-AF patients screening for mutations and counting SNP frequencies for the two noted SNPs in KCNJ5. Results: No mutations were found in KCNJ2, KCNJ3 or KCNJ5. Both genotype distribution and allele frequencies of the SNPs rs6590357 and rs7118824 significantly differed between the AF and control group (pgenotype = 0.0067, pallele = 0.0021 and pgenotype = 0.014, pallele = 0.0101, respectively). On allele level, the OR for lone AF for rs6590357 was 1.77 (95% CI 1.16–2.73, p = 0.009) and for rs7118824 it was 1.71 (95% CI 1.13–2.57, p = 0.01) in a model adjusted for age and gender. Conclusions: Our findings indicate that rs6590357 and rs7118824 in KCNJ5 are associated with early-onset lone AF in Caucasians. No mutations were found in the exon or splice site of KCNJ2, KCNJ3 or KCNJ5.