Lena Refsgaard

New population-based exome data are questioning the pathogenicity of previously cardiomyopathy-associated genetic variants

Cardiomyopathies are a heterogeneous group of diseases with various etiologies. We focused on three genetically determined cardiomyopathies: hypertrophic (HCM), dilated (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC). Eighty-four genes have so far been associated with these cardiomyopathies, but the disease-causing effect of reported variants is often dubious. In order to identify possible false-positive variants, we investigated the prevalence of previously reported cardiomyopathy-associated variants in recently published exome data. We searched for reported missense and nonsense variants in the NHLBI-Go Exome Sequencing Project (ESP) containing exome data from 6500 individuals. In ESP, we identified 94 variants out of 687 (14%) variants previously associated with HCM, 58 out of 337 (17%) variants associated with DCM, and 38 variants out of 209 (18%) associated with ARVC. These findings correspond to a genotype prevalence of 1:4 for HCM, 1:6 for DCM, and 1:5 for ARVC. PolyPhen-2 predictions were conducted on all previously published cardiomyopathy-associated missense variants. We found significant overrepresentation of variants predicted as being benign among those present in ESP compared with the ones not present. In order to validate our findings, seven variants associated with cardiomyopathy were genotyped in a control population and this revealed frequencies comparable with the ones found in ESP. In conclusion, we identified genotype prevalences up to more than one thousand times higher than expected from the phenotype prevalences in the general population (HCM 1:500, DCM 1:2500, and ARVC 1:5000) and our data suggest that a high number of these variants are not monogenic causes of cardiomyopathy.

High prevalence of genetic variants previously associated with LQT syndrome in new exome data.

To date, hundreds of variants in 13 genes have been associated with long QT syndrome (LQTS). The prevalence of LQTS is estimated to be between 1:2000 and 1:5000. The knowledge of genetic variation in the general population has until recently been limited, but newly published data from NHLBI GO Exome Sequencing Project (ESP) has provided important knowledge on this topic. We aimed to investigate the prevalence of previously LQTS-associated variants in ESP (5400 individuals), in order to identify possible false-positive LQTS variants. With this aim, we performed a search for previously published LQTS-associated variants in ESP. In addition, a PolyPhen-2 prediction was conducted, and the four most prevalent LQTS-associated variants with significant functional effects present in ESP were genotyped in a second control population. We identified 33 missense variants previously associated with LQTS in ESP. These 33 variants affected 173 alleles and this corresponded to a LQTS prevalence of 1:31 in the ESP population. PolyPhen-2 predicted 30% of the 33 variants present in ESP to be benign compared with 13% among LQTS-associated variants not present in ESP (P=0.019). Genotyping of the four variants KCNH2 P347S; SCN5A: S216L, V1951L; and CAV3 T78M in the control population (n=704) revealed prevalences comparable to those of ESP. Thus, we identified a much higher prevalence of previously LQTS-associated variants than expected in exome data from population studies. Great caution regarding the possible disease causation of some of these variants has to be taken, especially when used for risk stratification in family members.

A Novel KCND3 Gain-of-Function Mutation Associated with Early-Onset of Persistent Lone Atrial Fibrillation

AIMS Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current (Ito) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K(+) Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. METHODS AND RESULTS Two hundred and nine unrelated early-onset lone AF patients (<40 years) were recruited. The entire coding sequence of KCND3 and KCNIP2 was bidirectionally sequenced. One novel non-synonymous mutation A545P was found in KCND3 and was neither present in the control group (n = 432 alleles) nor in any publicly available database. The proband had onset of persistent AF at the age of 22, and no mutations in genes previously associated with AF were found. Electrophysiological analysis of KV4.3-A545P expressed in CHO-K1 cells, revealed that peak-current density was increased and the onset of inactivation was slower compared with WT, resulting in a significant gain-of-function both in the absence and the presence of KChIP2. CONCLUSION Gain-of-function mutations in KV4.3 have previously been described in Brugada Syndrome, however, this is the first report of a KV4.3 gain-of-function mutation in early-onset lone AF. This association of KV4.3 gain-of-function and early-onset lone AF further supports the hypothesis that increased potassium current enhances AF susceptibility.

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.

Sodium Current and Potassium Transient Outward Current Genes in Brugada Syndrome: Screening and Bioinformatics

BACKGROUND Brugada syndrome (BrS) is a primary arrhythmia syndrome characterized by the occurrence of malignant ventricular arrhythmias. Previously, the genes SCN1B, SCN3B, MOG1, and KCND3 have been associated with BrS. Recent data from exome screening efforts permit better discrimination between low-frequency genetic variants and true monogenetic disease-causing variants. We aimed to screen the genes SCN1B through SCN4B, MOG1, CAV3, and KCND3 for variations in a population of SCN5A negative Danish and Iranian BrS patients, as well as research prior associations using newly released exome data. METHODS Screening of all exons and splice sites was performed using Sanger sequencing. Bioinformatic searches were performed in the Single-nucleotide polymorphism database (build 132) and in the National Heart, Lung, and Blood Institute Grand Opportunity Exome Sequencing Project (ESP) for both previously published variant-BrS associations and newly uncovered variations within the noted genes. RESULTS A total of 42 BrS patients were screened, and 2 different nonsynonymous mutations in SCN1Bb (H162P and R214Q) were found in 2 different Danish patients. The variants were not found in 216 Danish controls, but R214Q was present in ESP data (5 of 841 alleles). No other mutations were found. Previously BrS-associated mutations in KNCD3 and SCN3B were also present in ESP data. This was not the case for MOG1, but a nonsense polymorphism was present in 0.5% of alleles. CONCLUSIONS Our study supports the association of SCN1Bb with BrS. However, recently released exome data make some of the prior associations of BrS with genes SCN3B, MOG1, and KCND3 less likely.

Brugada syndrome risk loci seem protective against atrial fibrillation.

Several studies have shown an overlap between genes involved in the pathophysiological mechanisms of atrial fibrillation (AF) and Brugada Syndrome (BrS). We investigated whether three single-nucleotide polymorphisms (SNPs) (rs11708996; G>C located intronic to SCN5A, rs10428132; T>G located in SCN10A, and rs9388451; T>C located downstream to HEY2) at loci associated with BrS in a recent genome-wide association study (GWAS) also were associated with AF. A total of 657 patients diagnosed with AF and a control group comprising 741 individuals free of AF were included. The three SNPs were genotyped using TaqMan assays. The frequencies of risk alleles in the AF population and the control population were compared in two-by-two models. One variant, rs10428132 at SCN10A, was associated with a statistically significant decreased risk of AF (odds ratio (OR)=0.77, P=0.001). A meta-analysis was performed by enriching the control population with allele frequencies from controls in the recently published BrS GWAS (2230 alleles). In this meta-analysis, both rs10428132 at SCN10A (OR=0.73, P=5.7 × 10−6) and rs11708996 at SCN5A (OR=0.80, P=0.02) showed a statistically significant decreased risk of AF. When assessing the additive effect of the three loci, we found that the risk of AF decreased in a dose-responsive manner with increasing numbers of risk alleles (OR=0.50, P=0.001 for individuals carrying ≥4 risk alleles vs ≤1 allele). In conclusion, the prevalence of three risk alleles previously associated with BrS was lower in AF patients than in patients free of AF, suggesting a protective role of these loci in developing AF.

Screening of the Ito Regulatory Subunit Klf15 in Patients with Early-Onset Lone Atrial Fibrillation

Several studies have associated mutations in genes encoding potassium channels and accessory subunits involved in cardiac repolarization with increased susceptibility of atrial fibrillation (AF). Recently, the Krüppel-like factor 15 (Klf15) was found to transcriptionally control rhythmic expression of KChIP2, a critical subunit required for generating the transient outward potassium current (Ito), and that deficiency or excess of Klf15 increased the susceptibility of arrhythmias. On this basis we hypothesized that mutations in Klf15 could be associated with AF. A total of 209 unrelated Caucasian lone AF patients were screened for mutations in Klf15 by direct sequencing. No mutations in the lone AF cohort were found. In one patient we found a synonymous variant (c.36C > T). In NHLBI GO Exome Sequencing Project (ESP) the variant was present in 31 of 4269 Caucasian individuals and in 3 of 2200 African Americans. In our cohort Klf15 was not associated with lone AF.

Mutation analysis of the candidate genes SCN1B-4B, FHL1, and LMNA in patients with arrhythmogenic right ventricular cardiomyopathy

Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically determined heart disease characterized by fibrofatty infiltrations in the myocardium, right and/or left ventricular involvement, and ventricular tachyarrhythmias. Although ten genes have been associated with ARVC, only about 40% of the patients have an identifiable disease-causing mutation. In the present study we aimed at investigating the involvement of the genes SCN1B-SCN4B, FHL1, and LMNA in the pathogenesis of ARVC. Methods Sixty-five unrelated patients (55 fulfilling ARVC criteria and 10 borderline cases) were screened for variants in SCN1B-4B, FHL1, and LMNA by direct sequencing and LightScanner melting curve analysis. Results A total of 28 sequence variants were identified: seven in SCN1B, three in SCN2B, two in SCN3B, two in SCN4B, four in FHL1, and ten in LMNA. Three of the variants were novel. One of the variants was non-synonymous. No disease-causing mutations were identified. Conclusions In our limited sized cohort the six studied candidate genes were not associated with ARVC.

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a PITX2 p.M200V mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the specific PITX2 gene have been identified in patients with early-onset AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a PITX2 p.M200V mutation, using a commercially available non-integrating expression system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the PITX2 p.M200V mutation.

Rare truncating variants in the sarcomeric protein titin associate with familial and early-onset atrial fibrillation

A family history of atrial fibrillation constitutes a substantial risk of developing the disease, however, the pathogenesis of this complex disease is poorly understood. We perform whole-exome sequencing on 24 families with at least three family members diagnosed with atrial fibrillation (AF) and find that titin-truncating variants (TTNtv) are significantly enriched in these patients (P = 1.76 × 10−6). This finding is replicated in an independent cohort of early-onset lone AF patients (n = 399; odds ratio = 36.8; P = 4.13 × 10−6). A CRISPR/Cas9 modified zebrafish carrying a truncating variant of titin is used to investigate TTNtv effect in atrial development. We observe compromised assembly of the sarcomere in both atria and ventricle, longer PR interval, and heterozygous adult zebrafish have a higher degree of fibrosis in the atria, indicating that TTNtv are important risk factors for AF. This aligns with the early onset of the disease and adds an important dimension to the understanding of the molecular predisposition for AF.Common genetic variants in structural proteins contribute to risk of atrial fibrillation (AF). Here, using whole-exome sequencing, the authors identify rare truncating variants in TTN that associate with familial and early-onset AF and show defects in cardiac sarcomere assembly in ttn.2-mutant zebrafish.