A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

REduction of THRomboembolic EVents during Ablation using the laserballoon: The RETHREVA registry.

INTRODUCTION: Cerebral events (CEs), including silent (SCEs), are a known complication of left atrial catheter ablation (LACA) in patients with atrial fibrillation. The aim of this prospective registry was to gain more information about CEs during laserballoon LACA and to reduce the risk of their occurrence. METHODS AND RESULTS: We enrolled 74 patients (age 61 ± 11 years; 74% male; CHA2 DS2 -VASc 1.9 ± 1.4). Cerebral MRI (1.5 Tesla) was performed to detect CEs. ASPItest identified aspirin-resistant patients (ARPs). All bleeding complications were recorded. Due to an initial high CE rate, we evolved our clinical procedure step-by-step arriving at an optimized protocol: -Group 1: heparin after single transseptal puncture (TP), activated clotting time (ACT) > 300 seconds (CE: 64.3%). -Group 2: heparin after double TP, ACT > 300 seconds; 2a without (CE: 45.5%, RRR: -29.2%) and 2b with additional intravenous aspirin (CE: 36.4%, RRR: -43.4%; excluding ARP: 30%, RRR: -53.3%). -Group 3: heparin before double TP, ACT > 350 seconds; 3a without (CE: 54.5%, RRR: -15.2%) and 3b with aspirin (CE: 18.5%, RRR: -71.2%; excluding ARP: 8.7%, RRR: -86.5%). Larger LA diameter > 44 mm (OR: 1.149, P  =  0.005) and no aspirin use (OR: 4.308, P  =  0.008) were CE risk factors in multivariate logistic regression. In those patients receiving aspirin, aspirin resistance (OR: 22.4, P  =  0.011) was an exceptionally strong risk factor. CONCLUSION: These data support the use of intravenous aspirin including monitoring of aspirin resistance in addition to ACT-guided heparin. An optimized protocol of heparin before TP, double TP, and intravenous aspirin in non-ARP resulted in a significantly lowered CE incidence and severity.

NFAT5 and SLC4A10 Loci Associate with Plasma Osmolality.

Disorders of water balance, an excess or deficit of total body water relative to body electrolyte content, are common and ascertained by plasma hypo- or hypernatremia, respectively. We performed a two-stage genome-wide association study meta-analysis on plasma sodium concentration in 45,889 individuals of European descent (stage 1 discovery) and 17,637 additional individuals of European descent (stage 2 replication), and a transethnic meta-analysis of replicated single-nucleotide polymorphisms in 79,506 individuals (63,526 individuals of European descent, 8765 individuals of Asian Indian descent, and 7215 individuals of African descent). In stage 1, we identified eight loci associated with plasma sodium concentration at P<5.0 × 10-6 Of these, rs9980 at NFAT5 replicated in stage 2 meta-analysis (P=3.1 × 10-5), with combined stages 1 and 2 genome-wide significance of P=5.6 × 10-10 Transethnic meta-analysis further supported the association at rs9980 (P=5.9 × 10-12). Additionally, rs16846053 at SLC4A10 showed nominally, but not genome-wide, significant association in combined stages 1 and 2 meta-analysis (P=6.7 × 10-8). NFAT5 encodes a ubiquitously expressed transcription factor that coordinates the intracellular response to hypertonic stress but was not previously implicated in the regulation of systemic water balance. SLC4A10 encodes a sodium bicarbonate transporter with a brain-restricted expression pattern, and variant rs16846053 affects a putative intronic NFAT5 DNA binding motif. The lead variants for NFAT5 and SLC4A10 are cis expression quantitative trait loci in tissues of the central nervous system and relevant to transcriptional regulation. Thus, genetic variation in NFAT5 and SLC4A10 expression and function in the central nervous system may affect the regulation of systemic water balance.

An enhancer polymorphism at the cardiomyocyte intercalated disc protein NOS1AP locus is a major regulator of the QT interval.

QT interval variation is assumed to arise from variation in repolarization as evidenced from rare Na- and K-channel mutations in Mendelian QT prolongation syndromes. However, in the general population, common noncoding variants at a chromosome 1q locus are the most common genetic regulators of QT interval variation. In this study, we use multiple human genetic, molecular genetic, and cellular assays to identify a functional variant underlying trait association: a noncoding polymorphism (rs7539120) that maps within an enhancer of NOS1AP and affects cardiac function by increasing NOS1AP transcript expression. We further localized NOS1AP to cardiomyocyte intercalated discs (IDs) and demonstrate that overexpression of NOS1AP in cardiomyocytes leads to altered cellular electrophysiology. We advance the hypothesis that NOS1AP affects cardiac electrical conductance and coupling and thereby regulates the QT interval through propagation defects. As further evidence of an important role for propagation variation affecting QT interval in humans, we show that common polymorphisms mapping near a specific set of 170 genes encoding ID proteins are significantly enriched for association with the QT interval, as compared to genome-wide markers. These results suggest that focused studies of proteins within the cardiomyocyte ID are likely to provide insights into QT prolongation and its associated disorders.

Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics.

Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes involved in the Mendelian disorder long QT syndrome (LQTS). We integrated the LQTS network with GWAS loci from the corresponding common complex trait, QT-interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LQTS protein network to filter weak GWAS signals by identifying single-nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy to propose candidates in GWAS loci for functional studies and to systematically filter subtle association signals using tissue-specific quantitative interaction proteomics.

Electrophysiological characterization of a large set of novel variants in the SCN5A-gene: identification of novel LQTS3 and BrS mutations.

SCN5A encodes for the α-subunit of the cardiac voltage-gated sodium channel Nav1.5. Gain-of-function mutations in SCN5A are related to congenital long QT syndrome (LQTS3) characterized by delayed cardiac repolarization, leading to a prolonged QT interval in the ECG. Loss-of-function mutations in SCN5A are related to Brugada syndrome (BrS), characterized by an ST-segment elevation in the right precordial leads (V1-V3). The aim of this study was the characterization of a large set of novel SCN5A variants found in patients with different cardiac phenotypes, mainly LQTS and BrS. SCN5A variants of 13 families were functionally characterized in Xenopus laevis oocytes using the two-electrode voltage-clamp technique. We found in most of the cases, but not all, that the electrophysiology of the variants correlated with the clinically diagnosed phenotype. A susceptibility to develop LQTS can be suggested in patients carrying the variants S216L, K480N, A572D, F816Y, and G983D. However, taking the phenotype into account, the presence of the variants in genomic data bases, the mutational segregation, combined with our in vitro and in silico experiments, the variants S216L, S262G, K480N, A572D, F816Y, G983D, and T1526P remain as variants of unknown significance. However, the SCN5A variants R568H and A993T can be classified as pathogenic LQTS3 causing mutations, while R222stop and R2012H are novel BrS causing mutations.

SNiPA: an interactive, genetic variant-centered annotation browser.

MOTIVATION: Linking genes and functional information to genetic variants identified by association studies remains difficult. Resources containing extensive genomic annotations are available but often not fully utilized due to heterogeneous data formats. To enhance their accessibility, we integrated many annotation datasets into a user-friendly webserver. AVAILABILITY AND IMPLEMENTATION: http://www.snipa.org/ CONTACT: g.kastenmueller@helmholtz-muenchen.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Candidate gene variants of the immune system and sudden infant death syndrome.

BACKGROUND: Sudden infant death syndrome (SIDS) causes early infant death with an incidence between 0.5 and 2.5 cases among 1000 live births. Besides central sleep apnea and thermal dysregulation, infections have been repeatedly suggested to be implicated in SIDS etiology. METHODS: To test the risk contribution of common genetic variants related to infection, we genotyped 40 single-nucleotide polymorphisms (SNPs) from 15 candidate genes for association with SIDS in a total of 579 cases and 1124 controls from Germany and the UK in a two-stage case control design. RESULTS: The discovery-stage series (267 SIDS cases and 303 controls) revealed nominally significant associations for variants in interleukin 6 (IL6) (rs1880243), interleukin 10 (IL10) (rs1800871, rs1800872), and mannose-binding lectin 2 (MBL2) (rs930506), and for several other variants in subgroups. Meta-analyses were then performed in adding genotype information from a genome-wide association study of another 312 European SIDS cases and 821 controls. Overall associations were observed for two independent variants in MBL2: rs930506 in a co-dominant model (odds ratio (OR) = 0.82, p = 0.04) and rs1838065 in a dominant model (OR = 1.27, p = 0.03). CONCLUSION: Our study did not replicate published associations of IL10 variants with SIDS. However, the evidence for two independent MBL2 variants in the combined analysis of two large series seems consistent with the hypothesis that infection may play a role in SIDS pathogenesis.

The cost-effectiveness of UGT1A1 genotyping before colorectal cancer treatment with irinotecan from the perspective of the German statutory health insurance.

BACKGROUND: The evidence concerning the cost-effectiveness of UGT1A1*28 genotyping is ambiguous and does not allow drawing valid conclusions for Germany. This study evaluates the cost-effectiveness of UGT1A1 genotyping in patients with metastatic colorectal cancer undergoing irinotecan-based chemotherapy compared to no testing from the perspective of the German statutory health insurance. MATERIAL AND METHODS: A decision-analytic Markov model with a life time horizon was developed. No testing was compared to two genotype-dependent therapy strategies: 1) dose reduction by 25%; and 2) administration of a prophylactic G-CSF growth factor analog for homozygous and heterozygous patients. Probability, quality of life and cost parameters used in this study were based on published literature. Deterministic and probabilistic sensitivity analyses were performed to account for parameter uncertainties. RESULTS: Strategy 1 dominated all remaining strategies. Compared to no testing, it resulted in only marginal QALY increases (0.0002) but a cost reduction of €580 per patient. Strategy 2 resulted in the same health gains but increased costs by €10 773. In the probabilistic analysis, genotyping and dose reduction was the optimal strategy in approximately 100% of simulations at a threshold of €50 000 per QALY. Deterministic sensitivity analysis shows that uncertainty for this strategy originated primarily from costs for irinotecan-based chemotherapy, from the prevalence of neutropenia among heterozygous patients, and from whether dose reduction is applied to both homozygotes and heterozygotes or only to the former. CONCLUSION: This model-based synthesis of the most recent evidence suggests that pharmacogenetic UGT1A1 testing prior to irinotecan-based chemotherapy dominates non-personalized colon cancer care in Germany. However, as structural uncertainty remains high, these results require validation in clinical practice, e.g. based on a managed-entry agreement.

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations.

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10(-204)) and 10 loci for sphingolipids (smallest P-value = 3.10×10(-57)). After a correction for multiple comparisons (P-value<2.2×10(-9)), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.

The metabochip, a custom genotyping array for genetic studies of metabolic, cardiovascular, and anthropometric traits.

Genome-wide association studies have identified hundreds of loci for type 2 diabetes, coronary artery disease and myocardial infarction, as well as for related traits such as body mass index, glucose and insulin levels, lipid levels, and blood pressure. These studies also have pointed to thousands of loci with promising but not yet compelling association evidence. To establish association at additional loci and to characterize the genome-wide significant loci by fine-mapping, we designed the "Metabochip," a custom genotyping array that assays nearly 200,000 SNP markers. Here, we describe the Metabochip and its component SNP sets, evaluate its performance in capturing variation across the allele-frequency spectrum, describe solutions to methodological challenges commonly encountered in its analysis, and evaluate its performance as a platform for genotype imputation. The metabochip achieves dramatic cost efficiencies compared to designing single-trait follow-up reagents, and provides the opportunity to compare results across a range of related traits. The metabochip and similar custom genotyping arrays offer a powerful and cost-effective approach to follow-up large-scale genotyping and sequencing studies and advance our understanding of the genetic basis of complex human diseases and traits.

A genome-wide association study identifies 6p21 as novel risk locus for dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) is one of the leading causes for cardiac transplantations and accounts for up to one-third of all heart failure cases. Since extrinsic and monogenic causes explain only a fraction of all cases, common genetic variants are suspected to contribute to the pathogenesis of DCM, its age of onset, and clinical progression. By a large-scale case-control genome-wide association study we aimed here to identify novel genetic risk loci for DCM. METHODS AND RESULTS: Applying a three-staged study design, we analysed more than 4100 DCM cases and 7600 controls. We identified and successfully replicated multiple single nucleotide polymorphism on chromosome 6p21. In the combined analysis, the most significant association signal was obtained for rs9262636 (P = 4.90 × 10(-9)) located in HCG22, which could again be replicated in an independent cohort. Taking advantage of expression quantitative trait loci (eQTL) as molecular phenotypes, we identified rs9262636 as an eQTL for several closely located genes encoding class I and class II major histocompatibility complex heavy chain receptors. CONCLUSION: The present study reveals a novel genetic susceptibility locus that clearly underlines the role of genetically driven, inflammatory processes in the pathogenesis of idiopathic DCM.

A common genetic variant in the NOS1 regulator NOS1AP modulates cardiac repolarization.

Extremes of the electrocardiographic QT interval, a measure of cardiac repolarization, are associated with increased cardiovascular mortality. We identified a common genetic variant influencing this quantitative trait through a genome-wide association study on 200 subjects at the extremes of a population-based QT interval distribution of 3,966 subjects from the KORA cohort in Germany, with follow-up screening of selected markers in the remainder of the cohort. We validated statistically significant findings in two independent samples of 2,646 subjects from Germany and 1,805 subjects from the US Framingham Heart Study. This genome-wide study identified NOS1AP (CAPON), a regulator of neuronal nitric oxide synthase, as a new target that modulates cardiac repolarization. Approximately 60% of subjects of European ancestry carry at least one minor allele of the NOS1AP genetic variant, which explains up to 1.5% of QT interval variation.

Importance of different types of prior knowledge in selecting genome-wide findings for follow-up.

Biological plausibility and other prior information could help select genome-wide association (GWA) findings for further follow-up, but there is no consensus on which types of knowledge should be considered or how to weight them. We used experts' opinions and empirical evidence to estimate the relative importance of 15 types of information at the single-nucleotide polymorphism (SNP) and gene levels. Opinions were elicited from 10 experts using a two-round Delphi survey. Empirical evidence was obtained by comparing the frequency of each type of characteristic in SNPs established as being associated with seven disease traits through GWA meta-analysis and independent replication, with the corresponding frequency in a randomly selected set of SNPs. SNP and gene characteristics were retrieved using a specially developed bioinformatics tool. Both the expert and the empirical evidence rated previous association in a meta-analysis or more than one study as conferring the highest relative probability of true association, whereas previous association in a single study ranked much lower. High relative probabilities were also observed for location in a functional protein domain, although location in a region evolutionarily conserved in vertebrates was ranked high by the data but not by the experts. Our empirical evidence did not support the importance attributed by the experts to whether the gene encodes a protein in a pathway or shows interactions relevant to the trait. Our findings provide insight into the selection and weighting of different types of knowledge in SNP or gene prioritization, and point to areas requiring further research.

SNP prioritization using a Bayesian probability of association.

Prioritization is the process whereby a set of possible candidate genes or SNPs is ranked so that the most promising can be taken forward into further studies. In a genome-wide association study, prioritization is usually based on the P-values alone, but researchers sometimes take account of external annotation information about the SNPs such as whether the SNP lies close to a good candidate gene. Using external information in this way is inherently subjective and is often not formalized, making the analysis difficult to reproduce. Building on previous work that has identified 14 important types of external information, we present an approximate Bayesian analysis that produces an estimate of the probability of association. The calculation combines four sources of information: the genome-wide data, SNP information derived from bioinformatics databases, empirical SNP weights, and the researchers' subjective prior opinions. The calculation is fast enough that it can be applied to millions of SNPS and although it does rely on subjective judgments, those judgments are made explicit so that the final SNP selection can be reproduced. We show that the resulting probability of association is intuitively more appealing than the P-value because it is easier to interpret and it makes allowance for the power of the study. We illustrate the use of the probability of association for SNP prioritization by applying it to a meta-analysis of kidney function genome-wide association studies and demonstrate that SNP selection performs better using the probability of association compared with P-values alone.

Genome-wide association studies of the PR interval in African Americans.

The PR interval on the electrocardiogram reflects atrial and atrioventricular nodal conduction time. The PR interval is heritable, provides important information about arrhythmia risk, and has been suggested to differ among human races. Genome-wide association (GWA) studies have identified common genetic determinants of the PR interval in individuals of European and Asian ancestry, but there is a general paucity of GWA studies in individuals of African ancestry. We performed GWA studies in African American individuals from four cohorts (n = 6,247) to identify genetic variants associated with PR interval duration. Genotyping was performed using the Affymetrix 6.0 microarray. Imputation was performed for 2.8 million single nucleotide polymorphisms (SNPs) using combined YRI and CEU HapMap phase II panels. We observed a strong signal (rs3922844) within the gene encoding the cardiac sodium channel (SCN5A) with genome-wide significant association (p<2.5 x 10⁻8) in two of the four cohorts and in the meta-analysis. The signal explained 2% of PR interval variability in African Americans (beta  = 5.1 msec per minor allele, 95% CI  = 4.1-6.1, p = 3 x 10⁻²³). This SNP was also associated with PR interval (beta = 2.4 msec per minor allele, 95% CI = 1.8-3.0, p = 3 x 10⁻¹6) in individuals of European ancestry (n = 14,042), but with a smaller effect size (p for heterogeneity <0.001) and variability explained (0.5%). Further meta-analysis of the four cohorts identified genome-wide significant associations with SNPs in SCN10A (rs6798015), MEIS1 (rs10865355), and TBX5 (rs7312625) that were highly correlated with SNPs identified in European and Asian GWA studies. African ancestry was associated with increased PR duration (13.3 msec, p = 0.009) in one but not the other three cohorts. Our findings demonstrate the relevance of common variants to African Americans at four loci previously associated with PR interval in European and Asian samples and identify an association signal at one of these loci that is more strongly associated with PR interval in African Americans than in Europeans.

Genome-wide association studies of serum magnesium, potassium, and sodium concentrations identify six Loci influencing serum magnesium levels.

Magnesium, potassium, and sodium, cations commonly measured in serum, are involved in many physiological processes including energy metabolism, nerve and muscle function, signal transduction, and fluid and blood pressure regulation. To evaluate the contribution of common genetic variation to normal physiologic variation in serum concentrations of these cations, we conducted genome-wide association studies of serum magnesium, potassium, and sodium concentrations using approximately 2.5 million genotyped and imputed common single nucleotide polymorphisms (SNPs) in 15,366 participants of European descent from the international CHARGE Consortium. Study-specific results were combined using fixed-effects inverse-variance weighted meta-analysis. SNPs demonstrating genome-wide significant (p<5 x 10(-8)) or suggestive associations (p<4 x 10(-7)) were evaluated for replication in an additional 8,463 subjects of European descent. The association of common variants at six genomic regions (in or near MUC1, ATP2B1, DCDC5, TRPM6, SHROOM3, and MDS1) with serum magnesium levels was genome-wide significant when meta-analyzed with the replication dataset. All initially significant SNPs from the CHARGE Consortium showed nominal association with clinically defined hypomagnesemia, two showed association with kidney function, two with bone mineral density, and one of these also associated with fasting glucose levels. Common variants in CNNM2, a magnesium transporter studied only in model systems to date, as well as in CNNM3 and CNNM4, were also associated with magnesium concentrations in this study. We observed no associations with serum sodium or potassium levels exceeding p<4 x 10(-7). Follow-up studies of newly implicated genomic loci may provide additional insights into the regulation and homeostasis of human serum magnesium levels.

Network-based SNP meta-analysis identifies joint and disjoint genetic features across common human diseases.

BACKGROUND: Genome-wide association studies (GWAS) have provided a large set of genetic loci influencing the risk for many common diseases. Association studies typically analyze one specific trait in single populations in an isolated fashion without taking into account the potential phenotypic and genetic correlation between traits. However, GWA data can be efficiently used to identify overlapping loci with analogous or contrasting effects on different diseases. RESULTS: Here, we describe a new approach to systematically prioritize and interpret available GWA data. We focus on the analysis of joint and disjoint genetic determinants across diseases. Using network analysis, we show that variant-based approaches are superior to locus-based analyses. In addition, we provide a prioritization of disease loci based on network properties and discuss the roles of hub loci across several diseases. We demonstrate that, in general, agonistic associations appear to reflect current disease classifications, and present the potential use of effect sizes in refining and revising these agonistic signals. We further identify potential branching points in disease etiologies based on antagonistic variants and describe plausible small-scale models of the underlying molecular switches. CONCLUSIONS: The observation that a surprisingly high fraction (>15%) of the SNPs considered in our study are associated both agonistically and antagonistically with related as well as unrelated disorders indicates that the molecular mechanisms influencing causes and progress of human diseases are in part interrelated. Genetic overlaps between two diseases also suggest the importance of the affected entities in the specific pathogenic pathways and should be investigated further.

Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 cause dilated cardiomyopathy.

AIMS: Cardiac energy requirement is met to a large extent by oxidative phosphorylation in mitochondria that are highly abundant in cardiac myocytes. Human mitochondrial thioredoxin reductase (TXNRD2) is a selenocysteine-containing enzyme essential for mitochondrial oxygen radical scavenging. Cardiac-specific deletion of Txnrd2 in mice results in dilated cardiomyopathy (DCM). The aim of this study was to investigate whether TXNRD2 mutations explain a fraction of monogenic DCM cases. METHODS AND RESULTS: Sequencing and subsequent genotyping of TXNRD2 in patients diagnosed with DCM (n = 227) and in DCM-free (n = 683) individuals from the general population sample KORA S4 was performed. The functional impact of observed mutations on Txnrd2 function was tested in mouse fibroblasts. We identified two novel amino acid residue-altering TXNRD2 mutations [175G > A (Ala59Thr) and 1124G > A (Gly375Arg)] in three heterozygous carriers among 227 patients that were not observed in the 683 DCM-free individuals. Both DCM-associated mutations result in amino acid substitutions of highly conserved residues in helices contributing to the flavin-adenine dinucleotide (FAD)-binding domain of TXNRD2. Functional analysis of both mutations in Txnrd2(-/-) mouse fibroblasts revealed that contrasting to wild-type (wt) Txnrd2, neither mutant did restore Txnrd2 function. Mutants even impaired the survival of Txnrd2 wt cells under oxidative stress by a dominant-negative mechanism. CONCLUSION: For the first time, we describe mutations in DCM patients in a gene involved in the regulation of cellular redox state. TXNRD2 mutations may explain a fraction of human DCM disease burden.

Independent susceptibility markers for atrial fibrillation on chromosome 4q25.

BACKGROUND: Genetic variants on chromosome 4q25 are associated with atrial fibrillation (AF). We sought to determine whether there is more than 1 susceptibility signal at this locus. METHODS AND RESULTS: Thirty-four haplotype-tagging single-nucleotide polymorphisms (SNPs) at the 4q25 locus were genotyped in 790 case and 1177 control subjects from Massachusetts General Hospital and tested for association with AF. We replicated SNPs associated with AF after adjustment for the most significantly associated SNP in 5066 case and 30 661 referent subjects from the German Competence Network for Atrial Fibrillation, Atherosclerosis Risk In Communities Study, Cleveland Clinic Lone AF Study, Cardiovascular Health Study, and Rotterdam Study. All subjects were of European ancestry. A multimarker risk score composed of SNPs that tagged distinct AF susceptibility signals was constructed and tested for association with AF, and all results were subjected to meta-analysis. The previously reported SNP, rs2200733, was most significantly associated with AF (minor allele odds ratio 1.80, 95% confidence interval 1.50 to 2.15, P=1.2 x 10(-20)) in the discovery sample. Adjustment for rs2200733 genotype revealed 2 additional susceptibility signals marked by rs17570669 and rs3853445. A graded risk of AF was observed with an increasing number of AF risk alleles at SNPs that tagged these 3 susceptibility signals. CONCLUSIONS: We identified 2 novel AF susceptibility signals on chromosome 4q25. Consideration of multiple susceptibility signals at chromosome 4q25 identifies individuals with an increased risk of AF and may localize regulatory elements at the locus with biological relevance in the pathogenesis of AF.