Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.

Germline Mutations in CIDEB and Protection against Liver Disease.

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).

A fast linkage method for population GWAS cohorts with related individuals.

Linkage analysis, a class of methods for detecting co-segregation of genomic segments and traits in families, was used to map disease-causing genes for decades before genotyping arrays and dense SNP genotyping enabled genome-wide association studies in population samples. Population samples often contain related individuals, but the segregation of alleles within families is rarely used because traditional linkage methods are computationally inefficient for larger datasets. Here, we describe Population Linkage, a novel application of Haseman-Elston regression as a method of moments estimator of variance components and their standard errors. We achieve additional computational efficiency by using modern methods for detection of IBD segments and variance component estimation, efficient preprocessing of input data, and minimizing redundant numerical calculations. We also refined variance component models to account for the biases in population-scale methods for IBD segment detection. We ran Population Linkage on four blood lipid traits in over 70,000 individuals from the HUNT and SardiNIA studies, successfully detecting 25 known genetic signals. One notable linkage signal that appeared in both was for low-density lipoprotein (LDL) cholesterol levels in the region near the gene APOE (LOD = 29.3, variance explained = 4.1%). This is the region where the missense variants rs7412 and rs429358, which together make up the ε2, ε3, and ε4 alleles each account for 2.4% and 0.8% of variation in circulating LDL cholesterol. Our results show the potential for linkage analysis and other large-scale applications of method of moments variance components estimation.

Genome-wide association study of cardiac troponin I in the general population.

Circulating cardiac troponin proteins are associated with structural heart disease and predict incident cardiovascular disease in the general population. However, the genetic contribution to cardiac troponin I (cTnI) concentrations and its causal effect on cardiovascular phenotypes are unclear. We combine data from two large population-based studies, the Trøndelag Health Study and the Generation Scotland Scottish Family Health Study, and perform a genome-wide association study of high-sensitivity cTnI concentrations with 48 115 individuals. We further use two-sample Mendelian randomization to investigate the causal effects of circulating cTnI on acute myocardial infarction (AMI) and heart failure (HF). We identified 12 genetic loci (8 novel) associated with cTnI concentrations. Associated protein-altering variants highlighted putative functional genes: CAND2, HABP2, ANO5, APOH, FHOD3, TNFAIP2, KLKB1 and LMAN1. Phenome-wide association tests in 1688 phecodes and 83 continuous traits in UK Biobank showed associations between a genetic risk score for cTnI and cardiac arrhythmias, metabolic and anthropometric measures. Using two-sample Mendelian randomization, we confirmed the non-causal role of cTnI in AMI (5948 cases, 355 246 controls). We found indications for a causal role of cTnI in HF (47 309 cases and 930 014 controls), but this was not supported by secondary analyses using left ventricular mass as outcome (18 257 individuals). Our findings clarify the biology underlying the heritable contribution to circulating cTnI and support cTnI as a non-causal biomarker for AMI in the general population. Using genetically informed methods for causal inference helps inform the role and value of measuring cTnI in the general population.

Ventricular rate in atrial fibrillation and the risk of heart failure and death.

AIMS: While clinical trials have suggested that a high ventricular rate is associated with increased risk of heart failure (HF) and mortality, all-comers studies are warranted. OBJECTIVE: To assess 1-year risk of new-onset diagnosed HF and all-cause mortality among rate-control treated patients presenting with atrial fibrillation (AF) on an electrocardiogram (ECG) according to ventricular rate. METHODS AND RESULTS: ECGs recorded at the Copenhagen General Practitioners Laboratory (2001-15) were used to identify patients with AF. Multivariate Cox proportional hazard regression models were used to compare risk of new-onset HF and all-cause mortality after first ECG presenting with AF according to ventricular rate on ECG [<60, 60-79, 80-99, and 100-110, > 110 beats per minute (bpm)]. We identified 7408 patients in treatment with rate control drugs at time of first ECG presenting with AF [median age 78 years (Q1,Q3 = 70-85 years)], 45.8% male, median ventricular rate 83 bpm, (Q1,Q3 = 71-101 bpm)]. During 1-year follow-up, 666 (9.0%) of all patients with AF developed HF and 858 (11.6%) died. Patients with AF ventricular rates 100-110 bpm and >110 bpm had a hazard ratio (HR) of 1.46 (CI: 1.10-1.95) and 2.41 (CI: 1.94-3.00) respectively for new-onset HF, compared with 60-79 bpm. Similarly, patients with AF ventricular rates 100-110 bpm and >110 bpm had a HR of 1.44 (CI: 1.13-1.82) and 1.34 (CI: 1.08-1.65) respectively for all-cause mortality, compared with 60-79 bpm. CONCLUSIONS: Ventricular rates ≥100 bpm among patients presenting with AF on ECG in treatment with rate control drugs were associated with greater risk of both new-onset HF and all-cause mortality.

Age-of-onset information helps identify 76 genetic variants associated with allergic disease.

Risk factors that contribute to inter-individual differences in the age-of-onset of allergic diseases are poorly understood. The aim of this study was to identify genetic risk variants associated with the age at which symptoms of allergic disease first develop, considering information from asthma, hay fever and eczema. Self-reported age-of-onset information was available for 117,130 genotyped individuals of European ancestry from the UK Biobank study. For each individual, we identified the earliest age at which asthma, hay fever and/or eczema was first diagnosed and performed a genome-wide association study (GWAS) of this combined age-of-onset phenotype. We identified 50 variants with a significant independent association (P<3x10-8) with age-of-onset. Forty-five variants had comparable effects on the onset of the three individual diseases and 38 were also associated with allergic disease case-control status in an independent study (n = 222,484). We observed a strong negative genetic correlation between age-of-onset and case-control status of allergic disease (rg = -0.63, P = 4.5x10-61), indicating that cases with early disease onset have a greater burden of allergy risk alleles than those with late disease onset. Subsequently, a multivariate GWAS of age-of-onset and case-control status identified a further 26 associations that were missed by the univariate analyses of age-of-onset or case-control status only. Collectively, of the 76 variants identified, 18 represent novel associations for allergic disease. We identified 81 likely target genes of the 76 associated variants based on information from expression quantitative trait loci (eQTL) and non-synonymous variants, of which we highlight ADAM15, FOSL2, TRIM8, BMPR2, CD200R1, PRKCQ, NOD2, SMAD4, ABCA7 and UBE2L3. Our results support the notion that early and late onset allergic disease have partly distinct genetic architectures, potentially explaining known differences in pathophysiology between individuals.

Implantable loop recorder detection of atrial fibrillation to prevent stroke (The LOOP Study): a randomised controlled trial.

BACKGROUND: It is unknown whether screening for atrial fibrillation and subsequent treatment with anticoagulants if atrial fibrillation is detected can prevent stroke. Continuous electrocardiographic monitoring using an implantable loop recorder (ILR) can facilitate detection of asymptomatic atrial fibrillation episodes. We aimed to investigate whether atrial fibrillation screening and use of anticoagulants can prevent stroke in individuals at high risk. METHODS: We did a randomised controlled trial in four centres in Denmark. We included individuals without atrial fibrillation, aged 70-90 years, with at least one additional stroke risk factor (ie, hypertension, diabetes, previous stroke, or heart failure). Participants were randomly assigned in a 1:3 ratio to ILR monitoring or usual care (control) via an online system in permuted blocks with block sizes of four or eight participants stratified according to centre. In the ILR group, anticoagulation was recommended if atrial fibrillation episodes lasted 6 min or longer. The primary outcome was time to first stroke or systemic arterial embolism. This study is registered with ClinicalTrials.gov, NCT02036450. FINDINGS: From Jan 31, 2014, to May 17, 2016, 6205 individuals were screened for inclusion, of whom 6004 were included and randomly assigned: 1501 (25·0%) to ILR monitoring and 4503 (75·0%) to usual care. Mean age was 74·7 years (SD 4·1), 2837 (47·3%) were women, and 5444 (90·7%) had hypertension. No participants were lost to follow-up. During a median follow-up of 64·5 months (IQR 59·3-69·8), atrial fibrillation was diagnosed in 1027 participants: 477 (31·8%) of 1501 in the ILR group versus 550 (12·2%) of 4503 in the control group (hazard ratio [HR] 3·17 [95% CI 2·81-3·59]; p<0·0001). Oral anticoagulation was initiated in 1036 participants: 445 (29·7%) in the ILR group versus 591 (13·1%) in the control group (HR 2·72 [95% CI 2·41-3·08]; p<0·0001), and the primary outcome occurred in 318 participants (315 stroke, three systemic arterial embolism): 67 (4·5%) in the ILR group versus 251 (5·6%) in the control group (HR 0·80 [95% CI 0·61-1·05]; p=0·11). Major bleeding occurred in 221 participants: 65 (4·3%) in the ILR group versus 156 (3·5%) in the control group (HR 1·26 [95% CI 0·95-1·69]; p=0·11). INTERPRETATION: In individuals with stroke risk factors, ILR screening resulted in a three-times increase in atrial fibrillation detection and anticoagulation initiation but no significant reduction in the risk of stroke or systemic arterial embolism. These findings might imply that not all atrial fibrillation is worth screening for, and not all screen-detected atrial fibrillation merits anticoagulation. FUNDING: Innovation Fund Denmark, The Research Foundation for the Capital Region of Denmark, The Danish Heart Foundation, Aalborg University Talent Management Program, Arvid Nilssons Fond, Skibsreder Per Henriksen, R og Hustrus Fond, The AFFECT-EU Consortium (EU Horizon 2020), Læge Sophus Carl Emil Friis og hustru Olga Doris Friis' Legat, and Medtronic.

Associations between primary care electrocardiography and non-Alzheimer dementia.

OBJECTIVES: To determine whether electrocardiogram (ECG) markers are associated with incident non-Alzheimer's dementia (non-AD) and whether these markers also improve risk prediction for non-AD. MATERIALS AND METHODS: We retrospectively included 170,605 primary care patients aged 60 years or older referred for an ECG by their general practitioner and followed them for a median of 7.6 years. Using Cox regression, we reported hazard ratios (HRs) for electrocardiogram markers. Subsequently, we evaluated if addition of these electrocardiogram markers to a clinical model improved risk prediction for non-AD using change in area under the receiver-operator characteristics curve (AUC). RESULTS: The 5-year cumulative incidence of non-AD was 3.4 %. Increased heart rate (HR=1.06 pr. 10 bpm [95% confidence interval: 1.04-1.08], p<0.001), shorter QRS duration (HR=1.07 pr. 10 ms [1.05-1.09], p<0.001), elevated J-amplitude (HR=1.16 pr. mm [1.08-1.24], p<0.001), decreased T-peak amplitude (HR=1.02 pr. mm [1.01-1.04], p=0.002), and increased QTc (HR=1.08 pr. 20 ms [1.05-1.10], p<0.001) were associated with an increased rate of non-AD. Atrial fibrillation on the ECG (HR=1.18 [1.08-1.28], p<0.001) Sokolow-Lyon index > 35 mm (HR=1.31 [1.18-1.46], p<0.001) and borderline (HR=1.18 [1.11-1.26], p<0.001) or abnormal (HR=1.40 [1.27-1.55], p<0.001) QRS-T angle were also associated with an increased rate of non-AD. Upon addition of ECG markers to the Cox model, 5-year and 10-year C-statistic (AUC) improved significantly (delta-AUC, 0.36 [0.18-0.50] and 0.20 [0.03-0.35] %-points, respectively). CONCLUSIONS: ECG markers typical of an elevated cardiovascular risk profile were associated with non-AD and improved both 5-year and 10-year risk predictions for non-AD.

Genome-wide Study of Atrial Fibrillation Identifies Seven Risk Loci and Highlights Biological Pathways and Regulatory Elements Involved in Cardiac Development.

Atrial fibrillation (AF) is a common cardiac arrhythmia and a major risk factor for stroke, heart failure, and premature death. The pathogenesis of AF remains poorly understood, which contributes to the current lack of highly effective treatments. To understand the genetic variation and biology underlying AF, we undertook a genome-wide association study (GWAS) of 6,337 AF individuals and 61,607 AF-free individuals from Norway, including replication in an additional 30,679 AF individuals and 278,895 AF-free individuals. Through genotyping and dense imputation mapping from whole-genome sequencing, we tested almost nine million genetic variants across the genome and identified seven risk loci, including two novel loci. One novel locus (lead single-nucleotide variant [SNV] rs12614435; p = 6.76 × 10-18) comprised intronic and several highly correlated missense variants situated in the I-, A-, and M-bands of titin, which is the largest protein in humans and responsible for the passive elasticity of heart and skeletal muscle. The other novel locus (lead SNV rs56202902; p = 1.54 × 10-11) covered a large, gene-dense chromosome 1 region that has previously been linked to cardiac conduction. Pathway and functional enrichment analyses suggested that many AF-associated genetic variants act through a mechanism of impaired muscle cell differentiation and tissue formation during fetal heart development.

Genomic and transcriptomic association studies identify 16 novel susceptibility loci for venous thromboembolism.

Venous thromboembolism (VTE) is a significant contributor to morbidity and mortality. To advance our understanding of the biology contributing to VTE, we conducted a genome-wide association study (GWAS) of VTE and a transcriptome-wide association study (TWAS) based on imputed gene expression from whole blood and liver. We meta-analyzed GWAS data from 18 studies for 30 234 VTE cases and 172 122 controls and assessed the association between 12 923 718 genetic variants and VTE. We generated variant prediction scores of gene expression from whole blood and liver tissue and assessed them for association with VTE. Mendelian randomization analyses were conducted for traits genetically associated with novel VTE loci. We identified 34 independent genetic signals for VTE risk from GWAS meta-analysis, of which 14 are newly reported associations. This included 11 newly associated genetic loci (C1orf198, PLEK, OSMR-AS1, NUGGC/SCARA5, GRK5, MPHOSPH9, ARID4A, PLCG2, SMG6, EIF5A, and STX10) of which 6 replicated, and 3 new independent signals in 3 known genes. Further, TWAS identified 5 additional genetic loci with imputed gene expression levels differing between cases and controls in whole blood (SH2B3, SPSB1, RP11-747H7.3, RP4-737E23.2) and in liver (ERAP1). At some GWAS loci, we found suggestive evidence that the VTE association signal for novel and previously known regions colocalized with expression quantitative trait locus signals. Mendelian randomization analyses suggested that blood traits may contribute to the underlying risk of VTE. To conclude, we identified 16 novel susceptibility loci for VTE; for some loci, the association signals are likely mediated through gene expression of nearby genes.

Robust meta-analysis of biobank-based genome-wide association studies with unbalanced binary phenotypes.

With the availability of large-scale biobanks, genome-wide scale phenome-wide association studies are being instrumental in discovering novel genetic variants associated with clinical phenotypes. As increasing number of such association results from different biobanks become available, methods to meta-analyse those association results is of great interest. Because the binary phenotypes in biobank-based studies are mostly unbalanced in their case-control ratios, very few methods can provide well-calibrated tests for associations. For example, traditional Z-score-based meta-analysis often results in conservative or anticonservative Type I error rates in such unbalanced scenarios. We propose two meta-analysis strategies that can efficiently combine association results from biobank-based studies with such unbalanced phenotypes, using the saddlepoint approximation-based score test method. Our first method involves sharing the overall genotype counts from each study, and the second method involves sharing an approximation of the distribution of the score test statistic from each study using cubic Hermite splines. We compare our proposed methods with a traditional Z-score-based meta-analysis strategy using numerical simulations and real data applications, and demonstrate the superior performance of our proposed methods in terms of Type I error control.

Mitochondrial genome-wide association study of migraine - the HUNT Study.

BACKGROUND: Variation in mitochondrial DNA (mtDNA) has been indicated in migraine pathogenesis, but genetic studies to date have focused on candidate variants, with sparse findings. We aimed to perform the first mitochondrial genome-wide association study of migraine, examining both single variants and mitochondrial haplogroups. METHODS: In total, 71,860 participants from the population-based Nord-Trøndelag Health Study were genotyped. We excluded samples not passing quality control for nuclear genotypes, in addition to samples with low call rate and closely maternally related. We analysed 775 mitochondrial DNA variants in 4021 migraine cases and 14,288 headache-free controls, using logistic regression. In addition, we analysed 3831 cases and 13,584 controls who could be reliably assigned to a mitochondrial haplogroup. Lastly, we attempted to replicate previously reported mitochondrial DNA candidate variants. RESULTS: Neither of the mitochondrial variants or haplogroups were associated with migraine. In addition, none of the previously reported mtDNA candidate variants replicated in our data. CONCLUSIONS: Our findings do not support a major role of mitochondrial genetic variation in migraine pathophysiology, but a larger sample is needed to detect rare variants and future studies should also examine heteroplasmic variation, epigenetic changes and copy-number variation.

Effect of diabetes duration on the relationship between glycaemic control and risk of death in older adults with type 2 diabetes.

AIM: To investigate the effect of diabetes duration on glycaemic control, measured using mean glycated haemoglobin (HbA1c) level, and mortality risk within different age, sex and clinically relevant, comorbidity-defined subgroups in an elderly population with type 2 diabetes (T2D). METHODS: We studied older (≥65 years) primary care patients with T2D, who had three successive annual measurements of HbA1c taken between 2005 and 2013. The primary exposure was the mean of all three HbA1c measurements. Follow-up began on the date of the third measurement. Individual mean HbA1c levels were categorized into clinically relevant groups (<6.5% [<48 mmol/mol]; 6.5%-6.9% [48-52 mmol/mol]; 7%-7.9% [53-63 mmol/mol]; 8%-8.9% [64-74 mmol/mol]; and ≥9% [≥75 mmol/mol]). We used multiple Cox regression to study the effect of glycaemic control on the hazard of all-cause mortality, adjusted for age, sex, use of concomitant medication, and age- and disease-related comorbidities. RESULTS: A total of 9734 individuals were included. During a median (interquartile range) follow-up of 7.3 (4.6-8.7) years, 3320 individuals died. We found that the effect of mean HbA1c on all-cause mortality depended on the duration of diabetes (P for interaction <.001). For individuals with short diabetes duration (<5 years), the risk of death increased with poorer glycaemic control (increasing HbA1c), whereas for individuals with longstanding diabetes (≥5 years), we found a J-shaped association, where a mean HbA1c level between 6.5% and 7.9% [48 and 63 mmol/mol] was associated with the lowest risk of death. For individuals with longstanding diabetes, both low (<6.5% [<48 mmol/mol]; hazard ratio [HR] 1.21, 95% confidence interval [CI] 1.07-1.37, P = .002) and high mean HbA1c levels (≥9.0% [≥75 mmol/mol]; HR 1.60, 95% CI 1.28-1.99, P < .001) were associated with an increased risk of death. We also calculated 5-year absolute risks of all-cause mortality, separately for short and long diabetes duration, and found similar risk patterns across different age groups, sex and comorbidity strata. CONCLUSIONS: In elderly individuals with T2D, the effect of glycaemic control (measured by HbA1c) on all-cause mortality depended on the duration of diabetes. Of particular clinical importance, we found that strict glycaemic control was associated with an increased risk of death among individuals with long (≥ 5 years) diabetes duration. Conversely, for individuals with short diabetes duration, strict glycaemic control was associated with the lowest risk of death. These results indicate that tight glycemic control may be beneficial in people with short duration of diabetes, whereas a less stringent target may be warranted with longer diabetes exposure.

Genetic variants on chromosomes 7p31 and 12p12 are associated with abnormal atrial electrical activation in patients with early-onset lone atrial fibrillation.

BACKGROUND: Abnormal P-wave morphology (PWM) has been associated with a history of atrial fibrillation (AF) in earlier studies. Although lone AF is believed to have substantial genetic basis, studies on associations between single nucleotide polymorphisms (SNP) linked to lone AF and PWM have not been reported. We aimed to assess whether SNPs previously associated with lone AF (rs2200733, rs13376333, rs3807989, and rs11047543) are also linked to P-wave abnormalities. METHODS: Four SNPs were studied in 176 unrelated individuals with early-onset lone AF (age at onset <50 years), median age 38 years (19-63 years), 149 men. Using sinus rhythm ECG, orthogonal PWM was classified as Type 1-positive in leads X and Y and negative in lead Z, Type 2-positive in leads X and Y and biphasic (-/+) in lead Z, Type 3-positive in lead X and biphasic in lead Y (+/-), and the remaining as atypical. RESULTS: Two SNPs were found to be significantly associated with altered P-wave morphology distribution: rs3807989 near the gene CAV1/CAV2 and rs11047543 near the gene SOX5. Both SNPs were associated with a higher risk of non-Type 1 P-wave morphology (rs3807989: OR = 4.8, 95% CI = 2.3-10.2, p < 0.001; rs11047543: OR = 4.7, 95% CI = 1.1-20.5, p = 0.04). No association was observed for rs2200733 and rs13376333. CONCLUSION: In this study, the two variants rs3807989 and rs11047543, previously associated with PR interval and lone AF, were associated with altered P-wave morphology distribution in patients with early-onset lone AF. These findings suggest that common genetic variants may modify atrial conduction properties.

Improving power of association tests using multiple sets of imputed genotypes from distributed reference panels.

The accuracy of genotype imputation depends upon two factors: the sample size of the reference panel and the genetic similarity between the reference panel and the target samples. When multiple reference panels are not consented to combine together, it is unclear how to combine the imputation results to optimize the power of genetic association studies. We compared the accuracy of 9,265 Norwegian genomes imputed from three reference panels-1000 Genomes phase 3 (1000G), Haplotype Reference Consortium (HRC), and a reference panel containing 2,201 Norwegian participants from the population-based Nord Trøndelag Health Study (HUNT) from low-pass genome sequencing. We observed that the population-matched reference panel allowed for imputation of more population-specific variants with lower frequency (minor allele frequency (MAF) between 0.05% and 0.5%). The overall imputation accuracy from the population-specific panel was substantially higher than 1000G and was comparable with HRC, despite HRC being 15-fold larger. These results recapitulate the value of population-specific reference panels for genotype imputation. We also evaluated different strategies to utilize multiple sets of imputed genotypes to increase the power of association studies. We observed that testing association for all variants imputed from any panel results in higher power to detect association than the alternative strategy of including only one version of each genetic variant, selected for having the highest imputation quality metric. This was particularly true for lower frequency variants (MAF < 1%), even after adjusting for the additional multiple testing burden.

Numerous Brugada syndrome-associated genetic variants have no effect on J-point elevation, syncope susceptibility, malignant cardiac arrhythmia, and all-cause mortality.

PURPOSE: We investigated whether Brugada syndrome (BrS)-associated variants identified in the general population have an effect on J-point elevation as well as whether carriers of BrS variants were more prone to experience syncope and malignant ventricular arrhythmia and had increased mortality compared with noncarriers. METHODS: All BrS-associated variants were identified using the Human Gene Mutation Database (HGMD). Individuals were randomly selected from a general population study using whole-exome sequencing data (n = 870) and genotype array data (n = 6,161) and screened for BrS-associated variants. Electrocardiograms (ECG) were analyzed electronically, and data on syncope, ventricular arrhythmias, and mortality were obtained from administrative health-care registries. RESULTS: In HGMD, 382 BrS-associated genetic variants were identified. Of these, 28 variants were identified in the study cohort. None of the carriers presented with type 1 BrS ECG pattern. Mean J-point elevation in V1 and V2 were within normal guideline limits for carriers and noncarriers. There was no difference in syncope susceptibility (carriers 8/624; noncarriers 98/5,562; P = 0.51), ventricular arrhythmia (carriers 4/620; noncarriers 9/5,524; P = 0.24), or overall mortality (hazard ratio 0.93, 95% CI 0.63-1.4). CONCLUSIONS: Our data indicate that a significant number of BrS-associated variants are not the monogenic cause of BrS.Genet Med advance online publication 06 October 2016.

Integrating genetic, transcriptional, and functional analyses to identify 5 novel genes for atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) affects >30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood. METHODS AND RESULTS: To identify new AF-related genes, we used a multifaceted approach, combining large-scale genotyping in 2 ethnically distinct populations, cis-eQTL (expression quantitative trait loci) mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501; relative risk [RR]=1.18; 95% confidence interval [CI], 1.13-1.23; P=6.5×10(-16)), GJA1 (rs13216675; RR=1.10; 95% CI, 1.06-1.14; P=2.2×10(-8)), TBX5 (rs10507248; RR=1.12; 95% CI, 1.08-1.16; P=5.7×10(-11)), and CAND2 (rs4642101; RR=1.10; 95% CI, 1.06-1.14; P=9.8×10(-9)). In Japanese, novel loci were identified near NEURL (rs6584555; RR=1.32; 95% CI, 1.26-1.39; P=2.0×10(-25)) and CUX2 (rs6490029; RR=1.12; 95% CI, 1.08-1.16; P=3.9×10(-9)). The top single-nucleotide polymorphisms or their proxies were identified as cis-eQTLs for the genes CAND2 (P=2.6×10(-19)), GJA1 (P=2.66×10(-6)), and TBX5 (P=1.36×10(-5)). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively). CONCLUSIONS: We have identified 5 novel loci for AF. Our results expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.

Risk prediction of cardiovascular death based on the QTc interval: evaluating age and gender differences in a large primary care population.

AIMS: Using a large, contemporary primary care population we aimed to provide absolute long-term risks of cardiovascular death (CVD) based on the QTc interval and to test whether the QTc interval is of value in risk prediction of CVD on an individual level. METHODS AND RESULTS: Digital electrocardiograms from 173 529 primary care patients aged 50-90 years were collected during 2001-11. The Framingham formula was used for heart rate-correction of the QT interval. Data on medication, comorbidity, and outcomes were retrieved from administrative registries. During a median follow-up period of 6.1 years, 6647 persons died from cardiovascular causes. Long-term risks of CVD were estimated for subgroups defined by age, gender, cardiovascular disease, and QTc interval categories. In general, we observed an increased risk of CVD for both very short and long QTc intervals. Prolongation of the QTc interval resulted in the worst prognosis for men whereas in women, a very short QTc interval was equivalent in risk to a borderline prolonged QTc interval. The effect of the QTc interval on the absolute risk of CVD was most pronounced in the elderly and in those with cardiovascular disease whereas the effect was negligible for middle-aged women without cardiovascular disease. The most important improvement in prediction accuracy was noted for women aged 70-90 years. In this subgroup, a total of 9.5% were reclassified (7.2% more accurately vs. 2.3% more inaccurately) within clinically relevant 5-year risk groups when the QTc interval was added to a conventional risk model for CVD. CONCLUSION: Important differences were observed across subgroups when the absolute long-term risk of CVD was estimated based on QTc interval duration. The accuracy of the personalized CVD prognosis can be improved when the QTc interval is introduced to a conventional risk model for CVD.

Genetic variation in the two-pore domain potassium channel, TASK-1, may contribute to an atrial substrate for arrhythmogenesis.

The two-pore domain potassium channel, K2P3.1 (TASK-1) modulates background conductance in isolated human atrial cardiomyocytes and has been proposed as a potential drug target for atrial fibrillation (AF). TASK-1 knockout mice have a predominantly ventricular phenotype however, and effects of TASK-1 inactivation on atrial structure and function have yet to be demonstrated in vivo. The extent to which genetic variation in KCNK3, that encodes TASK-1, might be a determinant of susceptibility to AF is also unknown. To address these questions, we first evaluated the effects of transient knockdown of the zebrafish kcnk3a and kcnk3b genes and cardiac phenotypes were evaluated using videomicroscopy. Combined kcnk3a and kcnk3b knockdown in 72 hour post fertilization embryos resulted in lower heart rate (p<0.001), marked increase in atrial diameter (p<0.001), and mild increase in end-diastolic ventricular diameter (p=0.01) when compared with control-injected embryos. We next performed genetic screening of KCNK3 in two independent AF cohorts (373 subjects) and identified three novel KCNK3 variants. Two of these variants, present in one proband with familial AF, were located at adjacent nucleotides in the Kozak sequence and reduced expression of an engineered reporter. A third missense variant, V123L, in a patient with lone AF, reduced resting membrane potential and altered pH sensitivity in patch-clamp experiments, with structural modeling predicting instability in the vicinity of the TASK-1 pore. These in vitro data suggest that the double Kozak variants and V123L will have loss-of-function effects on ITASK. Cardiac action potential modeling predicted that reduced ITASK prolongs atrial action potential duration, and that this is potentiated by reciprocal changes in activity of other ion channel currents. Our findings demonstrate the functional importance of ITASK in the atrium and suggest that inactivation of TASK-1 may have diverse effects on atrial size and electrophysiological properties that can contribute to an arrhythmogenic substrate.

New population-based exome data question the pathogenicity of some genetic variants previously associated with Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is a rare autosomal dominantly inherited connective tissue disorder with an estimated prevalence of 1:5,000. More than 1000 variants have been previously reported to be associated with MFS. However, the disease-causing effect of these variants may be questionable as many of the original studies used low number of controls. To study whether there are possible false-positive variants associated with MFS, four in silico prediction tools (SIFT, Polyphen-2, Grantham score, and conservation across species) were used to predict the pathogenicity of these variant. RESULTS: Twenty-three out of 891 previously MFS-associated variants were identified in the ESP. These variants were distributed on 100 heterozygote carriers in 6494 screened individuals. This corresponds to a genotype prevalence of 1:65 for MFS. Using a more conservative approach (cutoff value of >2 carriers in the EPS), 10 variants affected a total of 82 individuals. This gives a genotype prevalence of 1:79 (82:6494) in the ESP. A significantly higher frequency of MFS-associated variants not present in the ESP were predicted to be pathogenic with the agreement of ≥3 prediction tools, compared to the variants present in the ESP (p = 3.5 × 10-15). CONCLUSIONS: This study showed a higher genotype prevalence of MFS than expected from the phenotype prevalence in the general population. The high genotype prevalence suggests that these variants are not the monogenic cause of MFS. Therefore, caution should be taken with regard to disease stratification based on these previously reported MFS-associated variants.