Arne Pfeufer

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5 million genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N ≤ 71,225 European ancestry, N ≤ 12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N = 29,136). We identified association between systolic or diastolic blood pressure and common variants in eight regions near the CYP17A1 (P = 7 × 10(-24)), CYP1A2 (P = 1 × 10(-23)), FGF5 (P = 1 × 10(-21)), SH2B3 (P = 3 × 10(-18)), MTHFR (P = 2 × 10(-13)), c10orf107 (P = 1 × 10(-9)), ZNF652 (P = 5 × 10(-9)) and PLCD3 (P = 1 × 10(-8)) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

Obesity is a heritable trait and a risk factor for many common diseases such as type 2 diabetes, heart disease, and hypertension. We used a dense whole-genome scan of DNA samples from the Framingham Heart Study participants to identify a common genetic variant near the INSIG2 gene associated with obesity. We have replicated the finding in four separate samples composed of individuals of Western European ancestry, African Americans, and children. The obesity-predisposing genotype is present in 10% of individuals. Our study suggests that common genetic polymorphisms are important determinants of obesity.

Atrial fibrillation is a highly prevalent arrhythmia and a major risk factor for stroke, heart failure and death. We conducted a genome-wide association study (GWAS) in individuals of European ancestry, including 6,707 with and 52,426 without atrial fibrillation. Six new atrial fibrillation susceptibility loci were identified and replicated in an additional sample of individuals of European ancestry, including 5,381 subjects with and 10,030 subjects without atrial fibrillation (P < 5 × 10(-8)). Four of the loci identified in Europeans were further replicated in silico in a GWAS of Japanese individuals, including 843 individuals with and 3,350 individuals without atrial fibrillation. The identified loci implicate candidate genes that encode transcription factors related to cardiopulmonary development, cardiac-expressed ion channels and cell signaling molecules.

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.

Patrick Ellinor and colleagues report a genome-wide association study identifying variants in KCNN3 associated to lone atrial fibrillation. Atrial fibrillation (AF) is the most common sustained arrhythmia. Previous studies have identified several genetic loci associated with typical AF. We sought to identify common genetic variants underlying lone AF. This condition affects a subset of individuals without overt heart disease and with an increased heritability of AF. We report a meta-analysis of genome-wide association studies conducted using 1,335 individuals with lone AF (cases) and 12,844 unaffected individuals (referents). Cases were obtained from the German AF Network, Heart and Vascular Health Study, the Atherosclerosis Risk in Communities Study, the Cleveland Clinic and Massachusetts General Hospital. We identified an association on chromosome 1q21 to lone AF (rs13376333, adjusted odds ratio = 1.56; P = 6.3 × 10−12), and we replicated this association in two independent cohorts with lone AF (overall combined odds ratio = 1.52, 95% CI 1.40–1.64; P = 1.83 × 10−21). rs13376333 is intronic to KCNN3, which encodes a potassium channel protein involved in atrial repolarization.

The electrocardiographic PR interval (or PQ interval) reflects atrial and atrioventricular nodal conduction, disturbances of which increase risk of atrial fibrillation. We report a meta-analysis of genome-wide association studies for PR interval from seven population-based European studies in the CHARGE Consortium: AGES, ARIC, CHS, FHS, KORA, Rotterdam Study, and SardiNIA (N = 28,517). We identified nine loci associated with PR interval at P < 5 x 10(-8). At the 3p22.2 locus, we observed two independent associations in voltage-gated sodium channel genes, SCN10A and SCN5A. Six of the loci were near cardiac developmental genes, including CAV1-CAV2, NKX2-5 (CSX1), SOX5, WNT11, MEIS1, and TBX5-TBX3, providing pathophysiologically interesting candidate genes. Five of the loci, SCN5A, SCN10A, NKX2-5, CAV1-CAV2, and SOX5, were also associated with atrial fibrillation (N = 5,741 cases, P < 0.0056). This suggests a role for common variation in ion channel and developmental genes in atrial and atrioventricular conduction as well as in susceptibility to atrial fibrillation.

Arne Pfeufer, Aravinda Chakravarti and colleagues from the QTSCD consortium report genetic associations influencing the QT interval duration, a measure of cardiac repolarization which is a risk factor for sudden cardiac death, in five genome-wide association studies. The QT interval, a measure of cardiac repolarization, predisposes to ventricular arrhythmias and sudden cardiac death (SCD) when prolonged or shortened. A common variant in NOS1AP is known to influence repolarization. We analyze genome-wide data from five population-based cohorts (ARIC, KORA, SardiNIA, GenNOVA and HNR) with a total of 15,842 individuals of European ancestry, to confirm the NOS1AP association and identify nine additional loci at P < 5 × 10−8. Four loci map near the monogenic long-QT syndrome genes KCNQ1, KCNH2, SCN5A and KCNJ2. Two other loci include ATP1B1 and PLN, genes with established electrophysiological function, whereas three map to RNF207, near LITAF and within NDRG4-GINS3-SETD6-CNOT1, respectively, all of which have not previously been implicated in cardiac electrophysiology. These results, together with an accompanying paper from the QTGEN consortium, identify new candidate genes for ventricular arrhythmias and SCD.

We conducted meta-analyses of genome-wide association studies for atrial fibrillation (AF) in participants from five community-based cohorts. Meta-analyses of 896 prevalent (15,768 referents) and 2,517 incident (21,337 referents) AF cases identified a new locus for AF (ZFHX3, rs2106261, risk ratio RR = 1.19; P = 2.3 x 10(-7)). We replicated this association in an independent cohort from the German AF Network (odds ratio = 1.44; P = 1.6 x 10(-11); combined RR = 1.25; combined P = 1.8 x 10(-15)).

The QRS interval, from the beginning of the Q wave to the end of the S wave on an electrocardiogram, reflects ventricular depolarization and conduction time and is a risk factor for mortality, sudden death and heart failure. We performed a genome-wide association meta-analysis in 40,407 individuals of European descent from 14 studies, with further genotyping in 7,170 additional Europeans, and we identified 22 loci associated with QRS duration (P < 5 × 10(-8)). These loci map in or near genes in pathways with established roles in ventricular conduction such as sodium channels, transcription factors and calcium-handling proteins, but also point to previously unidentified biologic processes, such as kinase inhibitors and genes related to tumorigenesis. We demonstrate that SCN10A, a candidate gene at the most significantly associated locus in this study, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN10A blocker prolongs QRS duration. These findings extend our current knowledge of ventricular depolarization and conduction.

Using principal component (PC) analysis, we studied the genetic constitution of 3,112 individuals from Europe as portrayed by more than 270,000 single nucleotide polymorphisms (SNPs) genotyped with the Illumina Infinium platform. In cohorts where the sample size was >100, one hundred randomly chosen samples were used for analysis to minimize the sample size effect, resulting in a total of 1,564 samples. This analysis revealed that the genetic structure of the European population correlates closely with geography. The first two PCs highlight the genetic diversity corresponding to the northwest to southeast gradient and position the populations according to their approximate geographic origin. The resulting genetic map forms a triangular structure with a) Finland, b) the Baltic region, Poland and Western Russia, and c) Italy as its vertexes, and with d) Central- and Western Europe in its centre. Inter- and intra- population genetic differences were quantified by the inflation factor lambda (lambda) (ranging from 1.00 to 4.21), fixation index (F(st)) (ranging from 0.000 to 0.023), and by the number of markers exhibiting significant allele frequency differences in pair-wise population comparisons. The estimated lambda was used to assess the real diminishing impact to association statistics when two distinct populations are merged directly in an analysis. When the PC analysis was confined to the 1,019 Estonian individuals (0.1% of the Estonian population), a fine structure emerged that correlated with the geography of individual counties. With at least two cohorts available from several countries, genetic substructures were investigated in Czech, Finnish, German, Estonian and Italian populations. Together with previously published data, our results allow the creation of a comprehensive European genetic map that will greatly facilitate inter-population genetic studies including genome wide association studies (GWAS).

Early repolarization pattern (ERP) on electrocardiogram was associated with idiopathic ventricular fibrillation and sudden cardiac arrest in a case-control study and with cardiovascular mortality in a Finnish community-based sample. We sought to determine ERP prevalence and its association with cardiac and all-cause mortality in a large, prospective, population-based case-cohort study (Monitoring of Cardiovascular Diseases and Conditions [MONICA]/KORA [Cooperative Health Research in the Region of Augsburg]) comprised of individuals of Central-European descent.Electrocardiograms of 1,945 participants aged 35-74 y, representing a source population of 6,213 individuals, were analyzed applying a case-cohort design. Mean follow-up was 18.9 y. Cause of death was ascertained by the 9th revision of the International Classification of Disease (ICD-9) codes as documented in death certificates. ERP-attributable effects on mortality were determined by a weighted Cox proportional hazard model adjusted for covariables. Prevalence of ERP was 13.1% in our study. ERP was associated with cardiac and all-cause mortality, most pronounced in those of younger age and male sex; a clear ERP-age interaction was detected (p = 0.005). Age-stratified analyses showed hazard ratios (HRs) for cardiac mortality of 1.96 (95% confidence interval [CI] 1.05-3.68, p = 0.035) for both sexes and 2.65 (95% CI 1.21-5.83, p = 0.015) for men between 35-54 y. An inferior localization of ERP further increased ERP-attributable cardiac mortality to HRs of 3.15 (95% CI 1.58-6.28, p = 0.001) for both sexes and to 4.27 (95% CI 1.90-9.61, p<0.001) for men between 35-54 y. HRs for all-cause mortality were weaker but reached significance.We found a high prevalence of ERP in our population-based cohort of middle-aged individuals. ERP was associated with about a 2- to 4-fold increased risk of cardiac mortality in individuals between 35 and 54 y. An inferior localization of ERP was associated with a particularly increased risk. Please see later in the article for the Editors' Summary.

Abstract 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.

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.

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.

A recent genome-wide association study identified a haplotype block on chromosome 4q25 associated with atrial fibrillation (AF). We sought to replicate this association in four independent cohorts.The Framingham Heart Study and Rotterdam Study are community-based longitudinal studies. The Vanderbilt AF Registry and German AF Network (AFNet) are case-control studies. Participants with AF (n = 3508) were more likely to be male and were older than referent participants (n = 12 173; Framingham 82 +/- 10 vs. 71 +/- 13 years; Rotterdam 73 +/- 8 vs. 69 +/- 9 years; Vanderbilt 54 +/- 14 vs. 57 +/- 14 years; AFNet 62 +/- 12 vs. 49 +/- 14 years). Single nucleotide polymorphism (SNP) rs2200733 was associated with AF in all four cohorts, with odds ratios (ORs) ranging from 1.37 in Rotterdam [95% confidence interval (CI) 1.18-1.59; P = 3.1 x 10(-5)] to 2.52 in AFNet (95% CI 2.22-2.8; P = 1.8 x 10(-49)). There also was a significant association between AF and rs10033464 in Framingham (OR 1.34; 95% CI 1.03-1.75; P = 0.031) and AFNet (OR 1.30; 95% CI 1.13-1.51; P = 0.0002), but not Vanderbilt (OR 1.16; 95% CI 0.86-1.56; P = 0.33). A meta-analysis of the current and prior AF studies revealed an OR of 1.90 (95% CI 1.60-2.26; P = 3.3 x 10(-13)) for rs2200733 and of 1.36 (95% CI 1.26-1.47; P = 6.7 x 10(-15)) for rs10033464.The non-coding SNPs rs2200733 and rs10033464 are strongly associated with AF in four cohorts of European descent. These results confirm the significant relations between AF and intergenic variants on chromosome 4.

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.

Connie Bezzina and colleagues report a genome-wide association study for ventricular fibrillation in individuals with acute myocardial infarction, a leading cause of total and cardiovascular mortality. Sudden cardiac death from ventricular fibrillation during acute myocardial infarction is a leading cause of total and cardiovascular mortality. To our knowledge, we here report the first genome-wide association study for this trait, conducted in a set of 972 individuals with a first acute myocardial infarction, 515 of whom had ventricular fibrillation and 457 of whom did not, from the Arrhythmia Genetics in The Netherlands (AGNES) study. The most significant association to ventricular fibrillation was found at 21q21 (rs2824292, odds ratio = 1.78, 95% CI 1.47–2.13, P = 3.3 × 10−10). The association of rs2824292 with ventricular fibrillation was replicated in an independent case-control set consisting of 146 out-of-hospital cardiac arrest individuals with myocardial infarction complicated by ventricular fibrillation and 391 individuals who survived a myocardial infarction (controls) (odds ratio = 1.49, 95% CI 1.14–1.95, P = 0.004). The closest gene to this SNP is CXADR, which encodes a viral receptor previously implicated in myocarditis and dilated cardiomyopathy and which has recently been identified as a modulator of cardiac conduction. This locus has not previously been implicated in arrhythmia susceptibility.

Brugada syndrome is a genetic disease associated with sudden cardiac death that is characterized by ventricular fibrillation and right precordial ST segment elevation on ECG. Loss-of-function mutations in SCN5A, which encodes the predominant cardiac sodium channel alpha subunit NaV1.5, can cause Brugada syndrome and cardiac conduction disease. However, SCN5A mutations are not detected in the majority of patients with these syndromes, suggesting that other genes can cause or modify presentation of these disorders. Here, we investigated SCN1B, which encodes the function-modifying sodium channel beta1 subunit, in 282 probands with Brugada syndrome and in 44 patients with conduction disease, none of whom had SCN5A mutations. We identified 3 mutations segregating with arrhythmia in 3 kindreds. Two of these mutations were located in a newly described alternately processed transcript, beta1B. Both the canonical and alternately processed transcripts were expressed in the human heart and were expressed to a greater degree in Purkinje fibers than in heart muscle, consistent with the clinical presentation of conduction disease. Sodium current was lower when NaV1.5 was coexpressed with mutant beta1 or beta1B subunits than when it was coexpressed with WT subunits. These findings implicate SCN1B as a disease gene for human arrhythmia susceptibility.

Background— Today, mutations in more than 30 different genes have been found to cause inherited cardiomyopathies, some associated with very poor prognosis. However, because of the genetic heterogeneity and limitations in throughput and scalability of current diagnostic tools up until now, it is hardly possible to genetically characterize patients with cardiomyopathy in a fast, comprehensive, and cost-efficient manner. Methods and Results— We established an array-based subgenomic enrichment followed by next-generation sequencing to detect mutations in patients with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). With this approach, we show that the genomic region of interest can be enriched by a mean factor of 2169 compared with the coverage of the whole genome, resulting in high sequence coverage of selected disease genes and allowing us to define the genetic pathogenesis of cardiomyopathies in a single sequencing run. In 6 patients, we detected disease-causing mutations, 2 microdeletions, and 4 point mutations. Furthermore, we identified several novel nonsynonymous variants, which are predicted to be harmful, and hence, might be potential disease mutations or modifiers for DCM or HCM. Conclusions— The approach presented here allows for the first time a comprehensive genetic screening in patients with hereditary DCM or HCM in a fast and cost-efficient manner.

Background— Drug-induced long-QT syndrome (diLQTS) is an adverse drug effect that has an important impact on drug use, development, and regulation. We tested the hypothesis that common variants in key genes controlling cardiac electric properties modify the risk of diLQTS. Methods and Results— In a case-control setting, we included 176 patients of European descent from North America and Europe with diLQTS, defined as documented torsades de pointes during treatment with a QT-prolonging drug. Control samples were obtained from 207 patients of European ancestry who displayed &lt;50 ms QT lengthening during initiation of therapy with a QT-prolonging drug and 837 control subjects from the population-based KORA study. Subjects were successfully genotyped at 1424 single-nucleotide polymorphisms (SNPs) in 18 candidate genes including 1386 SNPs tagging common haplotype blocks and 38 nonsynonymous ion channel gene SNPs. For validation, we used a set of cases (n=57) and population-based control subjects of European descent. The SNP KCNE1 D85N (rs1805128), known to modulate an important potassium current in the heart, predicted diLQTS with an odds ratio of 9.0 (95% confidence interval, 3.5–22.9). The variant allele was present in 8.6% of cases, 2.9% of drug-exposed control subjects, and 1.8% of population control subjects. In the validation cohort, the variant allele was present in 3.5% of cases and in 1.4% of control subjects. Conclusions— This high-density candidate SNP approach identified a key potassium channel susceptibility allele that may be associated with the rare adverse drug reaction torsades de pointes.

Stress-dependent regulation of cardiac action potential duration is mediated by the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. It is accompanied by an increased magnitude of the slow outward potassium ion current, I(Ks). KCNQ1 and KCNE1 subunits coassemble to form the I(Ks) channel. Mutations in either subunit cause long QT syndrome, an inherited cardiac arrhythmia associated with an increased risk of sudden cardiac death. Here we demonstrate that exocytosis of KCNQ1 proteins to the plasma membrane requires the small GTPase RAB11, whereas endocytosis is dependent on RAB5. We further demonstrate that RAB-dependent KCNQ1/KCNE1 exocytosis is enhanced by the serum- and glucocorticoid-inducible kinase 1, and requires phosphorylation and activation of phosphoinositide 3-phosphate 5-kinase and the generation of PI(3,5)P(2). Identification of KCNQ1/KCNE1 recycling and its modulation by serum- and glucocorticoid-inducible kinase 1-phosphoinositide 3-phosphate 5-kinase -PI(3,5)P(2) provides a mechanistic insight into stress-induced acceleration of cardiac repolarization.

Altered myocardial repolarization is one of the important substrates of ventricular tachycardia and fibrillation. The influence of rare gene variants on repolarization is evident in familial long QT syndrome. To investigate the influence of common gene variants on the QT interval we performed a linkage disequilibrium based SNP association study of four candidate genes. Using a two-step design we analyzed 174 SNPs from the KCNQ1, KCNH2, KCNE1, and KCNE2 genes in 689 individuals from the population-based KORA study and 14 SNPs with results suggestive of association in a confirmatory sample of 3277 individuals from the same survey. We detected association to a gene variant in intron 1 of the KCNQ1 gene (rs757092, +1.7 ms/allele, P=0.0002) and observed weaker association to a variant upstream of the KCNE1 gene (rs727957, +1.2 ms/allele, P=0.0051). In addition we detected association to two SNPs in the KCNH2 gene, the previously described K897T variant (rs1805123, -1.9 ms/allele, P=0.0006) and a gene variant that tags a different haplotype in the same block (rs3815459, +1.7 ms/allele, P=0.0004). The analysis of additive effects by an allelic score explained a 10.5 ms difference in corrected QT interval length between extreme score groups and 0.951 of trait variance (P<0.00005). These results confirm previous heritability studies indicating that repolarization is a complex trait with a significant heritable component and demonstrate that high-resolution SNP-mapping in large population samples can detect and fine map quantitative trait loci even if locus specific heritabilities are small.

A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples.

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.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.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.

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.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.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.

Higher resting heart rate is associated with increased cardiovascular disease and mortality risk. Though heritable factors play a substantial role in population variation, little is known about specific genetic determinants. This knowledge can impact clinical care by identifying novel factors that influence pathologic heart rate states, modulate heart rate through cardiac structure and function or by improving our understanding of the physiology of heart rate regulation. To identify common genetic variants associated with heart rate, we performed a meta-analysis of 15 genome-wide association studies (GWAS), including 38,991 subjects of European ancestry, estimating the association between age-, sex- and body mass-adjusted RR interval (inverse heart rate) and approximately 2.5 million markers. Results with P < 5 × 10(-8) were considered genome-wide significant. We constructed regression models with multiple markers to assess whether results at less stringent thresholds were likely to be truly associated with RR interval. We identified six novel associations with resting heart rate at six loci: 6q22 near GJA1; 14q12 near MYH7; 12p12 near SOX5, c12orf67, BCAT1, LRMP and CASC1; 6q22 near SLC35F1, PLN and c6orf204; 7q22 near SLC12A9 and UfSp1; and 11q12 near FADS1. Associations at 6q22 400 kb away from GJA1, at 14q12 MYH6 and at 1q32 near CD34 identified in previously published GWAS were confirmed. In aggregate, these variants explain approximately 0.7% of RR interval variance. A multivariant regression model including 20 variants with P < 10(-5) increased the explained variance to 1.6%, suggesting that some loci falling short of genome-wide significance are likely truly associated. Future research is warranted to elucidate underlying mechanisms that may impact clinical care.

Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death. This meta-analysis sought to gain insights into the genetic determinants of myocardial mass. We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10−8. These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo. Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets.

Understanding the genetic architecture of cardiac structure and function may help to prevent and treat heart disease. This investigation sought to identify common genetic variations associated with inter-individual variability in cardiac structure and function.A GWAS meta-analysis of echocardiographic traits was performed, including 46,533 individuals from 30 studies (EchoGen consortium). The analysis included 16 traits of left ventricular (LV) structure, and systolic and diastolic function.The discovery analysis included 21 cohorts for structural and systolic function traits (n = 32,212) and 17 cohorts for diastolic function traits (n = 21,852). Replication was performed in 5 cohorts (n = 14,321) and 6 cohorts (n = 16,308), respectively. Besides 5 previously reported loci, the combined meta-analysis identified 10 additional genome-wide significant SNPs: rs12541595 near MTSS1 and rs10774625 in ATXN2 for LV end-diastolic internal dimension; rs806322 near KCNRG, rs4765663 in CACNA1C, rs6702619 near PALMD, rs7127129 in TMEM16A, rs11207426 near FGGY, rs17608766 in GOSR2, and rs17696696 in CFDP1 for aortic root diameter; and rs12440869 in IQCH for Doppler transmitral A-wave peak velocity. Findings were in part validated in other cohorts and in GWAS of related disease traits. The genetic loci showed associations with putative signaling pathways, and with gene expression in whole blood, monocytes, and myocardial tissue.The additional genetic loci identified in this large meta-analysis of cardiac structure and function provide insights into the underlying genetic architecture of cardiac structure and warrant follow-up in future functional studies.For detailed information per study, see Acknowledgments.

Electrocardiographic PR interval measures atrio-ventricular depolarization and conduction, and abnormal PR interval is a risk factor for atrial fibrillation and heart block. Our genome-wide association study of over 92,000 European-descent individuals identifies 44 PR interval loci (34 novel). Examination of these loci reveals known and previously not-yet-reported biological processes involved in cardiac atrial electrical activity. Genes in these loci are over-represented in cardiac disease processes including heart block and atrial fibrillation. Variants in over half of the 44 loci were associated with atrial or blood transcript expression levels, or were in high linkage disequilibrium with missense variants. Six additional loci were identified either by meta-analysis of ~105,000 African and European-descent individuals and/or by pleiotropic analyses combining PR interval with heart rate, QRS interval, and atrial fibrillation. These findings implicate developmental pathways, and identify transcription factors, ion-channel genes, and cell-junction/cell-signaling proteins in atrio-ventricular conduction, identifying potential targets for drug development.

Polymorphisms of the gene encoding apolipoprotein E have been implicated in the pathogenesis of peripheral and coronary artery disease and neurodegenerative disorders such as sporadic and late-onset familial forms of Alzheimer's disease. We have developed TaqMan assay systems for the single nucleotide polymorphisms -219G/T, located in the promoter of the apolipoprotein E gene, 113G/C, present in the transcriptional enhancer element of intron 1, 334T/C, determining Cys or Arg as amino acid residue 112 of mature apolipoprotein E, and 472C/T, determining Arg or Cys as residue 158. The accuracy of genotype determination with the TaqMan systems was demonstrated by analyses with restriction endonucleases. We determined the genotypes of the apolipoprotein E polymorphisms in 2349 study subjects. The genotypes were distributed as: -219GG = 27.3%, -219GT = 49.1%, and -219TT = 23.6% (p = 0.435); 113GG = 41.3%, 113GC = 45.2%, and 113CC = 13.5% (p = 0.343); 334TT = 73.4%, 334TC = 24.7%, and 334CC = 1.9% (p = 0.539); 472CC = 86.3%, 472CT=12.8%, and 472TT= 0.9% ( p = 0.004) (Hardy-Weinberg equilibrium estimates are given in parentheses). The allele combinations which define the three major isoforms of apolipoprotein E, namely apoE2, apoE3, and apoE4, had the following allele frequencies: 334T/472T (epsilon2; 112Cys/158Cys) = 7.3%, 334T/472C (epsilon3; 112Cys/158Arg) = 78.4%, and 334C/472C (epsilon4; 112Arg/158Arg) = 14.2%, respectively. ApoE genotypes were distributed as: epsilon2epsilon2 = 0.9%, epsilon2epsilon3 = 11.2%, epsilon2epsilon4 = 1.6%, epsilon3epsilon3 = 61.3%, epsilon3epsilon4 = 23.1%, and epsilon4epsilon4 = 1.9% (p = 0.014). The TaqMan assays allow for fast and sensitive genotyping and are especially suitable for studies including large numbers of participants.

The pattern of linkage disequilibrium (LD) is critical for association studies, in which disease-causing variants are identified by allelic association with adjacent markers. The aim of this study is to compare the LD patterns in several distinct European populations. We analyzed four genomic regions (in total, 749 kb) containing candidate genes for complex traits. Individuals were genotyped for markers that are evenly distributed at an average spacing of approximately 2-4 kb in eight population-based samples from ongoing epidemiological studies across Europe. The Centre d'Etude du Polymorphisme Humain (CEPH) trios of the HapMap project were included and were used as a reference population. In general, we observed a conservation of the LD patterns across European samples. Nevertheless, shifts in the positions of the boundaries of high-LD regions can be demonstrated between populations, when assessed by a novel procedure based on bootstrapping. Transferability of LD information among populations was also tested. In two of the analyzed gene regions, sets of tagging single-nucleotide polymorphisms (tagSNPs) selected from the HapMap CEPH trios performed surprisingly well in all local European samples. However, significant variation in the other two gene regions predicts a restricted applicability of CEPH-derived tagging markers. Simulations based on our data set show the extent to which further gain in tagSNP efficiency and transferability can be achieved by increased SNP density.

Torsades de pointes arrhythmias (TdP) occur by definition in the setting of prolonged QT intervals. Animal models of drug induced Long-QT syndrome (dLQTS) have shown higher predictive value for proarrhythmia with beat-to-beat variability of repolarization duration (BVR) when compared with QT intervals. Here, we evaluate variability of QT intervals in patients with a history of drug-induced long QT syndrome (dLQTS) and TdP in absence of a mutation in any of the major LQTS genes.Twenty patients with documented TdP under drugs with QT-prolonging potential were compared with 20 matched control individuals. An observer blinded to diagnosis manually measured lead-II, RR, and QT intervals from 30 consecutive beats. BVR was determined from Poincaré plots of QT intervals as short-term variability (STV(QT) = Sigma|QT(n)(+1) - QT(n)|/[30 x radical2]). QRS interval and cycle length was comparable between study groups and controls. No difference was found in QTc between dLQTS and controls (428 +/- 25 vs. 421 +/- 34 ms, P = 0.26), whereas STV(QT) was significantly higher in dLQTS when compared with controls (8.1 +/- 3.7 vs. 3.6 +/- 1.3 ms, P = 0.001). Proarrhythmic predictive power of STV(QT) was superior to that of the QTc interval (AUC: 0.89 vs. 0.57, 95% CI: 0.79-0.99 vs. 0.39-0.75).In the absence of QTc prolongation, baseline STV(QT) characterized patients with documented drug-induced proarrhythmia. STV(QT) could prove to be a useful non-invasive, easily obtainable parameter aiding the identification of the patient at risk for potentially life threatening arrhythmia in the context of drugs with QT prolonging potential.

Cardiac repolarization, the process by which cardiomyocytes return to their resting potential after each beat, is a highly regulated process that is critical for heart rhythm stability. Perturbations of cardiac repolarization increase the risk for life-threatening arrhythmias and sudden cardiac death. Although genetic studies of familial long-QT syndromes have uncovered several key genes in cardiac repolarization, the major heritable contribution to this trait remains unexplained. Identification of additional genes may lead to a better understanding of the underlying biology, aid in identification of patients at risk for sudden death, and potentially enable new treatments for susceptible individuals.We extended and refined a zebrafish model of cardiac repolarization by using fluorescent reporters of transmembrane potential. We then conducted a drug-sensitized genetic screen in zebrafish, identifying 15 genes, including GINS3, that affect cardiac repolarization. Testing these genes for human relevance in 2 concurrently completed genome-wide association studies revealed that the human GINS3 ortholog is located in the 16q21 locus, which is strongly associated with QT interval.This sensitized zebrafish screen identified 15 novel myocardial repolarization genes. Among these genes is GINS3, the human ortholog of which is a major locus in 2 concurrent human genome-wide association studies of QT interval. These results reveal a novel network of genes that regulate cardiac repolarization.

Atrial fibrillation (AF) is the most common adverse event following coronary artery bypass graft surgery. A recent study identified chromosome 4q25 variants associated with AF in ambulatory populations. However, their role in postoperative AF is unknown. We hypothesized that genetic variants in the 4q25 chromosomal region are independently associated with postoperative AF after coronary artery bypass graft surgery.Two prospectively collected cohorts of patients undergoing coronary artery bypass graft surgery, with or without concurrent valve surgery, at 3 US centers. From a discovery cohort of 959 patients, clinical and genomic multivariate predictors of postoperative AF were identified by genotyping 45 single-nucleotide polymorphisms (SNPs) encompassing the 4q25 locus. Three SNPs were then assessed in a separately collected validation cohort of 494 patients. After adjustment for clinical predictors of postoperative AF and multiple comparisons, rs2200733, rs13143308, and 5 other linked SNPs independently predicted postoperative AF in the discovery cohort. Additive odds ratios for the 7 associated 4q25 SNPs ranged between 1.57 and 2.17 (P=8.0x10(-4) to 3.4x10(-6)). Association with postoperative AF were measured and replicated for rs2200733 and rs13143308 in the validation cohort.In 2 independently collected cardiac surgery cohorts, noncoding SNPs within the chromosome 4q25 region are independently associated with postoperative AF after coronary artery bypass graft surgery after adjusting for clinical covariates and multiple comparisons.

Atrial fibrillation (AF) is the most frequent arrhythmia in humans. Rare familial forms exist. Recent evidence indicates a genetic susceptibility to common forms of AF. The alpha-subunit of the myocardial I(Kr)-channel, encoded by the KCNH2 gene, is crucial to ventricular and atrial repolarization. Patients with mutations in KCNH2 present with higher incidence of AF. Common variants in KCNH2 have been shown to modify ventricular repolarization. We intended to investigate, whether such variants may also modulate atrial repolarization and predispose to AF.In a two-stage association study we analysed 1207 AF-cases and 2475 controls. In stage I 40 tagSNPs (single nucleotide polymorphisms) from the KCNH2 genomic region were genotyped in 671 AF-cases and 694 controls. Of five associated variants, the common K897-allele of the KCNH2-K897T variant was replicated in n = 536 independent AF cases and n = 1781 controls in stage II [overall odds ratio 1.25, 95% confidence interval 1.11-1.41, P = 0.00033]. This association remained significant after adjustment for gender and age.We report a genetic association finding including positive replication between the K897-allele and higher incidence of AF. This provides a molecular correlate for complex genetic predispositions to AF. The consequences of the K897T variant at the atrial level will require further functional investigations.

The high incidence of sudden cardiac death in heart failure (HF) reflects electrophysiologic changes in response to myocardial failure. We previously showed that short-term variability of QT intervals (STV(QT)) identifies latent repolarization disorders in patients with drug-induced or congenital long QT syndrome. This study sought to determine (1) if STV(QT) is increased in patients with dilated cardiomyopathy (DC) and moderate congestive HF and (2) if increased STV(QT) is associated with ventricular arrhythmia in patients with HF. Sixty patients (53 +/- 12 years of age, 14 women) with DC and moderate HF (New York Heart Association classes II to III) were compared to matched controls. Twenty patients had implantable cardiac defibrillators secondary to a history of ventricular tachycardia (VT). Two cardiologists blinded to diagnosis manually measured QT intervals. Beat-to-beat variability of repolarization was determined from Poincaré plots of 30 consecutive QT intervals as was STV(QT). QTc intervals were comparable in patients and controls (419 +/- 36 vs 415 +/- 32 ms, respectively, p >0.05), whereas STV(QT) was significantly higher in patients with HF (7.8 +/- 3 vs 4.1 +/- 2 ms, respectively, p <0.05). STV(QT) was more increased in patients with a history of VT compared to those without VT (10.1 +/- 2 vs 6.6 +/- 2 ms, respectively, p <0.05). Increased STV(QT) and decreased ejection fraction were associated with a history of VT; however, STV(QT) was the strongest indicator. In conclusion, the present study demonstrates for the first time that STV(QT) is increased in patients with DC with HF. Patients with DC and HF and implantable cardiac defibrillators for secondary prevention had the highest STV(QT). Thus, increased STV(QT) in the context of moderate HF may reflect a latent repolarization disorder and increased susceptibility to sudden death in patients with DC, which is not identified by a prolonged QT interval.

Apart from clinical symptoms the diagnosis and risk stratification in long-QT syndrome (LQTS) is usually based on the surface electrocardiogram. Studies have indicated that not only prolongation of the QT interval but also an increased short-term variability of QT interval (STV(QT)) is a marker for a decreased repolarization reserve in patients with drug-induced LQTS. The aims of this study were to determine if STV(QT) (1) is higher in patients with LQTS compared with controls, (2) if this effect is more pronounced in a high-risk LQTS population, and (3) could increase the diagnostic power of the surface electrocardiogram in identifying mutation carriers. Forty mutation carriers were compared with age- and gender-matched control subjects in the absence of beta-receptor-blocking agents. Lead II or V(5) RR and QT intervals from 30 consecutive beats were manually measured. STV(QT) was determined from Poincaré plots of QT intervals (STV(QT) = Sigma|QTn + 1 - QTn|/[30 x radical2]). Compared with controls, patients with LQTS had a prolonged QTc interval (449 +/- 41 vs 411 +/- 32 ms, p = 0.00049) and increased STV(QT) (6.4 +/- 3.2 vs 4.1 +/- 1.6 ms, p = 0.005). In patients with the highest risk of clinical events, defined as a QTc interval >500 ms or symptoms before beta-blocker therapy, STV(QT) was 9 +/- 4 ms. QTc interval had a sensitivity of 43% and a specificity of 97% in identifying mutation carriers (thresholds 450 ms for men and 460 ms for women). Receiver operator characteristic analysis showed that an STV(QT) of 4.9 ms was the optimal cut-off value to predict mutation carriers. When incorporating an STV(QT) >4.9 ms for those whose QTc interval was within the normal limits, sensitivity to distinguish mutation carriers increased to 83% with a specificity of 68%, so that another 15 mutation carriers could be identified. In conclusion, these are the first results in humans showing that STV(QT) is increased in congenital LQTS, this effect is increased in patients with symptoms before therapy, and, hence, STV(QT) could prove to be a useful noninvasive additive marker for diagnostic screening to bridge the gap before results of genetic testing are available.

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<2.5×10−8) 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 = 3×10−23). This SNP was also associated with PR interval (beta = 2.4 msec per minor allele, 95% CI = 1.8–3.0, p = 3×10−16) 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.

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.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.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.

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. 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.

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.

Physical and emotional stress is accompanied by release of stress hormones such as the glucocorticoid cortisol. This hormone upregulates the serum- and glucocorticoid-inducible kinase (SGK)1, which in turn stimulates I(Ks), a slow delayed rectifier potassium current that mediates cardiac action potential repolarization. Mutations in I(Ks) channel alpha (KCNQ1, KvLQT1, Kv7.1) or beta (KCNE1, IsK, minK) subunits cause long QT syndrome (LQTS), an inherited cardiac arrhythmia associated with increased risk of sudden death. Together with the GTPases RAB5 and RAB11, SGK1 facilitates membrane recycling of KCNQ1 channels. Here, we show altered SGK1-dependent regulation of LQTS-associated mutant I(Ks) channels. Whereas some mutant KCNQ1 channels had reduced basal activity but were still activated by SGK1, currents mediated by KCNQ1(Y111C) or KCNQ1(L114P) were paradoxically reduced by SGK1. Heteromeric channels coassembled of wild-type KCNQ1 and the LQTS-associated KCNE1(D76N) mutant were similarly downregulated by SGK1 because of a disrupted RAB11-dependent recycling. Mutagenesis experiments indicate that stimulation of I(Ks) channels by SGK1 depends on residues H73, N75, D76, and P77 in KCNE1. Identification of the I(Ks) recycling pathway and its modulation by stress-stimulated SGK1 provides novel mechanistic insight into potentially fatal cardiac arrhythmias triggered by physical or psychological stress.

Background— We set out to identify common genetic determinants of the length of the RR and QT intervals in 2325 individuals from isolated European populations. Methods and Results— We analyzed the heart rate at rest, measured as the RR interval, and the length of the corrected QT interval for association with 318 237 single-nucleotide polymorphisms. The RR interval was associated with common variants within GPR133 , a G-protein–coupled receptor (rs885389, P =3.9�10 −8 ). The QT interval was associated with the earlier reported NOS1AP gene (rs2880058, P =2.00�10 −10 ) and with a region on chromosome 13 (rs2478333, P =4.34�10 −8 ), which is 100 kb from the closest known transcript LOC730174 and has previously not been associated with the length of the QT interval. Conclusion— Our results suggested an association between the RR interval and GPR133 and confirmed an association between the QT interval and NOS1AP .

To identify loci affecting the electrocardiographic QT interval, a measure of cardiac repolarisation associated with risk of ventricular arrhythmias and sudden cardiac death, we conducted a meta-analysis of three genome-wide association studies (GWAS) including 3,558 subjects from the TwinsUK and BRIGHT cohorts in the UK and the DCCT/EDIC cohort from North America. Five loci were significantly associated with QT interval at P<1x10(-6). To validate these findings we performed an in silico comparison with data from two QT consortia: QTSCD (n = 15,842) and QTGEN (n = 13,685). Analysis confirmed the association between common variants near NOS1AP (P = 1.4x10(-83)) and the phospholamban (PLN) gene (P = 1.9x10(-29)). The most associated SNP near NOS1AP (rs12143842) explains 0.82% variance; the SNP near PLN (rs11153730) explains 0.74% variance of QT interval duration. We found no evidence for interaction between these two SNPs (P = 0.99). PLN is a key regulator of cardiac diastolic function and is involved in regulating intracellular calcium cycling, it has only recently been identified as a susceptibility locus for QT interval. These data offer further mechanistic insights into genetic influence on the QT interval which may predispose to life threatening arrhythmias and sudden cardiac death.

Considerable interest exists in the identification of genetic modifiers of disease severity in the long-QT syndrome (LQTS) as their identification may contribute to refinement of risk stratification.We searched for single-nucleotide polymorphisms (SNPs) that modulate the corrected QT (QTc)-interval and the occurrence of cardiac events in 639 patients harboring different mutations in KCNH2. We analyzed 1201 SNPs in and around 18 candidate genes, and in another approach investigated 22 independent SNPs previously identified as modulators of QTc-interval in genome-wide association studies in the general population. In an analysis for quantitative effects on the QTc-interval, 3 independent SNPs at NOS1AP (rs10494366, P=9.5×10(-8); rs12143842, P=4.8×10(-7); and rs2880058, P=8.6×10(-7)) were strongly associated with the QTc-interval with marked effects (>12 ms/allele). Analysis of patients versus general population controls uncovered enrichment of QTc-prolonging alleles in patients for 2 SNPs, located respectively at NOS1AP (rs12029454; odds ratio, 1.85; 95% confidence interval, 1.32-2.59; P=3×10(-4)) and KCNQ1 (rs12576239; odds ratio, 1.84; 95% confidence interval, 1.31-2.60; P=5×10(-4)). An analysis of the cumulative effect of the 6 NOS1AP SNPs by means of a multilocus genetic risk score (GRS(NOS1AP)) uncovered a strong linear relationship between GRS(NOS1AP) and the QTc-interval (P=4.2×10(-7)). Furthermore, patients with a GRS(NOS1AP) in the lowest quartile had a lower relative risk of cardiac events compared with patients in the other quartiles combined (P=0.039).We uncovered unexpectedly large effects of NOS1AP SNPs on the QTc-interval and a trend for effects on risk of cardiac events. For the first time, we linked common genetic variation at KCNQ1 with risk of long-QT syndrome.

To the Editor: Diaphragmatic spinal muscular atrophy (SMA) has been delineated as a variant of infantile SMA (SMA1 [MIM 253300]) (Mellins et al. Mellins et al., 1974Mellins RB Hays AP Gold AP Berdon WE Bowdler JD Respiratory distress as the initial manifestation of Werdnig-Hoffmann disease.Pediatrics. 1974; 53: 33-40PubMed Google Scholar; Bertini et al. Bertini et al., 1989Bertini E Gadisseux JL Palmieri G Ricci E Di Capua M Ferriere G Lyon G Distal infantile spinal muscular atrophy associated with paralysis of the diaphragm: a variant of infantile spinal muscular atrophy.Am J Med Genet. 1989; 33: 328-335Crossref PubMed Scopus (50) Google Scholar). The most prominent symptoms are severe respiratory distress resulting from diaphragmatic paralysis with eventration shown on chest x-ray and predominant involvement of the upper limbs and distal muscles. In contrast to classic SMA1, in diaphragmatic SMA the upper spinal cord is more severely affected than the lower section. The pmn mouse presents with progressive motor neuronopathy and a disease that closely resembles diaphragmatic SMA (Schmalbruch et al. Schmalbruch et al., 1991Schmalbruch H Jensen H-J Bjærg M Kamieniecka Z Kurland L A new mouse mutant with progressive motor neuronopathy.J Neuropathol Exp Neurol. 1991; 50: 192-204Crossref PubMed Scopus (156) Google Scholar). The pmn locus has been mapped to murine chromosome 13 (Brunialti et al. Brunialti et al., 1995Brunialti AL Poirier C Schmalbruch H Guénet J-L The mouse mutation progressive motor neuronopathy (pmn) maps to chromosome 13.Genomics. 1995; 29: 131-135Crossref PubMed Scopus (23) Google Scholar). Here we report on nine patients from three families with diaphragmatic SMA following autosomal recessive inheritance. The diagnosis of diaphragmatic SMA was made on the basis of clinical criteria (Rudnik-Schöneborn et al. Rudnik-Schöneborn et al., 1996Rudnik-Schöneborn S Forkert R Hahnen E Wirth B Zerres K Clinical spectrum and diagnostic criteria of infantile spinal muscular atrophy: further delineation on the basis of SMN gene deletion findings.Neuropediatrics. 1996; 27: 8-15Crossref PubMed Scopus (109) Google Scholar). Family 1 is of Lebanese origin; family 2, German origin; and family 3, Italian origin. We obtained DNA samples from these families after receiving informed consent, in accordance with the Declaration of Helsinki. In family 1 (fig. 1A), the parents are first cousins. The first affected son died, at the age of 10 wk, of suspected sudden infant death syndrome (SIDS). One daughter presented, at the age of 6 wk, with feeding difficulties and progressive respiratory distress. Chest x-ray showed eventration of the diaphragm. Mechanical ventilation was initiated at the age of 8 wk. She developed progressive muscular atrophy with complete paralysis of the upper and lower limbs and mild contractures of the knee and ankle joints. Three other children, nonidentical twin daughters and the youngest daughter, died of respiratory failure—the twins at the age of 8 and 9 wk and the youngest daughter at the age of 8 wk. Autopsy specimens were taken from gastrocnemius muscle in both twins and from the upper spinal cord in one twin. Skeletal-muscle histology revealed neurogenic atrophy without signs of reinnervation. Ultrastructurally, the motor end plates lacked nerve terminals and showed postsynaptic degenerative changes characterized by deep invaginations. The diameter of anterior spinal roots was reduced in the upper spinal cord. The remaining motor neurons showed chromatolysis. These findings offer two different pathophysiological concepts: (1) degeneration of the anterior horn cells of the spinal cord with neurogenic muscular atrophy suggests dying-forward atrophy, and (2) presynaptic and postsynaptic signs of motor end-plate degeneration suggest dying-back atrophy. In family 2 (fig. 1B), the first child had severe muscular hypotonia and died, at the age of 9 wk, of cardiorespiratory failure. The third child has been mechanically ventilated since the age of 3 mo. In family 3 (fig. 1C), which has been reported in detail elsewhere (Novelli et al. Novelli et al., 1995Novelli G Capon F Tamisari L Grandi E Angelini C Guerrini P Dallapiccola B Neonatal spinal muscular atrophy with diaphragmatic paralysis is unlinked to 5q11.2-q13.J Med Genet. 1995; 32: 216-219Crossref PubMed Scopus (30) Google Scholar), the gene locus for SMA1, on chromosome 5q, has been excluded. Both affected sibs presented with respiratory insufficiency right after birth and with the typical signs of diaphragmatic SMA. First, we confirmed that, in families 1 and 2, there is no linkage of the trait to markers of the SMA locus on 5q11.2-q13.3, as there is in family 3. Second, the orthologous regions corresponding to the murine pmn gene region on human chromosomes 1q and 7p were excluded as gene loci responsible for the disease (Grohmann et al. Grohmann et al., 1998Grohmann K, Hübner C, Saar K, Stoltenburg-Didinger G, Wienker T (1998) Diaphragmatic spinal muscular atrophy (SMAD) is not the homologue counterpart to murine progressive motoneuron disease (pmn). Paper presented at the 5th Workshop Neurogenetics in Germany, Freiburg, Germany, October 22-24Google Scholar). To locate the gene locus for diaphragmatic SMA, a whole-genome scan was undertaken in family 1. Microsatellite analysis was performed, by standard semiautomated methods, by an ABI 377-Sequencer, and the results were processed by GENESCAN software, as described elsewhere (Saar et al. Saar et al., 1997Saar K Chrzanowska KH Stumm M Jung M Nürnberg G Wienker TF Seemanová E et al.The gene for the ataxia-telangiectasia variant, Nijmegen breakage syndrome, maps to a 1-cM interval on chromosome 8q21.Am J Hum Genet. 1997; 60: 605-610PubMed Google Scholar). The whole-genome linkage scan was performed with the use of 340 polymorphic fluorescence–labeled markers spaced at ∼10-cM intervals throughout the autosomal part of the genome. Subsequent fine mapping was performed with eight additional microsatellite markers. Markers were chosen from the Généthon final linkage map. Two-point parametric linkage analyses were performed with the LINKAGE package, version 5.2 (Lathrop and Lalouel Lathrop and Lalouel, 1984Lathrop GM Lalouel JM Easy calculations of LOD scores and genetic risks on small computers.Am J Hum Genet. 1984; 36: 460-465PubMed Google Scholar), under the following assumptions: a regular, fully penetrant autosomal recessive trait locus with a disease-allele frequency of .002 and no phenocopy rate, codominant marker loci with uniformly distributed allele frequencies, and standard recombination rates. Multipoint analysis was performed with the GENEHUNTER program, version 1.3 (Kruglyak et al. Kruglyak et al., 1996Kruglyak L Daly MJ Reeve-Daly MP Lander ES Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar). Genomewide linkage scanning of family 1 revealed linkage of diaphragmatic SMA only to markers on chromosome 11q13-q21. In the following, we name this subtype of diaphragmatic SMA “spinal muscular atrophy with respiratory distress” (SMARD). For the markers D11S1296, D11S4095, D11S901, D11S1358, and D11S1757, a maximum two-point LOD score of 3.16 at recombination fraction (θ) 0 was obtained. The two-point LOD scores for 13 markers on chromosome 11q are summarized in table 1. Haplotype analysis revealed a recombination event in individual 2.4 that placed the disease locus distal to marker D11S1883 (fig. 1A). The crossing-over in individual 2.1 placed the disease locus proximal to marker D11S917. Consistent with parental consanguinity, all affected siblings from family 1 were autozygous for all markers within the cosegregating segment. Multipoint linkage analysis with the use of 13 markers yielded a maximum LOD score of 3.86, which clearly places the disease locus between D11S1883 and D11S917 (Généthon map positions 68.5 cM and 100.9 cM).Table 1LOD-Score Values at Standard Recombination Rates for Markers on Chromosome 11q in Lebanese Family 1LOD Score at θ =MarkerPositionaSex-averaged genetic coordinates on chromosome 11 (cM), according to the Généthon map.HeterozygositybEstimated value..00.01.05.10.15.20.30D11S188368.5.73−∞−.87−.24−.03.06.09.09D11S91370.9.571.151.131.04.93.81.69.43D11S129671.0cEstimated from the genetic maps of the Marshfield Medical Research Foundation Center for Medical Genetics..503.163.102.862.552.231.911.22D11S409571.0.643.163.102.862.552.231.901.22D11S417871.5.671.751.721.581.401.221.04.66D11S131477.5.771.751.721.581.401.221.04.66D11S91680.1.722.962.902.672.382.071.761.09D11S90189.8.823.163.102.862.552.231.901.22D11S135896.3.753.163.112.882.592.281.961.28D11S131197.5.751.751.721.581.401.221.04.66D11S417697.5.821.751.721.581.401.221.04.66D11S175798.1.653.163.112.882.592.281.961.28D11S917100.9.80−∞−.28.30.45.47.44.30a Sex-averaged genetic coordinates on chromosome 11 (cM), according to the Généthon map.b Estimated value.c Estimated from the genetic maps of the Marshfield Medical Research Foundation Center for Medical Genetics. Open table in a new tab In family 2, the two affected sibs shared two identical parental haplotypes in the SMARD cosegregating segment on 11q13-q21, a finding that supports the assignment of the SMARD locus to this region (fig. 1B). In family 3, haplotype analysis was inconsistent with linkage to the markers tested (fig. 1C). Thus, this locus was excluded as being responsible for the disease in this family. Our finding that diaphragmatic SMA with onset at age 6–12 wk is linked to chromosome 11q markers in two apparently unrelated families from different countries (families 1 and 2) but that diaphragmatic SMA with onset at birth does not show such linkage (family 3) suggests that diaphragmatic SMA is both clinically and genetically heterogeneous. The prevalence of diaphragmatic SMA is unknown. However, in a series of >200 patients with early-onset SMA, ∼1% presented with diaphragmatic SMA and did not have a deletion of the survival motor-neuron gene (SMN) on chromosome 5q (Rudnik-Schöneborn et al. Rudnik-Schöneborn et al., 1996Rudnik-Schöneborn S Forkert R Hahnen E Wirth B Zerres K Clinical spectrum and diagnostic criteria of infantile spinal muscular atrophy: further delineation on the basis of SMN gene deletion findings.Neuropediatrics. 1996; 27: 8-15Crossref PubMed Scopus (109) Google Scholar). Considering the case history of the affected son from family 1 who had suspected SIDS, we presume that some of those infants with SIDS may possibly have been misdiagnosed. We are currently looking for further patients with SMARD, to refine the large cosegregating region on chromosome 11q. The authors wish to thank the families for participation in the study. The help and advice of Angela Huebner, Hubert Pöche, Franz Rüschendorf, Hans-Ludwig Spohr, Frank van Landeghem, and Angelika Zwirner are gratefully acknowledged. This study has been supported in part by the German Association for Muscle Diseases, by the parents' action group “Helft dem muskelkranken Kind” (Hamburg), by the “Deutsche Forschungsgemeinschaft,” and by the Italian National Council of Research, Scientific Project of Biotechnology. The “Mikrosatellitenzentrum” at the Max Delbrück Center for Molecular Medicine is supported by a grant-in-aid (to A.R. and T.F.W.) from the German Human Genome Project.

The toll-like receptor 4 (TLR4) is predominantly known for its role as an important mediator of immune reactions and has been implicated in the initiation, progression, and plaque destabilization stages of atherosclerosis. We investigated whether genotypes and haplotypes of the 896A/G (Asp299Gly; rs4986790) and 1196C/T (Thr399Ile; rs4986791) single nucleotide polymorphisms of the gene encoding the TLR4 were associated with myocardial infarction (MI) in a large Caucasian sample.The case group included 3657 patients with MI and the control group comprised 1211 individuals with angiographically normal coronary arteries and without signs or symptoms of MI. Genotypes were determined with the use of TaqMan assays. Genotype distributions of the 896A/G and 1196C/T polymorphisms were not significantly different between the control and patient groups (P> or =0.30). The frequencies of haplotypes defined by the 896A/G and 1196C/T polymorphisms were similar between the control group and the patient group (P> or =0.16). In addition, the distributions of haplotype-defined genotypes (diplotypes) were not significantly different between the control group and the patient group (P> or =0.12). Separate analyses in women and men did not reveal sex-related associations of specific genotypes or haplotypes of the polymorphisms with MI (P> or =0.11).The results indicate that the 896A/G and 1196C/T polymorphisms of the TLR4 gene or haplotypes based on these polymorphisms are not associated with MI.

AimsThe Brugada syndrome is a rare, potentially fatal primary cardiomyopathy. Patients are identified by symptoms and typical electrocardiogram (ECG) patterns. Prevalence of spontaneous Brugada ECG patterns in the general population is unknown.

We examined the insertion/deletion polymorphism in the angiotensin-converting enzyme gene and identified polymorphisms in the heart chymase gene to test the hypothesis that these angiotensin II-producing enzymes are associated with a monogenic cardiac disease (50 patients and 50 control subjects) as a model of cardiac hypertrophy. We found that the angiotensin-converting enzyme DD genotype was present more often in patients than in control subjects and identified a possible interaction with 1 of the chymase polymorphisms.

Genome-wide association studies (GWAS) have become an effective tool to map genes and regions contributing to multifactorial human diseases and traits. A comparably small number of variants identified by GWAS are known to have a direct effect on protein structure whereas the majority of variants is thought to exert their moderate influences on the phenotype through regulatory changes in mRNA expression. MicroRNAs (miRNAs) have been identified as powerful posttranscriptional regulators of mRNAs. Binding to their target sites, which are mostly located within the 3'-untranslated region (3'-UTR) of mRNA transcripts, they modulate mRNA expression and stability. Until today almost all human mRNA transcripts are known to harbor at least one miRNA target site with an average of over 20 miRNA target sites per transcript. Among 5,101 GWAS-identified sentinel single nucleotide polymorphisms (SNPs) that correspond to 18,884 SNPs in linkage disequilibrium (LD) with the sentinels (r2 ≥ 0.8) we identified a significant overrepresentation of SNPs that affect the 3'-UTR of genes (OR = 2.33, 95% CI = 2.12-2.57, P < 10(-52)). This effect was even stronger considering all SNPs in one LD bin a single signal (OR = 4.27, 95% CI = 3.84-4.74, P < 10(-114)). Based on crosslinking immunoprecipitation data we identified four mechanisms affecting miRNA regulation by 3'-UTR mutations: (i) deletion or (ii) creation of miRNA recognition elements within validated RNA-induced silencing complex binding sites, (iii) alteration of 3'-UTR splicing leading to a loss of binding sites, and (iv) change of binding affinity due to modifications of 3'-UTR folding. We annotated 53 SNPs of a total of 288 trait-associated 3'-UTR SNPs as mediating at least one of these mechanisms. Using a qualitative systems biology approach, we demonstrate how our findings can be used to support biological interpretation of GWAS results as well as to provide new experimentally testable hypotheses.

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.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.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.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.

The effects of estrogen on blood vessels are partly due to changes in vascular cell gene expression and protein synthesis that are mediated by estrogen receptors. In previous association studies, the -397T/C (rs2234693) and -351A/G (rs9340799) single nucleotide polymorphisms in the estrogen receptor alpha gene (ESR1) have been implicated in the risk of coronary atherosclerosis and myocardial infarction. To test these findings, we examined the relationship of the polymorphisms to myocardial infarction in a large sample of white patients and control individuals of predominantly European descent.The case group included 3657 patients with myocardial infarction, and the control group comprised 1211 individuals with angiographically normal coronary arteries and without signs or symptoms of myocardial infarction. TaqMan assays were used for the determination of genotypes. Genotype distributions of the -397T/C and -351A/G polymorphisms were not significantly different between the control and patient groups (P> or =0.85). The frequencies of haplotypes defined by the -397T/C and -351A/G polymorphisms were similar in the control group and the patient group (P=0.42). In addition, the distributions of haplotype-defined genotypes (diplotypes) were not significantly different between the control group and the patient group (P=0.81). Separate analyses in women and men did not reveal sex-related associations of specific genotypes or haplotypes of the polymorphisms with myocardial infarction (P> or =0.25).The results indicate that the -397T/C and -351A/G polymorphisms of ESR1 or haplotypes based on these polymorphisms are not associated with myocardial infarction in a white population.

Given the cost constraints of the European health-care systems, criteria are needed to decide which genetic services to fund from the public budgets, if not all can be covered. To ensure that high-priority services are available equitably within and across the European countries, a shared set of prioritization criteria would be desirable. A decision process following the accountability for reasonableness framework was undertaken, including a multidisciplinary EuroGentest/PPPC-ESHG workshop to develop shared prioritization criteria. Resources are currently too limited to fund all the beneficial genetic testing services available in the next decade. Ethically and economically reflected prioritization criteria are needed. Prioritization should be based on considerations of medical benefit, health need and costs. Medical benefit includes evidence of benefit in terms of clinical benefit, benefit of information for important life decisions, benefit for other people apart from the person tested and the patient-specific likelihood of being affected by the condition tested for. It may be subject to a finite time window. Health need includes the severity of the condition tested for and its progression at the time of testing. Further discussion and better evidence is needed before clearly defined recommendations can be made or a prioritization algorithm proposed. To our knowledge, this is the first time a clinical society has initiated a decision process about health-care prioritization on a European level, following the principles of accountability for reasonableness. We provide points to consider to stimulate this debate across the EU and to serve as a reference for improving patient management.

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.

This study compared metabolite shifts induced by training for, participation in, and recovery from a marathon race competition among athletes divided into three groups based on fitness (relative maximum oxygen uptake (VO2max)) and performance levels (net running time). Plasma samples from 76 male runners participating in the Munich Marathon were analyzed for metabolite shifts using a targeted metabolomics panel. For the entire cohort of runners, pronounced increases were measured immediately after the race for plasma concentrations of acylcarnitines (AC), the ratio (palmitoylcarnitine + stearoylcarnitine)/free carnitine that is used as a proxy for the activity of the mitochondrial enzyme carnitine palmitoyltransferase, and arginine-related metabolites, with decreases in most amino acids (AA) and phospholipids. Plasma levels of AA and phospholipids were strongly increased 24 and 72 h post-race. Post-race plasma concentrations of AC and arginine-related metabolites were higher in the low compared to top performers, indicating an accumulation of fatty acids and a reliance on protein catabolism to provide energy after the marathon event. This study showed that marathon race competition is associated with an extensive and prolonged perturbation in plasma metabolite concentrations with a strong AC signature that is greater in the slower, less aerobically fit runners. Furthermore, changes in the arginine-related metabolites were observed.

Neonatal TSH screening has been a major achievement for the early detection and treatment of primary congenital hypothyroidism. It nevertheless fails to reveal cases of central hypothyroidism caused by TSH levels in the low normal range. In the last 10 yr, homozygous mutations in the TSHbeta-subunit gene have been recognized as a cause of central hypothyroidism with isolated TSH deficiency. The most frequent TSHbeta mutation 313DeltaT (C105V) has been described in six apparently unrelated families. We investigated the frequency and possible monophyletic origin of the different TSHbeta 313DeltaT alleles of the three affected families. Haplotype analysis of five polymorphic single-nucleotide polymorphism loci in the TSHbeta region revealed the presence of seven different haplotypes in the general population. In all six parental lines, the mutation occurred on the same haplotype. Extending the haplotype by two flanking microsatellite markers led to a mutational age estimate of about 150 generations. In 500 unrelated individuals from the general population, we did not detect any TSHbeta 313DeltaT allele, suggesting a population heterozygote carrier frequency less than 1:170 with more than 95% probability. Accordingly, the disease risk in the general population because of homozygosity is low. Our data suggest a monophyletic origin of the TSHbeta 313DeltaT mutation from a common ancestor and no significant population prevalence. Therefore, identification and genetic counseling of heterozygous carriers in affected families seems to be more advisable than population-wide neonatal T(4) screening programs for an early detection of this rare condition.

Abstract Background New technologies have enabled genome-wide association studies to be conducted with hundreds of thousands of genotyped SNPs. Several different first-generation genome-wide panels of SNPs have been commercialized. The total amount of common genetic variation is still unknown; however, the coverage of commercial panels can be evaluated against reference population samples genotyped by the International HapMap project. Less information is available about coverage in samples from other populations. Results In this study we compare four commercial panels: the HumanHap 300 and HumanHap 550 Array Sets from the Illumina Infinium series and the Mapping 100 K and Mapping 500 K Array Sets from the Affymetrix GeneChip series. Tagging performance is compared among HapMap CEPH (CEU), Asian (JPT, CHB) and Yoruba (YRI) population samples. It is also evaluated in an Estonian population sample with more than 1000 individuals genotyped in two 500-kbp ENCODE regions of chromosome 2: ENr112 on 2p16.3 and ENr131 on 2p37.1. Conclusion We found that in a non-reference Caucasian population, commercial SNP panels provide levels of coverage similar to those in the HapMap CEPH population sample. We present the proportions of universal and population-specific SNPs in all the commercial platforms studied.

Action myoclonus-renal failure syndrome is a hereditary form of progressive myoclonus epilepsy associated with renal failure. It is considered to be an autosomal-recessive disease related to loss-of-function mutations in SCARB2. We studied a German AMRF family, additionally showing signs of demyelinating polyneuropathy and dilated cardiomyopathy. To test the hypothesis whether isolated appearance of individual AMRF syndrome features could be related to heterozygote SCARB2 mutations, we screened for SCARB2 mutations in unrelated patients showing isolated AMRF features. In the AMRF family all exons of SCARB2 were analyzed by Sanger sequencing. The mutation screening of unrelated patients with isolated AMRF features affected by either epilepsy (n = 103, progressive myoclonus epilepsy or generalized epilepsy), demyelinating polyneuropathy (n = 103), renal failure (n = 192) or dilated cardiomyopathy (n = 85) was performed as high resolution melting curve analysis of the SCARB2 exons. A novel homozygous 1 bp deletion (c.111delC) in SCARB2 was found by sequencing three affected homozygous siblings of the affected family. A heterozygous sister showed generalized seizures and reduction of nerve conduction velocity in her legs. No mutations were found in the epilepsy, renal failure or dilated cardiomyopathy samples. In the polyneuropathy sample two individuals with demyelinating disease were found to be carriers of a SCARB2 frameshift mutation (c.666delCCTTA). Our findings indicate that demyelinating polyneuropathy and dilated cardiomyopathy are part of the action myoclonus-renal failure syndrome. Moreover, they raise the possibility that in rare cases heterozygous SCARB2 mutations may be associated with PNP features.

Background Atrial fibrillation (AF) is the most common sustained arrhythmia and has a substantial heritable component. Numerous associations between single nucleotide polymorphisms (SNPs) and AF have been described, but few have been replicated. Objective We sought to systematically replicate SNPs that are reported to be associated with AF in two large study samples of European descent. Methods We searched PubMed for studies reporting associations between SNPs and AF published before July 1, 2007. SNPs were genotyped in two independent case-control samples from Germany and the United States. Associations between SNPs and AF were assessed using logistic regression models adjusting for age, sex, and hypertension. A meta-analysis of the results from the two studies was performed. Results We identified 21 SNPs and the angiotensin-converting enzyme insertion/deletion polymorphism that were reported to be associated with AF in the literature. Nine of these genetic variants were not represented on common genome-wide SNP arrays. We successfully genotyped 21 of these 22 variants in 2,145 cases with AF from the German Competence Network for Atrial Fibrillation and 4,073 controls from the KORA S4 study and 16 variants in 790 cases and 1,330 controls from the Massachusetts General Hospital. None of the SNPs replicated in independent populations with AF. Conclusion Our results suggest that previously reported associations to AF were likely false positives and highlight the need for systematic replication of genetic associations in large, independent cohorts to accurately detect variants associated with disease. Atrial fibrillation (AF) is the most common sustained arrhythmia and has a substantial heritable component. Numerous associations between single nucleotide polymorphisms (SNPs) and AF have been described, but few have been replicated. We sought to systematically replicate SNPs that are reported to be associated with AF in two large study samples of European descent. We searched PubMed for studies reporting associations between SNPs and AF published before July 1, 2007. SNPs were genotyped in two independent case-control samples from Germany and the United States. Associations between SNPs and AF were assessed using logistic regression models adjusting for age, sex, and hypertension. A meta-analysis of the results from the two studies was performed. We identified 21 SNPs and the angiotensin-converting enzyme insertion/deletion polymorphism that were reported to be associated with AF in the literature. Nine of these genetic variants were not represented on common genome-wide SNP arrays. We successfully genotyped 21 of these 22 variants in 2,145 cases with AF from the German Competence Network for Atrial Fibrillation and 4,073 controls from the KORA S4 study and 16 variants in 790 cases and 1,330 controls from the Massachusetts General Hospital. None of the SNPs replicated in independent populations with AF. Our results suggest that previously reported associations to AF were likely false positives and highlight the need for systematic replication of genetic associations in large, independent cohorts to accurately detect variants associated with disease.

Sequence variation at Ch9p21 is a predisposing genetic factor for a number of diseases, including myocardial infarction (MI) and diabetes. We determined the risk of MI associated with various alleles and haplotypes, established and compared the predictive values of risk alleles, tested for the independence of associations between different risk alleles and MI, and sought to provide evidence for dual association of alleles with MI and diabetes.With the use of 35 single nucleotide polymorphisms, together capturing common variation seen in the associated interval, we genotyped 3657 MI cases and 1211 controls prospectively sampled in a European population.Polymorphisms rs10757278 and rs1333049 both exhibited the strongest individual risk signal (OR, 1.45; 95% CI, 1.32-1.59). Two haplotype blocks were established, each of which was mainly represented by a pair of a risk-conferring and a protective haplotype, but none of the risk-associated haplotypes exhibited stronger effects than rs10757278 or rs1333049 alone. Specific polymorphisms (rs7865618, rs1537378, rs7857345, rs1333049) were identified as independent predictors of MI in multivariable models adjusted for conventional cardiovascular risk factors. In specific instances, the presence of two or three polymorphisms in a model, instead of only one, improved the discriminating power. Finally, evidence was provided to suggest dual association of rs7865618 with MI and diabetes.In keeping with published results, this work was consistent with the association of alleles and haplotypes at Ch9p21 with MI and extended prior knowledge by also showing independence of associations among different risk alleles and an association of a specific polymorphism with both MI and diabetes.

Spatial and timely variations in QT interval, even within its normal range, may underlie susceptibility to cardiac arrhythmias and sudden cardiac death. Given its important role in cardiac electrophysiology, we hypothesized that common genetic variation in ankyrin-B gene (ANK2) might modify QT interval length.The study population consisted of 1188 participants of the World Health Organizational Multinational Monitoring of Trends and Determinants in Cardiovascular Disease (WHO MONICA) general population survey Cooperative Health Research in the Region of Augsburg (KORA S3). Corrected QT interval was calculated using population specific linear regression formulas. A total of 22 single-nucleotide polymorphisms in the genomic region of ANK2 gene were genotyped using TaqMan technology. In a replication study, 6 single nucleotide polymorphisms were genotyped in 3890 individuals from a second population study (KORA S4). The rare variant of the single-nucleotide polymorphism rs6850768 (allele frequency, 0.28) significantly influenced duration of the QT interval, both in KORA S3 and KORA S4 populations. In homozygotes, the shortening of the QT interval was 3.79 ms (95% CI, 1.48 to 5.58; P=0.001 and P=0.0008 for log-additive and dominant model, respectively) in KORA S3 and 2.94 ms (95% CI, 1.11 to 4.77; P=0.001 and P=0.006 for log-additive and dominant genetic model, respectively) in KORA S4. A common 2-locus haplotype (rs11098171-rs6850768; population frequency, 28%) was associated with a QT interval difference of 2.85 ms (permutation; P=0.006) in KORA S3 and 1.23 ms (permutation; P=0.009) in KORA S4. Reverse transcription-polymerase chain reaction expression analysis of the human ANK2 5' genomic region in the human left ventricular tissue revealed 2 previously unidentified ANK2 5' exons in the proximity of the identified variants.Common genetic variants juxtaposed with novel exons in the distant 5' genomic region of ANK2 influence the QT interval length in the general population. These findings support the role of ankyrin-B in normal cardiac electric activity.

Variable transcription of the cardiac sodium channel gene is a candidate mechanism determining arrhythmia susceptibility. We have previously cloned and characterized the core promoter and flanking region of SCN5A, encoding the cardiac sodium channel. Loss-of-function mutations in this gene have been reported in approximately 20% of patients with Brugada syndrome, an inherited cardiac electrical disorder associated with a high incidence of life-threatening arrhythmias. In this study, we identified DNA variants in the proximal 2.8 kb promoter region of SCN5A and determined their frequency in 1,121 subjects. This population consisted of 88 Brugada syndrome patients with no SCN5A coding region mutation, and 1,033 anonymized subjects from various ethnicities. Variant promoter activity was assayed in CHO cells and neonatal cardiomyocytes by transient transfection of promoter-reporter constructs. Single-nucleotide polymorphisms (SNPs) were identified at approximately 1/200 base pairs which are: 11 in the 5'-flanking region, 1 in exon 1, and 5 in intron 1. In addition, a haplotype consisting of two SNPs in complete linkage disequilibrium was identified. Minor allele frequencies were >5% in at least one ethnic panel at 5/19 polymorphic sites. In vitro functional analysis in cardiomyocytes identified four variants with significantly (P<0.05) reduced reporter activity (up to 63% reduction). The largest changes were seen with c.-225-1790 G>A, which reduced reporter activity by 62.8% in CHO cells and 55% in cardiomyocytes. From these results, we can conclude that the SCN5A core promoter includes multiple DNA polymorphisms with altered in vitro activity, further supporting the concept of interindividual variability in transcription of this cardiac ion channel gene.

The QT interval in the general population is a complex trait with 30-50% heritability. QT prolongation is associated with an increased risk of sudden death. A recent family-based study found an association between QT interval and the common non-synonymous Glycin 38 Serine variant (G38S, rs1805127) of the KCNE1 gene coding for the minK-potassium channel subunit. We intended to replicate this finding in a large population sample of central European Caucasian ancestry as part of our ongoing search for genetic variants predisposing to arrhythmias.We studied 3966 unrelated individuals from the KORA S4 population-based study without atrial fibrillation, pacemaker implant, or pregnancy. Individuals were genotyped by MALDI-TOF mass spectrometry. We did not detect any significant association between the genotypes of the G38S variant and the QT interval in the entire population or in any gender.Unlike the common Lysine 897 Threonine variant of KCNH2 (K897T, rs1805123) the G38S variant of KCNE1 does not appear to have a strong modifying effect on QT interval. However, we cannot rule out an effect of G38S on QT in other ethnic groups, under exercise or medications or on the risk for arrhythmias and sudden death.

Single-nucleotide polymorphisms within the BAT1-NFKBIL1-LTA genomic region (6p21.3) and the LGALS2 gene (22q13.1), encoding a regulator for lymphotoxin-alpha, the product of the LTA gene, have been reported to be linked with the risk of myocardial infarction in Japanese. We employed nine polymorphisms from the BAT1-NFKBIL1-LTA region and one polymorphism from the LGALS2 gene, and investigated whether such associations were also present in Europeans. The study included 3657 patients with myocardial infarction and 1211 control individuals with angiographically normal coronary arteries. Minor homozygous genotypes of polymorphisms in BAT1 (rs2239527, -23C/G), NFKBIL1 (rs2071592, -63T/A) and LTA (rs1800683, -162G/A; rs909253, 252G/A; rs1041981, Thr26Asn) were associated with moderately protective effects against myocardial infarction (P </= 0.045). The most abundant 9-marker haplotype of the BAT1-NFKBIL1-LTA region, named haplotype 1 (28% frequency in the study population), included the alleles of the five protective genotypes and was related with a significantly lower risk of myocardial infarction (OR 0.88, 95% CI 0.80-0.98; P = 0.015). Moreover, homozygosity for haplotype 1 was associated with an OR 0.72 (95% CI 0.57-0.90; P = 0.0047). Multiple logistic regression analysis revealed an independent protective effect against myocardial infarction in the homozygous carriers of haplotype 1 (adjusted OR 0.78, 95% CI 0.62-0.99; P = 0.043). A putative risk genotype of the polymorphism in the LGALS2 gene (rs7291467; 3279T/C) was not associated with myocardial infarction (OR 0.98, 95% CI 0.83-1.16; P = 0.84). Our finding that protective effects are linked with minor homozygous genotypes and haplotype 1 of the BAT1-NFKBIL1-LTA region in Europeans is opposite to the observation of associated risks in Japanese.

Background: The incidence of sudden cardiac death in persons under age 40 is roughly 3 per 100 000 persons per year in Germany and North America.Many of these deaths are found to be due to hereditary heart diseases, often a primary structural heart disease associated with arrhythmia or else a primary arrhythmia syndrome in a structurally normal heart.Such diseases are usually of autosomal dominant inheritance, often affect otherwise healthy persons, and can generally be well treated if recognized early.Patients commonly have affected relatives who are still asymptomatic.Methods: This review is based on articles up to May 2010 that were retrieved by a selective search of the Medline database via PubMed, with additional consideration of the relevant European and American guidelines and the German Law on Genetic Diagnosis.Results and conclusion: Hereditary arrhythmia syndromes are now found in more than half of all initially unexplained cases of sudden cardiac death in young persons.Among such cases, the hereditary arrhythmia syndrome is primary in 70% and caused by an arrhythmogenic structural heart disease in 30%.In addition to autopsy findings, a thorough family history, relevant medical findings obtained during life (if available), the examination of relatives, and directed molecular testing where appropiate enabled establishing the diagnosis.Arrthymia syndromes that can cause sudden death are often detectable during life if physicians and the public are appropriately sensitized.

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.

The modifying effects of tachykinins substance P, neurokinin A and neurokinin B on immunoglobulin production were analyzed in an in vitro culture system. Purified human T- and B-cells were stimulated with TGFbeta2 and IL-5 to induce preferential IgA production. Neuropeptides had the following effects. (1) The levels of IgA and IgG4 production were enhanced by IL-5 and TGFbeta2; IgA levels remained constant or were slightly augmented by neuropeptides, whereas IgG4 was further augmented. (2) IL-5 and TGFbeta2 did not alter IgG3 production, but neuropeptides stimulated secretion of this subclass. (3) IgG1 and IgM production were inhibited by IL-5 and TGFbeta2. This effect was prevented by neuropeptides. (4) Other isotypes including IgG2 and IgE remained unaffected. Except for IgM, these effects were blocked by specific receptor antagonists indicating specificity. The tachykinin receptor NK-1 mRNA was detected in B- and T-cells, whereas NK-3 mRNA was only present in T- and B-cell coculture following activation. Furthermore, neuropeptide effects depended on cytokine co-stimulation and the presence of T-cells. These results suggest that neuropeptides are potent modifiers of preferential IgA synthesis.

ABSTRACT Prioritization is the process whereby a set of possible candidate genes or SNP s 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 SNP s 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 B ayesian 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.

Experimental data support a protective function of apolipoprotein E (apoE) against restenosis, the main factor limiting the long-term benefit of percutaneous coronary interventions. We investigated the possibility that the single nucleotide polymorphisms (SNPs) –219G/T, 113G/C, 334T/C, and 472C/T of the gene encoding apoE (APOE) are associated with the incidence of death and myocardial infarction or restenosis after stenting in coronary arteries. In addition, we asked whether the apoE isotype-related ε2/ε3/ε4 polymorphism, defined by specific allele combinations (haplotypes) of the 334T/C and 472C/T polymorphism, and other APOE haplotypes, derived from all four SNPs investigated, are associated with adverse clinical and angiographic outcomes after stenting. Our study included 1,850 consecutive patients with symptomatic coronary artery disease (CAD) who underwent stent implantation. Follow-up angiography was performed in 1,556 patients (84.1%) at 6 months after the intervention. We found that none of the APOE SNPs is associated with death and myocardial infarction or restenosis after stenting. In addition, we observed no relationship between APOE haplotypes and adverse outcomes.In conclusion, the APOE –219G/T, 113G/C, 334T/C, and 472C/T polymorphisms, either alone or in combination, do not represent genetic markers of the risk of thrombotic and restenotic complications in patients with CAD treated with coronary stenting. Experimental data support a protective function of apolipoprotein E (apoE) against restenosis, the main factor limiting the long-term benefit of percutaneous coronary interventions. We investigated the possibility that the single nucleotide polymorphisms (SNPs) –219G/T, 113G/C, 334T/C, and 472C/T of the gene encoding apoE (APOE) are associated with the incidence of death and myocardial infarction or restenosis after stenting in coronary arteries. In addition, we asked whether the apoE isotype-related ε2/ε3/ε4 polymorphism, defined by specific allele combinations (haplotypes) of the 334T/C and 472C/T polymorphism, and other APOE haplotypes, derived from all four SNPs investigated, are associated with adverse clinical and angiographic outcomes after stenting. Our study included 1,850 consecutive patients with symptomatic coronary artery disease (CAD) who underwent stent implantation. Follow-up angiography was performed in 1,556 patients (84.1%) at 6 months after the intervention. We found that none of the APOE SNPs is associated with death and myocardial infarction or restenosis after stenting. In addition, we observed no relationship between APOE haplotypes and adverse outcomes. In conclusion, the APOE –219G/T, 113G/C, 334T/C, and 472C/T polymorphisms, either alone or in combination, do not represent genetic markers of the risk of thrombotic and restenotic complications in patients with CAD treated with coronary stenting. Compared with conventional balloon angioplasty, stenting has improved the outcome of patients with coronary artery disease (CAD) (1Lincoff A.M. Stent scrutiny.J. Am. Med. Assoc. 2000; 284: 1839-1841Crossref PubMed Scopus (13) Google Scholar). However, restenosis remains the principal factor limiting the long-term benefit of stenting (1Lincoff A.M. Stent scrutiny.J. Am. Med. Assoc. 2000; 284: 1839-1841Crossref PubMed Scopus (13) Google Scholar). Experimental data have pointed to a protective function of apolipoprotein E (apoE) against restenosis (2Ishigami M. Swertfeger D.K. Granholm N.A. Hui D.Y. Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase.J. Biol. Chem. 1998; 273: 20156-20161Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 3Zhu B. Kuhel D.G. Witte D.P. Hui D.Y. Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice.Am. J. Pathol. 2000; 157: 1839-1848Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 4De Geest B. Zhao Z. Collen D. Holvoet P. Effects of adenovirus-mediated human apo A-I gene transfer on neointima formation after endothelial denudation in apo E-deficient mice.Circulation. 1997; 96: 4349-4356Crossref PubMed Scopus (90) Google Scholar). ApoE was found to inhibit cell signaling events associated with growth factor-induced smooth muscle cell migration and proliferation and to limit neointimal hyperplasia after arterial injury (2Ishigami M. Swertfeger D.K. Granholm N.A. Hui D.Y. Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase.J. Biol. Chem. 1998; 273: 20156-20161Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 3Zhu B. Kuhel D.G. Witte D.P. Hui D.Y. Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice.Am. J. Pathol. 2000; 157: 1839-1848Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These results correspond to the observation that deficiency of apoE was associated with increased neointima formation after endothelial denudation (3Zhu B. Kuhel D.G. Witte D.P. Hui D.Y. Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice.Am. J. Pathol. 2000; 157: 1839-1848Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 4De Geest B. Zhao Z. Collen D. Holvoet P. Effects of adenovirus-mediated human apo A-I gene transfer on neointima formation after endothelial denudation in apo E-deficient mice.Circulation. 1997; 96: 4349-4356Crossref PubMed Scopus (90) Google Scholar). The apoE gene (APOE) is polymorphic, and some of its allelic forms are known to differentially affect transcriptional activity or give rise to structurally and functionally distinct protein isoforms (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar, 6Breslow J.L. Genetics of lipoprotein abnormalities associated with coronary heart disease susceptibility.Annu. Rev. Genet. 2000; 34: 233-254Crossref PubMed Scopus (95) Google Scholar, 7Artiga M.J. Bullido M.J. Sastre I. Recuero M. Garcia M.A. Aldudo J. Vásquez J. Valdivieso F. Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E.FEBS Lett. 1998; 421: 105-108Crossref PubMed Scopus (217) Google Scholar, 8Lambert J-C. Brousseau T. Defosse V. Evans A. Arveiler D. Ruidavets J-B. Haas B. Cambou J-P. Luc G. Ducimetière P. Cambien F. Chartier-Harlin M-C. Amouyel P. Independent association of an APOE gene promoter polymorphism with increased risk of myocardial infarction and decreased APOE plasma concentrations—the ECTIM study.Hum. Mol. Genet. 2000; 9: 57-61Crossref PubMed Google Scholar). Evidence exists to suggest that the variability of APOE has differential effects on the atheroprotective potential attributed to apoE (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar, 6Breslow J.L. Genetics of lipoprotein abnormalities associated with coronary heart disease susceptibility.Annu. Rev. Genet. 2000; 34: 233-254Crossref PubMed Scopus (95) Google Scholar). The ε2/ε3/ε4 polymorphism of APOE is caused by two single nucleotide polymorphisms (SNPs), 334T/C and 472C/T, which are in close physical proximity and absolute linkage disequilibrium (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar, 9Koch W. Ehrenhaft A. Griesser K. Pfeufer A. Müller J. Schömig A. Kastrati A. TaqMan systems for genotyping of disease-related polymorphisms present in the gene encoding apolipoprotein E.Clin. Chem. Lab. Med. 2002; 40: 1123-1131Crossref PubMed Scopus (0) Google Scholar). The 334T/C and 472C/T SNPs exclusively determine three haplotypes, known as the ε2 (334T/472T), ε3 (334C/472T), and ε4 (334C/472C) alleles of APOE (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar). This heterogeneity causes variation at amino acid positions 112 (cysteine or arginine) and 158 (arginine or cysteine) of apoE, resulting in three different isoforms of apoE (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar). The ε2/ε3/ε4 polymorphism is one of the most thoroughly studied polymorphisms, especially for its effects on lipid profiles and CAD risk (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar, 6Breslow J.L. Genetics of lipoprotein abnormalities associated with coronary heart disease susceptibility.Annu. Rev. Genet. 2000; 34: 233-254Crossref PubMed Scopus (95) Google Scholar). This polymorphism was found to be relevant for apoE plasma level, receptor binding affinity of apoE, plasma lipid and lipoprotein concentrations, and CAD (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar, 6Breslow J.L. Genetics of lipoprotein abnormalities associated with coronary heart disease susceptibility.Annu. Rev. Genet. 2000; 34: 233-254Crossref PubMed Scopus (95) Google Scholar). In particular, the 334C allele and the ε4 allele were observed to impose an increased risk of CAD (5Siest G. Pillot T. Régis-Bailly A. Leininger-Muller B. Steinmetz J. Galteau M-M. Visvikis S. Apolipoprotein E: an important gene and protein to follow in laboratory medicine.Clin. Chem. 1995; 41: 1068-1086Crossref PubMed Scopus (333) Google Scholar, 10Hirashiki A. Yamada Y. Murase Y. Suzuki Y. Kataoka H. Morimoto Y. Tajika T. Murohara T. Yokota M. Association of gene polymorphisms with coronary artery disease in low- or high-risk subjects defined by conventional risk factors.J. Am. Coll. Cardiol. 2003; 42: 1429-1437Crossref PubMed Scopus (124) Google Scholar), and, among patients with CAD, the ε4 allele was related to more severe and the ε2 allele was related to less severe disease (11Wang X.L. McCredie R.M. Wilcken D.E.L. Polymorphisms of the apolipoprotein E gene and severity of coronary artery disease defined by angiography.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1030-1034Crossref PubMed Scopus (109) Google Scholar). The presence of the ε4 allele has been associated with increased death rates in patients with CAD (12Stengård J.H. Weiss K.M. Sing C.F. An ecological study of association between coronary heart disease mortality rates in men and the relative frequencies of common allelic variations in the gene coding for apolipoprotein E.Hum. Genet. 1998; 103: 234-241Crossref PubMed Scopus (76) Google Scholar, 13Gerdes L.U. Gerdes C. Kervinen K. Savolainen M. Klausen I.C. Hansen P.S. Kesäniemi Y.A. Færgeman O. The apolipoprotein ε4 allele determines prognosis and the effect on prognosis of simvastatin in survivors of myocardial infarction: a substudy of the Scandinavian Simvastatin Survival Study.Circulation. 2000; 101: 1366-1371Crossref PubMed Scopus (223) Google Scholar). The cardiovascular risk attributed to the ε4 allele may be related to, at least in part, a lower antioxidant activity of the apoE4 isoform (112Arg/158Arg) compared with that of the apoE2 isoform (112Cys/158Cys) or the apoE3 isoform (112Cys/158Arg) (14Miyata M. Smith J.D. Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by oxidative insults and β-amyloid peptides.Nat. Genet. 1996; 14: 55-61Crossref PubMed Scopus (797) Google Scholar). Another SNP of APOE, –219G/T, located in the promoter of APOE, was reported to be significantly associated with APOE promoter activity, apoE plasma concentration, and CAD (7Artiga M.J. Bullido M.J. Sastre I. Recuero M. Garcia M.A. Aldudo J. Vásquez J. Valdivieso F. Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E.FEBS Lett. 1998; 421: 105-108Crossref PubMed Scopus (217) Google Scholar, 8Lambert J-C. Brousseau T. Defosse V. Evans A. Arveiler D. Ruidavets J-B. Haas B. Cambou J-P. Luc G. Ducimetière P. Cambien F. Chartier-Harlin M-C. Amouyel P. Independent association of an APOE gene promoter polymorphism with increased risk of myocardial infarction and decreased APOE plasma concentrations—the ECTIM study.Hum. Mol. Genet. 2000; 9: 57-61Crossref PubMed Google Scholar, 10Hirashiki A. Yamada Y. Murase Y. Suzuki Y. Kataoka H. Morimoto Y. Tajika T. Murohara T. Yokota M. Association of gene polymorphisms with coronary artery disease in low- or high-risk subjects defined by conventional risk factors.J. Am. Coll. Cardiol. 2003; 42: 1429-1437Crossref PubMed Scopus (124) Google Scholar). In addition, the 113G/C SNP of APOE may be relevant for APOE regulation because of its location in the APOE intron-1 enhancer element that constitutes a binding site for specific nuclear protein factors (15Mui S. Briggs M. Chung H. Wallace R.B. Gomez-Isla T. Rebeck G.W. Hyman B.T. A newly identified polymorphism in the apolipoprotein E enhancer gene region is associated with Alzheimer's disease and strongly with the ε4 allele.Neurology. 1996; 47: 196-201Crossref PubMed Scopus (61) Google Scholar, 16Paik Y-K. Chang D.J. Reardon C.A. Walker M.D. Taxman E. Taylor J.M. Identification and characterization of transcriptional regulatory regions associated with expression of the human apolipoprotein E gene.J. Biol. Chem. 1988; 263: 13340-13349Abstract Full Text PDF PubMed Google Scholar). Together, a number of findings suggest a significant impact of apoE and genetic variants of APOE on cardiovascular risk. APOE polymorphisms may also be associated with angiographic and clinical outcomes after subcutaneous interventions in coronary arteries. Inconsistent results were obtained regarding the relationship between the ε2/ε3/ε4 polymorphism and restenosis after balloon angioplasty in coronary arteries (17van Bockxmeer F.M. Mamotte C.D.S. Gibbons F.R. Taylor R.R. Apolipoprotein ε4 homozygosity—a determinant of restenosis after coronary angioplasty.Atherosclerosis. 1994; 110: 195-202Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 18van Bockxmeer F.M. Mamotte C.D.S. Gibbons F.A. Burke V. Taylor R.R. Angiotensin-converting enzyme and apolipoprotein E genotypes and restenosis after coronary angioplasty.Circulation. 1995; 92: 2066-2071Crossref PubMed Scopus (67) Google Scholar, 19Damaraju S. Yu Q-T. Safavi F. Marian A.J. Apolipoprotein ε4 is not a genetic risk factor for coronary artery disease or restenosis after percutaneous transluminal coronary angioplasty.Am. J. Cardiol. 1995; 75: 1181-1183Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 20Samani N.J. Martin D.S. Brack M. Cullen J. Wallis R. Lodwick D. Chauhan A. Harley A. Thompson J.R. Gershlick A.H. de Bono D.P. Apolipoprotein E polymorphism does not predict risk of restenosis after coronary angioplasty.Atherosclerosis. 1996; 125: 209-216Abstract Full Text PDF PubMed Scopus (12) Google Scholar). It has not been examined whether the APOE –219G/T, 113G/C, 334T/C, and 472C/T SNPs, or APOE haplotypes based on the ε2/ε3/ε4 polymorphism, or combinations of all four SNPs are related to thrombotic and restenotic complications after coronary stenting. We addressed this issue in a study that included a relatively large and consecutive series of patients with CAD. The study included a consecutive series of 1,850 Caucasian patients with symptomatic CAD who underwent stent implantation at Deutsches Herzzentrum München and 1. Medizinische Klinik rechts der Isar der Technischen Universität München. The protocols of stent placement and poststenting therapy were described in detail elsewhere (21Schömig A. Kastrati A. Mudra H. Blasini R. Schühlen H. Klauss V. Richardt G. Neumann F-J. Four-year experience with Palmaz-Schatz stenting in coronary angioplasty complicated by dissection with threatened or present vessel closure.Circulation. 1994; 90: 2716-2724Crossref PubMed Google Scholar, 22Schömig A. Neumann F-J. Kastrati A. Schühlen H. Blasini R. Hadamitzky M. Walter H. Zitzmann-Roth E-M. Richardt G. Alt E. Schmitt C. Ulm K. A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronary-artery stents.N. Engl. J. Med. 1996; 334: 1084-1089Crossref PubMed Scopus (1905) Google Scholar). Postprocedural pharmacologic therapy consisted of aspirin (100 mg twice daily, indefinitely) and ticlopidine (250 mg twice daily for 4 weeks). Patients who were considered at a higher risk for ischemic complications received additional therapy with the glycoprotein IIb/IIIa blocker abciximab, which was given as a bolus injection during the stent insertion procedure and as a 12 h continuous infusion thereafter. All patients were scheduled for angiographic follow-up at 6 months. Written informed consent was obtained from the patients for the intervention, follow-up angiography, and genotype determination. The study protocol was approved by the Institutional Ethics Committee, and the reported investigations were in accordance with the principles of the current version of the Declaration of Helsinki. Genotyping of the APOE –219G/T, 113G/C, 334T/C, and 472C/T SNPs was performed with TaqMan assays, as previously described (9Koch W. Ehrenhaft A. Griesser K. Pfeufer A. Müller J. Schömig A. Kastrati A. TaqMan systems for genotyping of disease-related polymorphisms present in the gene encoding apolipoprotein E.Clin. Chem. Lab. Med. 2002; 40: 1123-1131Crossref PubMed Scopus (0) Google Scholar). Lesion morphology was classified according to the modified American College of Cardiology/American Heart Association grading system in type A, B1, B2, and C; lesions of types B2 and C were considered complex lesions. Angiograms were recorded just before and immediately after the intervention and at 6 month follow-up. Matched projections of the target lesions were selected for quantitative computer-assisted off-line analysis of the angiograms with the automated edge-detection system CMS (Medis Medical Imaging Systems, Nuenen, The Netherlands). The angiographic parameters obtained were interpolated reference diameter, lesion length, diameter stenosis before and after stenting and at follow-up, diameter of the maximally inflated balloon during stent placement, and length of the stented segment. Quantitative analysis of angiograms was performed by operators not involved in the stenting procedure and unaware of the laboratory or genetic data. The primary end point of the study was restenosis. Two definitions of restenosis were used: the incidence of a diameter stenosis of ⩾50% at 6 month follow-up angiography (angiographic restenosis) and the need for target vessel revascularization (percutaneous transluminal coronary balloon angioplasty or aortocoronary bypass grafting) as a result of symptoms or signs of ischemia in the presence of angiographic restenosis at the stented site within 1 year after stent placement (clinical restenosis). A secondary end point was the combined incidence of all-cause death and nonfatal myocardial infarction at 1 year after stenting. The diagnosis of acute myocardial infarction was based on the presence of new pathological Q waves on the electrocardiogram or a value of creatine kinase or its MB isoenzyme at least three times the normal upper limit. Determination of haplotypes and haplotype frequencies was performed with the expectation-maximization (EM) algorithm and the Markov chain-Monte Carlo algorithm, as previously described (9Koch W. Ehrenhaft A. Griesser K. Pfeufer A. Müller J. Schömig A. Kastrati A. TaqMan systems for genotyping of disease-related polymorphisms present in the gene encoding apolipoprotein E.Clin. Chem. Lab. Med. 2002; 40: 1123-1131Crossref PubMed Scopus (0) Google Scholar). Discrete variables are expressed as counts and percentages and were compared with the Chi-square test or the Fisher exact test, as appropriate. Continuous variables are expressed as means ± SD and were compared by means of the unpaired, two-sided t-test or ANOVA for more than two groups. We tested for independent association of the APOE SNP-related genotypes and haplotype-related genotypes in multivariate models (multiple logistic regressions) of restenosis that included age, gender, arterial hypertension, hypercholesterolemia, current tobacco smoking, diabetes mellitus, unstable angina pectoris, acute myocardial infarction, previous myocardial infarction, previous bypass surgery, target coronary vessel, lesion complexity, ostial lesion, chronic occlusion, restenotic lesion, multivessel disease, reference diameter, lesion length, diameter stenosis before stenting, balloon-to-vessel ratio, maximal balloon pressure, length of the stented segment, diameter stenosis after stenting, and abciximab therapy as potentially confounding factors. Adjusted odds ratios and 95% Wald confidence intervals (CIs) were calculated on the basis of the multiple logistic regression models. Analyses were performed using the S-Plus statistical package (Mathsoft, Inc., Seattle, WA). P < 0.05 was considered statistically significant. We determined the genotypes of the –219G/T, 113G/C, 334T/C, and 472C/T SNPs of APOE in 1,850 patients with CAD who underwent stenting in coronary arteries. The distributions of the genotypes were 27.1% –219GG, 48.5% –219GT, 24.3% –219TT, 41.0% 113GG, 44.9% 113GC, 14.1% 113CC, 73.7% 334TT, 24.0% 334TC, 2.3% 334CC, and 86.8% 472CC, 12.4% 472CT, 0.8% 472TT. For comparisons with the carriers of the abundant 334TT and 472CC genotypes, patients with the rare 334CC and 472TT genotypes were combined with the patients who carried the 334TC and 472CT genotypes, respectively. Patients with the 334TC and 334CC genotypes represented the carriers of the APOE ε4 allele, and patients with the 472CT and 472TT genotypes represented the carriers of the APOE ε2 allele. Table 1 shows baseline clinical characteristics, lesion-related variables before stenting, and procedural parameters of the patients in relation to the genotypes of the APOE SNPs. We observed no statistically significant differences between the groups, with the following exceptions: i) patients with the –219GT genotype were younger than the carriers of the –219GG or –219TT genotype (P = 0.04); ii) reference diameter increased with the number of 113G alleles (P = 0.006); iii) in patients with the 334TT genotype, hypercholesterolemia (P = 0.04), acute myocardial infarction (P = 0.03), and periprocedural treatment with abciximab (P = 0.03) were less frequent and diabetes mellitus (P = 0.04) was more frequent than among carriers of the 334C allele (334TC genotype or 334CC genotype).TABLE 1Baseline clinical characteristics, lesion variables before stenting, and procedural parameters according to the genotypes of the APOE –219G/T, 113G/C, 334T/C, and 472C/T SNPs (n = 1,850)–219G/T113G/C334T/C472C/TVariableGG (n = 502)GT (n = 898)TT (n = 450)GG (n = 759)GC (n = 831)CC (n = 260)TT (n = 1,363)TC + CC (n = 487)CC (n = 1,606)CT + TT (n = 244)Age (years)63.6 ± 10.162.4 ± 10.063.6 ± 9.963.4 ± 10.362.5 ± 10.063.5 ± 9.263.0 ± 9.963.0 ± 10.462.9 ± 10.163.5 ± 9.4Women 112 (22.3) 174 (19.4) 105 (23.3) 158 (20.8) 184 (22.1) 49 (18.8)290 (21.3) 101 (20.7)336 (20.9) 55 (22.5)Arterial hypertension 336 (66.9) 614 (68.4) 309 (68.7) 510 (67.2) 564 (67.9) 185 (71.2)931 (68.3) 328 (67.4)1089 (67.8) 170 (69.7)Hypercholesterolemia 214 (42.6) 386 (43.0) 192 (42.7) 341 (44.9) 354 (42.6) 97 (37.3)564 (41.4) 228 (46.8)688 (42.8) 104 (42.6)Current smoking 146 (29.1) 289 (32.2) 135 (30.0) 234 (30.8) 253 (30.4) 83 (31.9)412 (30.2) 158 (32.4)506 (31.5) 64 (26.2)Diabetes mellitus 102 (20.3) 191 (21.3) 95 (21.1) 149 (19.6) 178 (21.4) 61 (23.5)302 (22.2) 86 (17.7)334 (20.8) 54 (22.1)Unstable angina pectoris 132 (26.3) 245 (27.3) 138 (30.7) 204 (26.9) 234 (28.2) 77 (29.6)372 (27.3) 143 (29.4)453 (28.2) 62 (25.4)Acute myocardial infarction 98 (19.5) 184 (20.5) 93 (20.7) 159 (20.9) 166 (20.0) 50 (19.2)260 (19.1) 115 (23.6)329 (20.5) 46 (18.9)Previous myocardial infarction 144 (28.7) 232 (25.8) 120 (26.7) 213 (28.1) 216 (26.0) 67 (25.8)362 (26.6) 134 (27.5)430 (26.8) 66 (27.0)Previous coronary bypass surgery 57 (11.4) 91 (10.1) 57 (12.7) 88 (11.6) 90 (10.8) 27 (10.4)149 (10.9) 56 (11.5)178 (11.1) 27 (11.1)Target coronary vesselLeft main coronary artery 8 (1.6) 13 (1.5) 6 (1.3) 10 (1.3) 14 (1.7) 3 (1.2)22 (1.6) 5 (1.0)24 (1.5) 3 (1.2)Left circumflex coronary artery 94 (18.7) 177 (19.7) 98 (21.8) 147 (19.4) 171 (20.6) 51 (19.6)269 (19.7) 100 (20.5)322 (20.1) 47 (19.3)Left anterior descending coronary artery 210 (41.8) 351 (39.1) 173 (38.4) 307 (40.5) 319 (38.4) 108 (41.5)548 (40.2) 186 (38.2)640 (39.9) 94 (38.5)Right coronary artery 159 (31.7) 300 (33.4) 137 (30.4) 240 (31.6) 275 (33.1) 81 (31.2)437 (32.1) 159 (32.7)516 (32.1) 80 (32.8)Venous bypass graft 31 (6.2) 57 (6.4) 36 (8.0) 55 (7.3) 52 (6.3) 17 (6.5)87 (6.4) 37 (7.6)104 (6.5) 20 (8.2)Complex lesion (ACC/AHA type B2 or C) 379 (75.5) 655 (72.9) 347 (77.1) 570 (75.1) 617 (74.2) 194 (74.6)1017 (74.6) 364 (74.7)1202 (74.8) 179 (73.4)Ostial lesion 36 (7.2) 64 (7.1) 27 (6.0) 51 (6.7) 64 (7.7) 12 (4.6)88 (6.5) 39 (8.0)108 (6.7) 19 (7.8)Chronic occlusion 40 (8.0) 49 (5.5) 29 (6.4) 57 (7.5) 47 (5.7) 14 (5.4)84 (6.2) 34 (7.0)99 (6.2) 19 (7.8)Restenotic lesion 133 (26.5) 213 (23.7) 103 (22.9) 200 (26.4) 189 (22.7) 60 (23.1)341 (25.0) 108 (22.2)381 (23.7) 68 (27.9)Multivessel disease 365 (72.7) 645 (71.8) 318 (70.7) 547 (72.1) 602 (72.4) 179 (68.8)980 (71.9) 348 (71.5)1143 (71.2) 185 (75.8)Reference diameter (mm)3.06 ± 0.533.04 ± 0.523.00 ± 0.563.08 ± 0.533.02 ± 0.542.97 ± 0.513.02 ± 0.523.08 ± 0.563.03 ± 0.533.07 ± 0.51Lesion length (mm)12.2 ± 6.912.1 ± 6.711.9 ± 6.712.3 ± 6.812.0 ± 6.611.9 ± 6.912.0 ± 6.812.4 ± 6.612.0 ± 6.712.4 ± 7.2Diameter stenosis before stenting (%)79.2 ± 15.778.2 ± 15.280.0 ± 14.979.1 ± 15.578.7 ± 15.379.4 ± 14.678.8 ± 15.279.3 ± 15.579.0 ± 15.278.2 ± 16.0Balloon-to-vessel ratio1.07 ± 0.091.07 ± 0.101.07 ± 0.101.07 ± 0.091.07 ± 0.101.08 ± 0.111.07 ± 0.101.07 ± 0.091.07 ± 0.101.06 ± 0.09Maximal balloon pressure (atmosphere)13.9 ± 3.213.8 ± 3.413.9 ± 3.213.8 ± 3.313.9 ± 3.313.8 ± 3.313.8 ± 3.313.9 ± 3.213.9 ± 3.313.8 ± 3.3Stented segment length (mm)20.2 ± 14.820.2 ± 13.820.0 ± 13.220.6 ± 14.820.0 ± 13.719.4 ± 12.019.9 ± 13.920.8 ± 14.020.2 ± 14.119.9 ± 13.1Diameter stenosis after stenting (%)5.2 ± 7.75.3 ± 9.15.4 ± 7.45.5 ± 9.25.2 ± 7.84.9 ± 7.65.1 ± 7.85.8 ± 9.85.2 ± 8.35.8 ± 8.5Periprocedural abciximab therapy 90 (17.9) 183 (20.4) 91 (20.2) 148 (19.5) 170 (20.5) 46 (17.7)252 (18.5) 112 (23.0)322 (20.0) 42 (17.2)Data are presented as mean values ± SD or the number (%) of patients. ACC/AHA, American College of Cardiology/American Heart Association grading system; APOE, gene encoding apolipoprotein E (apoE); SNP, single nucleotide polymorphism. Open table in a new tab Data are presented as mean values ± SD or the number (%) of patients. ACC/AHA, American College of Cardiology/American Heart Association grading system; APOE, gene encoding apolipoprotein E (apoE); SNP, single nucleotide polymorphism. The apoE isotype-related genotypes ε2ε2, ε2ε3, ε2ε4, ε3ε3, ε3ε4, and ε4ε4 were present at 0.8, 11.2, 1.2, 61.7, 22.8, and 2.3%, respectively. Genotype-based evaluation revealed that the alleles of the four APOE SNPs were arranged in eight different haplotypes: haplotype 1 = –219G/113G/334T/472T (GGTT; 7.0%), haplotype 2 = GGTC (41.6%), haplotype 3 = TCTC (36.3%), haplotype 4 = TGTC (0.6%), haplotype 5 = GCTC (0.2%), haplotype 6 = TGCC (11.7%), haplotype 7 = GGCC (2.6%), and haplotype 8 = TCCC (<0.001%). Haplotype 1 was the only haplotype that represented the ε2 allele (334T/472T); haplotypes 2–5 included the ε3 allele (334T/472C); and haplotypes 6–8 contained the ε4 allele (334C/472C). It was possible to assign a genotype, defined by a specific combination of two of the eight haplotypes, to each patient. In total, 23 different haplotype-related genotypes were present in the study population. Follow-up angiography of coronary arteries was performed in 1,556 (84.1%) of the patients 6 months after stenting. The proportions of the patients who underwent 6 month angiography were not substantially different among the SNP-related genotype groups (P ⩾ 0.60), apoE isotype-related genotypes (P ⩾ 0.80), and other haplotype-related genotypes (P ⩾ 0.63). Complete 1 year clinical follow-up data were available for all patients, irrespective of the presence or absence of follow-up angiography. The combined incidence of all-cause death and nonfatal myocardial infarction was not significantly different between patients with the –219GG, –219GT, and –219TT genotypes (P = 0.76), patients with the 113GG, 113GC, and 113CC genotypes (P = 0.78), patients with the 334TT genotype and carriers of the 334C allele (ε4 allele carriers) (P = 0.21), or patients with the 472CC genotype and carriers of the 472T

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations.The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease.Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples.Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

Two case/control studies with different phenotypes, marker densities, and microarrays were examined for the most significant single markers in defined regions. They show a pronounced bias toward exaggerated significance that increases with the number of observed markers and would increase further with imputed markers. This bias is eliminated by Bonferroni adjustment, thereby allowing combination by principal component analysis with a Malecot model composite likelihood evaluated by a permutation procedure to allow for multiple dependent markers. This intermediate value identifies the only demonstrated causal locus as most significant even in the preliminary analysis and clearly recognizes the strongest candidate in the other sample. Because the three metrics (most significant single marker, composite likelihood, and their principal component) are correlated, choice of the n smallest P values by each test gives &lt;3 n regions for follow-up in the next stage. In this way, methods with different response to marker selection and density are given approximately equal weight and economically compared, without expressing an untested prejudice or sacrificing the most significant results for any of them. Large numbers of cases, controls, and markers are by themselves insufficient to control type 1 and 2 errors, and so efficient use of multiple metrics with Bonferroni adjustment promises to be valuable in identifying causal variants and optimal design simultaneously.

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.

KCNE1 represents the regulatory beta-subunit of the slowly activating delayed rectifier potassium channel (IKs). Variants of KCNE1 have repeatedly been linked to the long-QT syndrome (LQTS), a disorder which predisposes to deafness, ventricular tachyarrhythmia, syncope, and sudden cardiac death.We here analyze the evolution of the common Gly38Ser variant (rs1805127), using genomic DNAs, complementary DNAs, and HEK293-expressed variants of altogether 19 mammalian species. The between species comparison reveals that the human-specific Gly38Ser polymorphism evolved under strong positive Darwinian selection, probably in adaptation to specific challenges in the fine-tuning of IKs channels. The involved amino acid exchanges (Asp > Gly, Gly > Ser) are moderately radical and do not induce apparent changes in posttranslational modification. According to population genetic analyses (HapMap phase II) a heterozygote advantage accounts for the maintenance of the Gly38Ser polymorphism in humans. On the other hand, the expression of the 38Ser allele seems to be disadvantageous under certain conditions, as suggested by the sporadic deficiency of 38Ser-coding mRNAs in heterozygote Central Europeans and the depletion of homozygotes 38Ser in the Yoruban sample.We speculate that individual differences in genomic imprinting or genomic recoding might have contributed to conflicting results of recent association studies between Gly38Ser polymorphism and QT phenotype. The findings thus highlight the relevance of mRNA data in future association studies of genotypes and clinical disorders. To the best of our knowledge, they moreover provide first time evidence for a unique pattern; i.e. coincidence of positive Darwinian selection and polymorphism with a sporadically suppressed expression of one allele.

Heritability studies have estimated the genetically attributable part of body mass index variance to be in the range of 30-70 %. Rs7566650 (G>C) single-nucleotide polymorphism (SNP) near the promoter of the INSIG2 gene has been identified as associated with body mass index. The gene product of INSIG2 is involved in regulation of fatty acid and cholesterol synthesis. In order to replicate this association we have analysed 2,559 unrelated individuals of Slavonic Caucasian origin from the populationbased Czech MONICA 3-year cohort. Body mass index, waist-hip ratio and plasma lipids (total-cholesterol, HDL-cholesterol, triglycerides) were measured at two independent examinations within three years. We could not detect any association between the SNP rs7566605 and body mass index, waist-hip ratio or lipid parameters, both with or without adjusting for age and gender. Neither the body mass index change nor lipid changes were significantly affected by the INSIG2 gene variant. Our results indicated that this INSIG2 polymorphism has no significant effect on body mass index and plasma lipids in the Czech Slavonic population.

We report a case of recurrent episodes of Torsades de Pointes arrhythmia in the setting of transiently impaired left ventricular ejection fraction, acute respiratory distress syndrome, transient hypokalaemia and QT-prolonging drugs, in a previously healthy 25-yr-old female patient. In the course of the clinical and genetic work-up this patient was newly diagnosed with a mutation in KCNH2 encoding the alpha-subunit of the human repolarizing potassium channel I(Kr). This case report illustrates the multivariate nature of long-QT syndrome, and emphasizes the usefulness of a pharmacological test for repolarization abnormalities.

Introduction A serious side-effect of antiarrhythmic and many other agents is drug-induced long QT syndrome (diLQTS) with torsades de pointes. To test the role of common genetic variation in diLQTS...

Identification of genetic variants affecting complex traits such as obesity is confounded by many types of bias, especially when effect sizes are small. Given our findings of a positive association between rs7566605 and body mass index in four out of five separate samples, a false positive finding cannot be ruled out with certainty but seems unlikely. Meta-analyses of multiple large studies will help refine the estimate of the effects of rs7566605 on body mass index.

Using principal component (PC) analysis, we studied the genetic constitution of 3,112 individuals from Europe as portrayed by more than 270,000 single nucleotide polymorphisms (SNPs) genotyped with the Illumina Infinium platform.In cohorts where the sample size was .100,one hundred randomly chosen samples were used for analysis to minimize the sample size effect, resulting in a total of 1,564 samples.This analysis revealed that the genetic structure of the European population correlates closely with geography.The first two PCs highlight the genetic diversity corresponding to the northwest to southeast gradient and position the populations according to their approximate geographic origin.The resulting genetic map forms a triangular structure with a) Finland, b) the Baltic region, Poland and Western Russia, and c) Italy as its vertexes, and with d) Central-and Western Europe in its centre.Inter-and intra-population genetic differences were quantified by the inflation factor lambda (l) (ranging from 1.00 to 4.21), fixation index (F st ) (ranging from 0.000 to 0.023), and by the number of markers exhibiting significant allele frequency differences in pair-wise population comparisons.The estimated lambda was used to assess the real diminishing impact to association statistics when two distinct populations are merged directly in an analysis.When the PC analysis was confined to the 1,019 Estonian individuals (0.1% of the Estonian population), a fine structure emerged that correlated with the geography of individual counties.With at least two cohorts available from several countries, genetic substructures were investigated in Czech, Finnish, German, Estonian and Italian populations.Together with previously published data, our results allow the creation of a comprehensive European genetic map that will greatly facilitate inter-population genetic studies including genome wide association studies (GWAS).

Abstract Motivation Genome-wide association studies have had great success in identifying human genetic variants associated with disease, disease risk factors, and other biomedical phenotypes. Many variants are associated with multiple traits, even after correction for trait-trait correlation. Discovering subsets of variants associated with a shared subset of phenotypes could help reveal disease mechanisms, suggest new therapeutic options, and increase the power to detect additional variants with similar pattern of associations. Here we introduce two methods based on a Bayesian framework, SNP And Pleiotropic PHenotype Organization (SAPPHO), one modeling independent phenotypes (SAPPHO-I) and the other incorporating a full phenotype covariance structure (SAPPHO-C). These two methods learn patterns of pleiotropy from genotype and phenotype data, using identified associations to discover additional associations with shared patterns. Results The SAPPHO methods, along with other recent approaches for pleiotropic association tests, were assessed using data from the Atherosclerotic Risk in Communities (ARIC) study of 8,000 individuals, whose gold-standard associations were provided by meta-analysis of 40,000 to 100,000 individuals from the CHARGE consortium. Using power to detect gold-standard associations at genome-wide significance (0.05 family-wise error rate) as a metric, SAPPHO performed best. The SAPPHO methods were also uniquely able to select the most significant variants in a parsimonious model, excluding other less likely variants within a linkage disequilibrium block. For meta-analysis, the SAPPHO methods implement summary modes that use sufficient statistics rather than full phenotype and genotype data. Meta-analysis applied to CHARGE detected 16 additional associations to the gold-standard loci, as well as 124 novel loci, at 0.05 false discovery rate. Reasons for the superior performance were explored by performing simulations over a range of scenarios describing different genetic architectures. With SAPPHO we were able to learn genetic structures that were hidden using the traditional univariate tests. Availability https://bitbucket.org/baderlab/fast/wiki/Home . SAPPHO software is available under the GNU General Public License, v2.

The chymase gene is said to be important for the generation of angiotensin II in the heart and therefore is a candidate gene for heart disease. However, we were unable to find an association between allelic variants of the chymase gene and acute myocardial infarction or linkage between the chymase gene locus and heart size.

Multiple single-nucleotide polymorphisms in the BAT1-NFKBIL1-LTA region on the short arm of chromosome 6 have been found to be associated with susceptibility to myocardial infarction in a recent case-control study including individuals from Japan. Specifically, the association relates to homozygosity for the minor alleles of five individual polymorphisms (one each in BAT1 and NFKBIL1 and three in LTA) and a haplotype defined by a particular allele combination of 10 polymorphisms (three each in BAT1 and NFKBIL1 and four in LTA). In the Japanese study, genotype determinations were carried out using a multiplex PCR-Invader assay or DNA sequencing. As an alternative to these methods, we have established TaqMan assays for genotyping of nine of the 10 polymorphisms. Accuracy of the genotyping results obtained with the TaqMan reactions was demonstrated in experiments involving restriction enzyme analysis or DNA sequencing. Using the new TaqMan assays, we have genotyped the polymorphisms in a group of 1211 Caucasians who presented without symptoms or signs of acute or previous myocardial infarction. Genotype distributions of each of the five myocardial infarction-associated polymorphisms were significantly different between the present study group and two Japanese control groups (p < or = 0.01). High linkage disequilibrium measures between polymorphisms suggested that the nine polymorphisms reside in a single haplotype block. Based on the nine polymorphisms, eight different haplotypes with allele frequencies above 1% were identified, which together represented 98.4% of the total number of chromosome 6 in the 1211 individuals. This Caucasian study group and the Japanese sample showed strong linkage disequilibrium in the myocardial infarction-associated BAT1-NFKBIL1-LTA genomic region and related haplotype content, but significantly different genotype frequencies. The new TaqMan systems provide relatively simple and fast assays to test the clinical relevance of these polymorphisms.

Genetics of the ECG: QT or not QT- A genetic analysis of a complex electrophysiological trait confirms several previously detected associations

Irinotecan is an anti-cancer agent that is used for the treatment of metastatic colorectal cancer. Although it prolongs survival, it can cause severe toxicity (e. g. diarrhea and neutropenia) in patients who carry the UGT1A1*28 allele. This study evaluates the cost-effectiveness of UGT1A1 genotyping prior to irinotecan-based chemotherapy from the perspective of the German statutory health insurance. We develop a decision-analytic Markov model to analyze costs and QALYs during a time horizon of six months (two-week cycles). No testing was compared to (1) change of chemotherapy to an irinotecan-free regimen, (2) dose reduction of irinotecan-based chemotherapy and (3) administration of a prophylactic G-CSF growth factor for patients with a UGT1A1*28 variant. Probability, utility and cost parameters used in this study were extracted from published literature. Uncertainty was assessed by deterministic and probabilistic sensitivity analyses. Strategy (2) was the cheapest strategy associated with costs of about €12,600 and effects of approx. 0.32 QALYs. All other three strategies were absolutely dominated. Compared to no testing, strategy (2) resulted in only marginal increases of QALYs (0.0003) but reduced costs by about €1,500 per patient. Strategy (1) resulted in smaller health gains (0.0002 QALYs) and smaller cost savings (about €60). Strategy (3) yielded approximately the same QALY gains as strategy (2) but at higher costs. In the probabilistic analysis, strategy (2) was the optimal strategy in 52% of simulations at a threshold of €50,000 per QALY. Uncertainty for this strategy originated primarily from the utility weights and the costs of chemotherapy. Our analysis suggests that UGT1A1 genotyping and subsequent reduction of irinotecan-based chemotherapy has a substantial cost-saving potential. Due to the promising results, further research, for example in the form of a managed entry agreement would be desirable to validate these findings.

Torsades de pointes arrhythmia (TdP) occur in the setting of a prolonged QT interval, however animal models have shown that beat-to-beat variability of repolarization duration (BVR) has more predictive power when identifying risk of proarrhythmia. Here, we evaluate variability of the QT interval in patients with a history of drug-induced long-QT syndrome and TdP (dLQTS), in patients with congenital long-QT syndromes (cLQTS), and in control individuals.

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.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.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.

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.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.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.

ABSTRACT Electrocardiographic PR interval measures atrial and atrioventricular depolarization and conduction, and abnormal PR interval is a risk factor for atrial fibrillation and heart block. We performed a genome-wide association study in over 92,000 individuals of European descent and identified 44 loci associated with PR interval (34 novel). Examination of the 44 loci revealed known and novel biological processes involved in cardiac atrial electrical activity, and genes in these loci were highly over-represented in several cardiac disease processes. Nearly half of the 61 independent index variants in the 44 loci were associated with atrial or blood transcript expression levels, or were in high linkage disequilibrium with one or more missense variants. Cardiac regulatory regions of the genome as measured by cardiac DNA hypersensitivity sites were enriched for variants associated with PR interval, compared to non-cardiac regulatory regions. Joint analyses combining PR interval with heart rate, QRS interval, and atrial fibrillation identified additional new pleiotropic loci. The majority of associations discovered in European-descent populations were also present in African-American populations. Meta-analysis examining over 105,000 individuals of African and European descent identified additional novel PR loci. These additional analyses identified another 13 novel loci. Together, these findings underscore the power of GWAS to extend knowledge of the molecular underpinnings of clinical processes.