SCN5A Variation is Associated with Electrocardiographic Traits in the Jackson Heart Study.

Jeff JM, Brown-Gentry K, Buxbaum SG, Sarpong DF, ... Roden DM, Crawford DC
Background: -Understanding variation in the normal electrical activity of the heart, assessed by the electrocardiogram (ECG), may provide a starting point for studies of susceptibility to serious arrhythmias, such as sudden cardiac death during myocardial infarction or drug therapy. Recent genetic association studies of one ECG trait, the QT interval, have identified common variation in European-descent populations, but little is known about the genetic determinants of ECG traits in populations of African-descent. Methods and results: -To identify genetic risk factors, we have undertaken a candidate gene study of ECG traits in collaboration with the Jackson Heart Study (JHS), a longitudinal study of 5,301 African Americans ascertained from the Jackson, Mississippi area. Nine quantitative ECG traits were evaluated: P, PR, QRS, QT, and QTc durations, heart rate and P, QRS and T axes. We genotyped 72 variations in the predominant sodium channel gene expressed in heart, SCN5A, encoding the Na(v)1.5 voltage-gated sodium channel in 4,558 subjects. Both rare and common variants in this gene have previously been associated with inherited arrhythmia syndromes and variable conduction. Adjusting for age, sex, and European ancestry, we performed tests of association in 3,054 unrelated participants and identified 14 significant associations (p<1.0x10(-4)), of which 13 are independent based upon linkage disequilibrium. These variants explain up to 2% of the variation in ECG traits in the JHS. Conclusions: -These results suggest that SCN5A variation contributes to ECG trait distributions in African Americans and these same variations may be risk or protective factors associated with susceptibility to arrhythmias.

Circ Cardiovasc Genet: 17 Feb 2011; epub ahead of print

Genetic Variations in the &alpha;2A-Adrenoreceptor Are Associated with Blood Pressure Response to the Agonist Dexmedetomidine.

Kurnik D, Muszkat M, Li C, Sofowora GG, ... Wood AJ, Stein CM
Background: -α(2A)-Adrenoceptors (α(2A)A-ARs) have important roles in sympathetic cardiovascular regulation. Variants of ADRA2A affect gene transcription and expression and are associated with insulin release and risk for type 2 diabetes. We examined whether ADRA2A variants are also associated with cardiovascular responses to the selective α(2)-AR-agonist, dexmedetomidine. Methods and results: -73 healthy subjects participated in a placebo-controlled single-blind study. After 3 infusions of placebo, subjects received 3 incremental infusions of dexmedetomidine (cumulative dose, 0.4 mcg/kg). Primary outcomes were changes in systolic blood pressure (SBP) and plasma norepinephrine concentrations, measured as difference of the area-under-the-curve during placebo and dexmedetomidine infusions (ΔAUC). We used multiple linear regression analysis to examine the associations between 9 ADRA2A tagging variants and 5 inferred haplotypes and ΔAUC after adjustment for covariates. Homozygous carriers of rs553668, and the corresponding haplotype 4, previously associated with increased α(2A)-AR expression, had a 2.2-fold greater decrease in AUCSBP after dexmedetomidine (adjusted P=0.006); similarly, the maximum decrease in SBP was 24.7±8.1 mmHg compared to 13.6±5.9 mmHg in carriers of the wildtype allele (P=0.007). Carriers of haplotype 3, previously associated with reduced α(2A)-AR expression, had a 44% smaller decrease in AUC(SBP) (P=0.013). Haplotype information significantly improved the model predicting the decrease in SBP (P<0.001). There were similar but non-significant trends for diastolic blood pressure and heart rate. Genotypes were not significantly associated with norepinephrine responses. Conclusions: -Common ADRA2A variants are associated with the hypotensive response to dexmedetomidine. Effects of specific variants/haplotypes in vivo are compatible with their known effects on gene expression in vitro.

Circ Cardiovasc Genet: 17 Feb 2011; epub ahead of print

Genome-Wide Association Study Pinpoints a New Functional ApoB Variant Influencing Oxidized LDL Levels but Not Cardiovascular Events: AtheroRemo Consortium.

Mäkelä KM, Seppälä I, Hernesniemi JA, Lyytikäinen LP, ... März W, Lehtimäki T
Background: -Oxidized low-density lipoprotein (oxLDL) may be a key factor in the development of atherosclerosis. We performed a genome-wide association study (GWAS) on oxLDL and tested the impact of the associated single-nucleotide polymorphisms (SNPs) on the risk factors of atherosclerosis, and cardiovascular events. Methods and results: -A discovery GWAS was performed on a population of young healthy Caucasian individuals (N=2,080), and the SNPs associated with a p value < 5 x 10(-8) were replicated in two independent samples (A: N=2,912; and B: N=1,326). Associations with cardiovascular endpoints were also assessed with two additional clinical cohorts (C: N=1,118; and D: N=808). We found 328 SNPs associated with oxLDL. The genetic variant rs676210 (Pro2739Leu) in apolipoprotein B (apoB) was the proxy SNP behind all associations (p=4.3 x 10(-136), effect size = 13.2 U/l per allele). This association was replicated in the two independent samples (A and B, p values = 2.5 x 10(-47) and 1.1 x 10(-11), effect sizes = 10.3 U/l and 7.8 U/l, respectively). In the meta-analyses of cohorts A, C, and D (excluding cohort B without angiographic data), the top SNP did not associate significantly with the age of onset of angiographically verified coronary artery disease (CAD, hazard ratio [HR]=1.00 [0.94-1.06] per allele), three vessel CAD (HR=1.03 [0.94-1.13]) or myocardial infarction (HR=1.04 [0.96-1.12]). Conclusions: -This novel genetic marker is an important factor regulating oxLDL levels but not a major genetic factor for the studied cardiovascular end-points.

Circ Cardiovasc Genet: 17 Dec 2012; epub ahead of print

Paraoxonase-1 Q192R Polymorphism and Antiplatelet Effects of Clopidogrel in Patients Undergoing Elective Coronary Stent Placement.

Trenk D, Hochholzer W, Fromm MF, Zolk O, ... Stratz C, Neumann FJ
Background: -Recently published data indicate that the paraoxonase-1 (PON1) Q192R genotype and not as previously shown activity of cytochrome P450 (CYP) 2C19 is the major determinant of metabolic bioactivation of clopidogrel and thereby variability of antiplatelet effect of clopidogrel. We sought to investigate whether the PON1 Q192R gene polymorphism impacts on platelet reactivity in patients undergoing elective coronary stent placement (PCI). Methods and results: -The study included 760 consecutive patients undergoing PCI after loading with clopidogrel 600mg. Platelet function was assessed by adenosine diphosphate-induced (ADP 5 and 20 μmol/L) platelet aggregation and by flow-cytometric analysis of platelet surface protein expression before clopidogrel, at the time of PCI and pre-discharge after PCI. PON1 Q192R genotype [NM_000446.5:c.575A>G SNP (rs662)] was analyzed by TaqMan PCR. Residual platelet aggregation (RPA ADP 5 μmol/L) at pre-discharge was 8.0% (3.0% - 17.0%) [median (interquartile range)] in PON1 QQ192 patients (n=384), 8.0% (3.0% - 15.0%) in PON1 QR192 (n=304) and 11.0% (3.0% - 18.0% ) in PON1 RR192 (n=72;p=0.603). By multivariable linear regression RPA was not associated with PON1 QQ192/QR192 (partial η(2): <0.001, p=0.728), but with CYP2C19*2 loss-of-function allele (partial η(2): 0.045, p<0.001) as well as any CYP2C19*17 gain-of-function allele (partial η(2): 0.012, p=0.004). All other platelet assays also showed no significant association between PON1 Q192R genotype and antiplatelet effect of clopidogrel. The 1-year incidence of death and myocardial infarction did not differ between PON1 Q192R genotypes. Conclusions: -On-treatment platelet reactivity in patients undergoing PCI after loading with clopidogrel 600mg was not associated with PON1 Q192R genotype. Clinical trial registration-URL: http://www.ClinicalTrials.gov. Unique identifier: NCT00457236.

Circ Cardiovasc Genet: 20 Jun 2011; epub ahead of print

A genome-wide association scan of RR and QT interval duration in 3 European genetically isolated populations: the EUROSPAN project.

Marroni F, Pfeufer A, Aulchenko YS, Franklin CS, ... Pramstaller PP,
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 x 10(-8)). The QT interval was associated with the earlier reported NOS1AP gene (rs2880058, P=2.00 x 10(-10)) and with a region on chromosome 13 (rs2478333, P=4.34 x 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. Conclusions: Our results suggested an association between the RR interval and GPR133 and confirmed an association between the QT interval and NOS1AP.

Circ Cardiovasc Genet: 24 Dec 2009; 2:322-8

Proteomic Identification of Matrix Metalloproteinase Substrates in the Human Vasculature.

Stegemann C, Didangelos A, Barallobre-Barreiro J, Langley S, ... Jahangiri M, Mayr M
Background: -Matrix metalloproteinases (MMPs) play a key role in cardiovascular disease, in particular aneurysm formation and plaque rupture. Surprisingly little is known about MMP substrates in the vasculature. Methods and results: -We used a proteomics approach to identify vascular substrates for three MMPs, one of each of the three major classes of MMPs: Human arteries were incubated with MMP-3 (a member of stromelysins), MMP-9 (considered a gelatinase) and MMP-14 (considered a member of the collagenases and of the membrane-bound MMPs). Candidate substrates were identified by mass spectrometry based on 1) increased release from the arterial tissue upon digestion, 2) spectral evidence for proteolytic degradation after gel separation, and 3) identification of non-tryptic cleavage sites. Using this approach, novel candidates were identified, including ECM proteins associated with the basement membrane, elastic fibers (emilin-1) and other extracellular proteins (periostin, tenascin-X). 74 non-tryptic cleavage sites were detected, many of which were shared among different MMPs. The proteomics findings were validated by immunoblotting and by digesting recombinant/purified proteins with exogenous MMPs. As proof-of-principle, results were related to in vivo pathology by searching for corresponding degradation products in human aortic tissue with different levels of endogenous MMP-9. Conclusions: -The application of proteomics to identify MMP targets is a new frontier in cardiovascular research. Our current classification of MMPs based on few substrates is an oversimplification of a complex area of biology. This study provides a more comprehensive assessment of potential MMP substrates in the vasculature and represents a valuable resource for future investigations.

Circ Cardiovasc Genet: 19 Dec 2012; epub ahead of print

High-dose simvastatin exhibits enhanced lipid-lowering effects relative to simvastatin/ezetimibe combination therapy.

Snowden SG, Grapov D, Settergren M, D\'Alexandri FL, ... Pernow J, Wheelock CE
Statins are the frontline in cholesterol reduction therapies; however, their use in combination with agents that possess complimentary mechanisms of action may achieve further reductions in low-density lipoprotein cholesterol. Thirty-nine patients were treated with either 80 mg simvastatin (n=20) or 10 mg simvastatin plus 10 mg ezetimibe (n=19) for 6 weeks. Dosing was designed to produce comparable low-density lipoprotein cholesterol reductions, while enabling assessment of potential simvastatin-associated pleiotropic effects. Baseline and post-treatment plasma were analyzed for lipid mediators (eg, eicosanoids and endocannabinoids) and structural lipids by liquid chromatography tandem mass spectrometry. After statistical analysis and orthogonal projections to latent structures multivariate modeling, no changes were observed in lipid mediator levels, whereas global structural lipids were reduced in response to both monotherapy (R(2)Y=0.74; Q(2)=0.66; cross-validated ANOVA P=7.0×10(-8)) and combination therapy (R(2)Y=0.67; Q(2)=0.54; cross-validated ANOVA P=2.6×10(-5)). Orthogonal projections to latent structures modeling identified a subset of 12 lipids that classified the 2 treatment groups after 6 weeks (R(2)Y=0.65; Q(2)=0.61; cross-validated ANOVA P=5.4×10(-8)). Decreases in the lipid species phosphatidylcholine (15:0/18:2) and hexosyl-ceramide (d18:1/24:0) were the strongest discriminators of low-density lipoprotein cholesterol reductions for both treatment groups (q<0.00005), whereas phosphatidylethanolamine (36:3e) contributed most to distinguishing treatment groups (q=0.017). Shifts in lipid composition were similar for high-dose simvastatin and simvastatin/ezetimibe combination therapy, but the magnitude of the reduction was linked to simvastatin dosage. Simvastatin therapy did not affect circulating levels of lipid mediators, suggesting that pleiotropic effects are not associated with eicosanoid production. Only high-dose simvastatin reduced the relative proportion of sphingomyelin and ceramide to phosphatidylcholine (q=0.008), suggesting a pleiotropic effect previously associated with a reduced risk of cardiovascular disease.

Circ Cardiovasc Genet: 16 Dec 2014; 7:955-64

Supravalvular aortic stenosis: elastin arteriopathy.

Merla G, Brunetti-Pierri N, Piccolo P, Micale L, Loviglio MN
Supravalvular aortic stenosis is a systemic elastin (ELN) arteriopathy that disproportionately affects the supravalvular aorta. ELN arteriopathy may be present in a nonsyndromic condition or in syndromic conditions such as Williams-Beuren syndrome. The anatomic findings include congenital narrowing of the lumen of the aorta and other arteries, such as branches of pulmonary or coronary arteries. Given the systemic nature of the disease, accurate evaluation is recommended to establish the degree and extent of vascular involvement and to plan appropriate interventions, which are indicated whenever hemodynamically significant stenoses occur. ELN arteriopathy is genetically heterogeneous and occurs as a consequence of haploinsufficiency of the ELN gene on chromosome 7q11.23, owing to either microdeletion of the entire chromosomal region or ELN point mutations. Interestingly, there is a prevalence of premature termination mutations resulting in null alleles among ELN point mutations. The identification of the genetic defect in patients with supravalvular aortic stenosis is essential for a definitive diagnosis, prognosis, and genetic counseling.

Circ Cardiovasc Genet: 18 Dec 2012; 5:692-6

Lipidomics: quest for molecular lipid biomarkers in cardiovascular disease.

Hinterwirth H, Stegemann C, Mayr M
Lipidomics is the comprehensive analysis of molecular lipid species, including their quantitation and metabolic pathways. The huge diversity of native lipids and their modifications make lipidomic analyses challenging. The method of choice for sensitive detection and quantitation of molecular lipid species is mass spectrometry, either by direct infusion (shotgun lipidomics) or coupled with liquid chromatography. Although shotgun lipidomics allows for high-throughput analysis, low-abundant lipid species are not detected. Previous separation of lipid species by liquid chromatography increases ionization efficiency and is better suited for quantifying low abundant and isomeric lipid species. In this review, we will discuss the potential of lipidomics for cardiovascular research. To date, cardiovascular research predominantly focuses on the role of lipid classes rather than molecular entities. An in-depth knowledge about the molecular lipid species that contribute to the pathophysiology of cardiovascular diseases may provide better biomarkers and novel therapeutic targets for cardiovascular disease.

Circ Cardiovasc Genet: 16 Dec 2014; 7:941-54

Cardiac Defects Are Infrequent Findings in Individuals with 8p23.1 Genomic Duplications Containing GATA4.

Yu S, Zhou XG, Fiedler SD, Brawner SJ, Joyce JM, Liu HY
Background: -GATA4 is a critical gene regulating myocardial differentiation and function. Haploinsufficiency of GATA4 are strongly associated with congenital heart defects (CHD). However it is inconclusive whether duplicated GATA4 may cause CHD. Methods and results: -We evaluated 1,645 consecutive pediatric patients with various developmental disorders by high-resolution microarray-based comparative genomic hybridization (aCGH) and found eight probands and two relatives with pathogenic genomic imbalances containing GATA4. Four probands contain a ~ 4.0 Mb interstitial duplication of 8p23.1 flanked by the two olfactory receptor gene clusters (REPD and REPP), representing approximately 0.24% (4/1,645) of the patients analyzed. None of the 4 patients has CHD or any other heart diseases while one mother who transmitted the duplication to his child has a history of aortic stenosis. Two patients who carried multiple genomic abnormalities including a duplication containing GATA4 have complex CHD. Only one of the three individuals carrying genomic deletion containing GATA4 has atrial septal defects (ASD) and ventricular septal defect (VSD). Conclusions: -Cardiac defects are infrequent findings in individuals with 8p23.1 genomic duplications containing GATA4. A 0.24% detection rate of this duplication in this study is significantly higher than previously estimated. Observation in two patients with multiple genomic abnormalities and complex CHD is consistent with a \'two-hit model\' emphasizing accumulative effects of more than one insult to the genome leading to a visible or more severe clinical manifestation. Haploinsufficient GATA4 may show variable expressivity with a wide spectrum of clinical findings including CHD.

Circ Cardiovasc Genet: 21 Sep 2011; epub ahead of print

A Complex Double Deletion in LMNA Underlies Progressive Cardiac Conduction Disease, Atrial Arrhythmias and Sudden Death.

Marsman RF, Bardai A, Postma AV, Res JC, ... Jordaens LJ, Bezzina CR
Background: -Cardiac conduction disease is a clinically and genetically heterogeneous disorder characterized by defects in electrical impulse generation and conduction, and associated with sudden cardiac death. Methods and results: -We here studied a four-generation family with autosomal dominant progressive cardiac conduction disease, including atrioventricular conduction block, sinus bradycardia, atrial arrhythmias and sudden death. Genome-wide linkage analysis mapped the disease locus to chromosome 1p22-q21. Multiplex ligation-dependent probe amplification (MLPA) analysis of the LMNA gene, which encodes the nuclear-envelop protein lamin A/C, revealed a novel gene rearrangement involving a 24-bp inversion flanked by a 3.8-kb deletion upstream and a 7.8-kb deletion downstream. The presence of short inverted sequence homologies at the breakpoint junctions suggested a mutational event involving serial replication slippage in trans during DNA replication. Conclusions: -We identified for the first time a complex LMNA gene rearrangement involving a double deletion in a four-generation Dutch family with progressive conduction system disease. Our findings underscore the fact that if conventional PCR-based direct sequencing approaches for LMNA analysis are negative in suggestive pedigrees, mutation detection techniques capable of detecting gross genomic lesions involving deletions and insertions should be considered.

Circ Cardiovasc Genet: 16 Mar 2011; epub ahead of print

A Common Variant at Chromosome 9P21.3 Is Associated with Age of Onset of Coronary Disease but Not Subsequent Mortality.

Ellis KL, Pilbrow AP, Frampton CM, Doughty RN, ... Richards AM, Cameron VA
Background: -Chromosome 9p21.3 (chr9p21.3) was recently identified by several genome-wide association studies as the genomic region most strongly associated with risk of coronary artery disease. Within the chr9p21.3 locus the single nucleotide polymorphism rs1333049 has been demonstrated to be most strongly associated with susceptibility to developing coronary artery disease. However, the impact of rs1333049 on clinical outcomes in patients with established coronary disease has yet to be determined. Methods and results: -Coronary Disease Cohort Study (CDCS, n=1054) and Post-Myocardial Infarction (PMI, n=816) study participants were genotyped for rs1333049. Clinical history, circulating lipids, neurohormones, cardiac function and discharge medications were documented. All-cause mortality and cardiovascular hospital readmissions were recorded over 4.0 years median follow-up for the CDCS cohort, and 9.1 years median follow-up for the PMI cohort. The CDCS patients homozygous for the high-risk \'C\' allele had an age of onset 2 to 5 years earlier for coronary disease (p=0.005), angina (p=0.025), myocardial infarction (p=0.022) and percutaneous transluminal coronary angioplasty (p=0.009). Patients with the \'CC\' genotype additionally had higher levels of total cholesterol (p=0.033) and triglycerides (p=0.003). PMI \'CC\' study participants were 3 years younger on entering (p=0.009). There was no significant association between rs1333049 genotype and mortality in either the CDCS (p=0.214) or PMI (p=0.696) cohorts when performing Cox proportional hazards analysis that adjusted for established predictors of increased risk. Conclusions: -The chr9p21.3 polymorphism, rs1333049, was associated with an earlier age of disease onset in two coronary disease cohorts, but not with poorer clinical outcome in either cohort.

Circ Cardiovasc Genet: 19 Apr 2010; epub ahead of print

Molecular Genetic and Functional Characterization Implicate Muscle-Restricted Coiled-Coil Gene (MURC) as a Causal Gene for Familial Dilated Cardiomyopathy.

Rodriguez G, Ueyama T, Ogata T, Czernuszewicz G, ... Willerson JT, Marian AJ
Background: -Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies. Methods and results: -We sequenced MURC in 1,199 individuals including 383 probands with DCM, 307 with HCM and 509 healthy controls. We found six heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L and p.S364L) in eight unrelated probands. Variants p.N128K and p.S307T segregated with inheritance of DCM in small families (χ(2)=8.5, p=0.003). Variants p.N128K, p.R140W, p.L153P and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in three independent probands, including one proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in three unrelated HCM probands but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L970V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in four patients, conduction defects and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared to wild type MURC. Conclusions: -MURC mutations impart loss-of-function effects on MURC functions and are likely causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain.

Circ Cardiovasc Genet: 06 Jun 2011; epub ahead of print

Causal Relevance of Blood Lipid Fractions in the Development of Carotid Atherosclerosis: Mendelian Randomization Analysis.

Shah S, Casas JP, Drenos F, Whittaker J, ... Humphries SE, Hingorani AD
Background: -Carotid intima-media thickness (CIMT), a subclinical measure of atherosclerosis, is associated with risk of coronary heart disease (CHD) events. Statins reduce progression of CIMT and CHD risk in proportion to the reduction in low-density lipoprotein cholesterol (LDL-C). However, interventions targeting triglycerides or high density lipoprotein cholesterol (HDL-C) have produced inconsistent effects on CIMT and CHD risk, making it uncertain whether such agents are ineffective for CHD prevention or whether CIMT is an inadequate marker of HDL-C or triglyceride-mediated effects. We aimed to determine the causal association between the three major blood lipid fractions and common CIMT using Mendelian randomisation (MR) analysis. Methods and results: -Gene scores specific for LDL-C, HDL-C and triglycerides were derived based on single nucleotide polymorphisms (SNPs) from a gene-centric array in around 5000 individuals (Cardiochip scores) and from a genome-wide association meta-analysis in over 100,000 individuals (Global Lipids Genetic Consortium (GLGC) scores). These were used as instruments in an MR analysis in two prospective cohort studies. A genetically-predicted 1 mmol/L higher LDL-C concentration was associated with a higher common CIMT by 0.03 mm (95% CI=0.01-0.04) and 0.04 mm (95% CI=0.02-0.06) based on the Cardiochip and GLGC scores, respectively. HDL-C and triglycerides were not causally associated with CIMT. Conclusions: -Our findings confirm a causal relationship between LDL-C and CIMT but not with HDL-C and triglycerides. At present, the suitability of CIMT as a surrogate marker in trials of cardiovascular therapies targeting HDL-C and triglycerides is questionable and requires further study.

Circ Cardiovasc Genet: 30 Dec 2012; epub ahead of print

Genetic Variation in PEAR1 is Associated with Platelet Aggregation and Cardiovascular Outcomes.

Lewis JP, Ryan K, O\'Connell JR, Horenstein RB, ... Gurbel PA, Shuldiner AR
Background: -Aspirin or dual antiplatelet therapy (DAPT) with aspirin and clopidogrel is standard therapy for patients at increased risk for cardiovascular events. However, the genetic determinants of variable response to aspirin (alone and in combination with clopidogrel) are not known. Methods and results: -We measured ex-vivo platelet aggregation before and after DAPT in individuals (n=565) from the Pharmacogenomics of Antiplatelet Intervention (PAPI) Study and conducted a genome-wide association study (GWAS) of drug response. Significant findings were extended by examining genotype and cardiovascular outcomes in two independent aspirin-treated cohorts: 227 percutaneous coronary intervention (PCI) patients, and 1,000 patients of the International VErapamil SR/trandolapril Study (INVEST) GENEtic Substudy (INVEST-GENES). GWAS revealed a strong association between single nucleotide polymorphisms on chromosome 1q23 and post-DAPT platelet aggregation. Further genotyping revealed rs12041331 in the platelet endothelial aggregation receptor-1 (PEAR1) gene to be most strongly associated with DAPT response (P=7.66x10(-9)). In Caucasian and African American patients undergoing PCI, A-allele carriers of rs12041331 were more likely to experience a cardiovascular event or death compared to GG homozygotes (hazard ratio = 2.62, 95%CI 0.96-7.10, P=0.059 and hazard ratio = 3.97, 95%CI 1.10-14.31, P=0.035 respectively). In aspirin-treated INVEST-GENES patients, rs12041331 A-allele carriers had significantly increased risk of myocardial infarction compared to GG homozygotes (OR=2.03, 95%CI 1.01-4.09, P=0.048). Conclusions: -Common genetic variation in PEAR1 may be a determinant of platelet response and cardiovascular events in patients on aspirin, alone and in combination with clopidogrel. Clinical Trial registration Information-clinicaltrials.gov; Identifiers: NCT00799396 and NCT00370045.

Circ Cardiovasc Genet: 07 Feb 2013; epub ahead of print

Dense Genotyping of Candidate Gene Loci Identifies Variants Associated with High-Density Lipoprotein Cholesterol.

Edmondson AC, Braund PS, Stylianou IM, Khera AV, ... Samani NJ, Rader DJ
Background: -Plasma levels of high density lipoprotein cholesterol (HDL-C) are known to be heritable, but only a fraction of the heritability is explained. We used a high density genotyping array containing SNPs from HDL-C candidate genes selected on known biology of HDL-C metabolism, mouse genetic studies, and human genetic association studies. SNP selection was based on tagging-SNPs but also included low-frequency nonsynonymous SNPs. Methods and results: -Association analysis in a cohort containing extremes of HDL-C (case-control, n=1733) provided a discovery phase, with replication in three additional populations for a total meta-analysis in 7,857 individuals. We replicated the majority of loci identified through genome wide association studies and present on the array (including ABCA1, APOA1/C3/A4/A5, APOB, APOE/C1/C2, CETP, CTCF-PRMT8, FADS1/2/3, GALNT2, LCAT, LILRA3, LIPC, LIPG, LPL, LRP4, SCARB1, TRIB1, ZNF664), and provide evidence suggestive of association in several previously unreported candidate gene loci (including ABCG1, GPR109A/B/81, NFKB1, PON1/2/3/4). There was evidence for multiple, independent association signals in five loci, including association with low frequency nonsynonymous variants. Conclusions: -Genetic loci associated with HDL-C are likely to harbor multiple, independent causative variants, frequently with opposite effects on the HDL-C phenotype. Cohorts composed of extreme individuals may be efficiently used in a case-control discovery of quantitative traits.

Circ Cardiovasc Genet: 09 Feb 2011; epub ahead of print

Joint Associations of 61 Genetic Variants in the Nicotinic Acetylcholine Receptor Genes with Subclinical Atherosclerosis in American Indians: A Gene-Family Analysis.

Yang J, Zhu Y, Lee ET, Zhang Y, ... Howard BV, Zhao J
Background: -Atherosclerosis is the underlying cause of cardiovascular disease, the leading cause of morbidity and mortality in all American populations including American Indians. Genetic factors play an important role in the etiology of atherosclerosis. While a single SNP may explain only a small portion of variability in disease, the joint effect of multiple variants in a pathway on disease susceptibility could be large. Methods and results: -Using a gene-family analysis, we investigated the joint associations of 61 tag SNPs in seven nicotinic acetylcholine receptors (nAChRs) genes with subclinical atherosclerosis, as measured by carotid intima-media thickness (IMT) and plaque score, in 3,665 American Indians from 94 families recruited by the Strong Heart Family Study (SHFS). Although multiple SNPs showed marginal association with IMT and/or plaque score individually, only a few survived adjustments for multiple testing. However, simultaneously modeling of the joint effect of all 61 SNPs in seven nAChRs genes revealed significant association of the nAChR gene family with both IMT and plaque score, independent of known coronary risk factors. Conclusions: -Genetic variants in the nicotinic acetylcholine receptors gene family jointly contribute to subclinical atherosclerosis in American Indians participated in the SHFS. These variants may influence the susceptibility of atherosclerosis through pathways other than cigarette smoking per se.

Circ Cardiovasc Genet: 23 Dec 2012; epub ahead of print

Common Variation at the 11-Beta Hydroxysteroid Dehydrogenase Type 1 (HSD11B1) Gene Is Associated with Left Ventricular Mass.

Rahman TJ, Mayosi BM, Hall D, Avery PJ, ... Watkins H, Keavney B
Background: -Polymorphisms in 11-β hydroxysteroid dehydrogenase type 1 (11βHSD-1, encoded by HSD11B1) have been reported to be associated with obesity-related cardiovascular risk factors, such as type II diabetes and hypertension. Left ventricular hypertrophy (LVH) is an independent risk factor for cardiovascular death that is associated with these factors, but has significant additional heritability the cause of which is undetermined. 11βHSD-1 is thought to maintain tonic inhibition of the mineralocorticoid receptor (MR) in cardiomyocytes, and MR activation is involved in the pathophysiology of LVH. We assessed association between polymorphisms in the HSD11B1 gene and left ventricular mass in an association study of 248 families ascertained through a proband with hypertension. Methods and results: -Left ventricular mass was measured by electrocardiography and echocardiography in 868 and 829 participants respectively. Single nucleotide polymorphisms (SNPs) tagging common variation in the HSD11B1 gene were genotyped by mass spectrometry. The rs846910 SNP which lies in the flanking region 5\' to exon 1B of HSD11B1 was associated with left ventricular mass both by electrocardiography (~5% lower LV mass per copy of the rare allele, p=0.02), and by echocardiography (~10% lower LV mass per copy of the rare allele, p=0.003). Genotype explained 1-2% of the population variability in left ventricular mass, or around 5% of the heritable fraction. There were no significant associations between any HSD11B1 SNP and blood pressure or body mass index that could have confounded the association with left ventricular mass. Conclusions: -Genotype at HSD11B1 has a small but significant effect on left ventricular mass, apparently independently of any effect on obesity-related traits. These findings suggest a novel action of 11βHSD-1 in the human cardiomyocyte which may be of therapeutic importance.

Circ Cardiovasc Genet: 15 Mar 2011; epub ahead of print

MOG1: A New Susceptibility Gene for Brugada Syndrome.

Kattygnarath D, Maugenre S, Neyroud N, Balse E, ... Hatem SN, Guicheney P
Background: -Brugada syndrome (BrS) is caused mainly by mutations in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel, Na(v)1.5. However, approximately 20% of probands have SCN5A mutations, suggesting the implication of other genes. MOG1 was recently described as a new partner of Na(v)1.5, playing a potential role in the regulation of its expression and trafficking. We investigated if mutations in MOG1 could cause BrS. Methods and results: -MOG1 was screened by direct sequencing in BrS and Idiopathic Ventricular Fibrillation (IVF) patients. A missense mutation p.Glu83Asp (E83D) was detected in a symptomatic female with a type-1 BrS ECG, but not in 281 controls. Wild type (WT) and mutant E83D MOG1 were expressed in HEK Na(v)1.5 stable cells and studied using patch-clamp. Over-expression of WT MOG1 alone doubled sodium current (I(Na)) density compared to control conditions (p<0.01). In contrast, over-expression of mutant E83D alone, or E83D+WT, failed to increase I(Na) (p<0.05), demonstrating the dominant negative effect of the mutant. Microscopy revealed that Na(v)1.5 channels failed to properly traffic to the cell membrane in the presence of the mutant. Silencing endogenous MOG1 demonstrated a 54% decrease in INa density. Conclusions: -Our results support the hypothesis that dominant negative mutations in MOG1 can impair the trafficking of Na(v)1.5 to the membrane, leading to I(Na) reduction and clinical manifestation of BrS. Moreover, silencing MOG1 reduced I(Na), demonstrating that MOG1 is likely to be important in the surface expression of Na(v)1.5 channels. Altogether, our data support MOG1 as a new susceptibility gene for BrS.

Circ Cardiovasc Genet: 30 Mar 2011; epub ahead of print

The Coronary Artery Disease Associated 9p21 Variant and Later Life 20 Year Survival to Cohort Extinction.

Dutta A, Henley W, Lang IA, Murray A, ... Wallace RB, Melzer D
Background: -Common variation at chromosome 9p21 (marked by rs10757278 or rs1333049) is associated with coronary artery disease (CAD) and peripheral vascular disease. A decreasing effect at older age was suggested, and impacts on long-term mortality are unclear. We estimated 9p21 associations with CAD and all cause mortality in a CAD diagnosis free older population. We also estimated classification gains on adding the variant to the Framingham Risk Score (FRS) for CAD. Methods and results: -DNA was from Iowa-EPESE cohort from 1988 (participants >71 years) with death certificates obtained to 2008 on 92% of participants. Cox regression models were adjusted for confounders and CAD risk factors. Of 1095 CAD diagnosis free participants, 52% were heterozygous (CG) and 22% were homozygous (CC) for the risk C-allele rs1333049. Unadjusted CAD-attributed death rates in the CC group were 30 versus 22/1000 person-years for the GG group. The C-allele was associated with all cause (HR/C-allele: 1.19, 95%CI: 1.08-1.30) and CAD mortality (HR/C-allele: 1.29, 95%CI 1.08-1.56) independent of CAD risk-factors. There was no association with stroke deaths. Variant associations with CAD mortality were attenuated after age 80 (age-interaction term p=0.05). In age-group 71-80, FRS classified as high-risk 21% respondents who died of CAD within 10 years: adding 9p21 identified 27% Conclusions: -In 71-80 year olds free of CAD diagnoses, 9p21 is associated with excess mortality mainly attributed to CAD mortality. Adding 9p21 to the FRS may improve the targeting of CAD prevention in older people, but validation in independent samples is needed to confirm this.

Circ Cardiovasc Genet: 19 Aug 2011; epub ahead of print

Recurrence of Discordant Congenital Heart Defects in Families.

Oyen N, Poulsen G, Wohlfahrt J, Boyd HA, Jensen PK, Melbye M
Background: -Variation within a single gene might produce different congenital heart defects (CHDs) within a family, which could explain the previously-reported familial aggregation of discordant CHDs. We investigated whether certain groups of discordant CHDs are more common in families than others. Methods and results: -Using Danish national population and health registers, we identified CHDs among all singletons born in Denmark during 1977-2005 and their first-degree relatives. In a cohort of 1,711,641 persons, 16,777 had CHDs, which we classified into 14 phenotypes. We estimated relative risks of discordant CHDs by history of specific CHDs in first-degree relatives. The relative risk of any dissimilar CHD given the specified CHD in first-degree relatives was as follows: heterotaxia, 2.00 (95% confidence interval 0.96-4.17); conotruncal defects, 2.78 (2.12-3.66); atrioventricular septal defects, 2.25 (1.39-3.66); anomalous pulmonary venous return, 1.76 (0.66-4.64); left- and right-ventricular outflow tract obstruction, 2.55 (1.87-3.48) and 3.09 (2.03-4.71), respectively; isolated atrial septal defects, 2.76 (2.11-3.61); isolated ventricular septal defects, 2.27 (1.75-2.94); persistent ductus arteriosus, 1.92 (1.32-2.79); other specified CHDs, 3.29 (2.51-4.32); and unspecified CHDs, 2.30 (1.76-3.00). Relative risks for all pair-wise combinations of discordant CHD phenotypes gave no indications that certain constellations of CHDs cluster more in families than others. Conclusions: -We documented strong familial aggregation of discordant CHD phenotypes. However, we observed no excess clustering of specific CHD phenotypes among first-degree relatives.

Circ Cardiovasc Genet: 22 Feb 2010; epub ahead of print

SuperTAG Methylation-Specific Digital Karyotyping (SMSDK) Reveals Uremia Induced Epigenetic Dysregulation of Atherosclerosis-Related Genes.

Zawada AM, Rogacev KS, Hummel B, Grün OS, ... Fliser D, Heine GH
Background: -Accelerated atherosclerosis is a hallmark of chronic kidney disease (CKD). While the role of epigenetic dysregulation in atherosclerosis is increasingly appreciated, only few studies focused on epigenetics in CKD-associated cardiovascular disease, virtually all of which assessed epigenetic dysregulation globally. We hypothesized that gene-specific epigenetic dysregulation in CKD exists, affecting genes pertinent to inflammation and atherosclerosis. Methods and results: -Ten clinically stable patients undergoing hemodialysis therapy and ten healthy, age and sex matched controls were recruited. Genome-wide analysis of DNA methylation was performed by SuperTAG methylation-specific digital karyotyping (SMSDK), in order to identify genes differentially methylated in CKD. Analysis of 27,043,436 tags revealed 4,288 genomic loci with differential DNA methylation (P < 10(-10)) between hemodialysis patients and control subjects. Annotation of UniTags to promoter databases allowed us to identify 52 candidate genes associated with cardiovascular disease and 97 candidate genes associated with immune / infection diseases. These candidate genes could be classified to distinct proatherogenic processes including lipid metabolism and transport (e.g. HMGCR, SREBF1, LRP5, EPHX2, FDPS), cell proliferation and cell-cycle regulation (e.g. MIK67, TP53, ALOX12), angiogenesis (e.g. ANGPT2, ADAMTS10, FLT4) and inflammation (e.g. TNFSF10, LY96, IFNGR1, HSPA1A, IL12RB1). Conclusions: -We provide a comprehensive analysis of genome wide epigenetic alterations in CKD, identifying candidate genes associated with proatherogenic and inflammatory processes. These results may spur further research in the field of epigenetics in kidney disease, and point to new therapeutic strategies in CKD-associated atherosclerotic disease.

Circ Cardiovasc Genet: 16 Oct 2012; epub ahead of print

Formin Homology 2 Domain Containing 3 (FHOD3) Variants Associated with Hypertrophic Cardiomyopathy.

Wooten EC, Hebl VB, Wolf MJ, Greytak SR, ... Ackerman MJ, Huggins GS
Background: -Incomplete penetrance and variable expression of Hypertrophic Cardiomyopathy (HCM) is well appreciated. Common genetic polymorphisms variants that may affect HCM penetrance and expression have been predicted but are not well established. Methods and results: -We performed a case-control genome wide association (GWA) study to identify common HCM-associated genetic polymorphisms and then asked whether such common variants were more represented in HCM or could explain the heterogeneity of HCM phenotypes. We identified an intronic FHOD3 variant (rs516514) associated with HCM (OR = 2.45 (95% CI 1.76-3.41), p=1.25 x 10(-7)) and validated this finding in an independent cohort. Next, we tested FHOD3-V1151I (rs2303510), a non-synonymous variant in partial linkage disequilibrium (LD) with rs516514, and we detected an even stronger association with HCM (p=1.76 x 10(-9)). While HCM patients were more likely to carry these FHOD3 alleles subjects homozygous for FHOD3-1151I had similar HCM phenotypes as carriers of the V1151 allele. FHOD3 expression is increased in the setting of HCM and both alleles of FHOD3-V1151I were detected in HCM myectomy tissue. Previously FHOD3 was found to be required for formation of the sarcomere and here we demonstrate that its fly homolog fhos is required for normal adult heart systolic contraction. Conclusions: -Here we demonstrate the association of a common non-synonymous FHOD3 genetic variant with HCM. This discovery further strengthens the potential role of gene mutations and polymorphisms that alter the amino acid sequence of sarcomere proteins and HCM.

Circ Cardiovasc Genet: 19 Dec 2012; epub ahead of print

Genome-wide Significance and Replication of the Chromosome 12p11.22 Locus Near the PTHLH Gene for Peripartum Cardiomyopathy.

Horne BD, Rasmusson KD, Alharethi R, Budge D, ... Renlund DG, Kfoury AG
Background: -Peripartum (PP) cardiomyopathy (CM) is a rare condition of unknown etiology that occurs in late pregnancy or early postpartum. Initial evidence suggests that genetic factors may influence PPCM. This study evaluated and replicated genome-wide association of single nucleotide polymorphisms (SNPs) with PPCM. Methods and results: -Genome-wide SNPs in females with verified PPCM diagnosis (n=41) were compared separately to Local Controls (n=49 post-menopausal age-discordant females with parity ≥1 and no heart failure) and iControls (n=654 females aged 30-84 with unknown phenotypes). A replication study of independent population samples utilized new cases (PPCM2, n=30) compared to new age-discordant controls (Local2, n=124) and to younger controls (YC, n=89) and obstetrical controls (OBC, n=90). A third case set of pregnancy-associated (PA) CM cases not meeting strict PPCM definitions (n=29) was also studied. In the genome-wide association study, one SNP (rs258415) met genome-wide significance for PPCM vs. Local Controls (p=2.06 x 10(-8), OR=5.96). This was verified vs. iControls (p=7.92 x 10(-19), OR=8.52). In the replication study for PPCM2 cases, rs258415 (ORs are per C allele) replicated at p=0.009 vs. Local2 Controls (OR=2.26). This replication was verified for PPCM2 vs. YC (p=0.029, OR=2.15) and vs. OBC (p=0.013, OR=2.44). In PACM cases, rs258415 had a similar effect vs. Local2 Controls (p=0.06, OR=1.79), YC (p=0.14, OR=1.65), and OBC (p=0.038, OR=1.99). Conclusions: -Genome-wide association with PPCM was discovered and replicated for rs258415 at chromosome 12p11.22 near PTHLH. This study indicates a role of genetic factors in PPCM and provides a new locus for further pathophysiological and clinical investigation.

Circ Cardiovasc Genet: 13 Jun 2011; epub ahead of print

Relative Familial Clustering of Cerebral Versus Coronary Ischaemic Events.

Banerjee A, Silver LE, Heneghan C, Welch SJ, ... Banning AP, Rothwell PM
Background: -Few population-based studies have ascertained both cerebral and coronary events or considered their relative heritability. Differences in heritability of transient ischaemic attack (TIA) and ischaemic stroke versus acute coronary syndromes (ACS) may inform risk prediction, genetic studies, and understanding of disease mechanisms. Methods and results: -In a population-based study of all acute vascular events, irrespective of age, we studied family history of myocardial infarction (MI), stroke and related risk factors in first degree relatives (FDR). To allow for differences in rates of affected FDRs due to differences in disease incidence, we looked at the extent to which parental history was associated with affected siblings within disease category. 906(604 males, mean age=70.0) probands with ACS and 1015(484 males,mean age=73.0) with cerebral events had complete family history data. In ACS probands, parental MI was associated with MI in ≥1 sibling: one parent with MI - OR=1.48, 1.04-2.10, p=0.03; both parents with MI - OR=5.97, 3.23-11.03; p<0.0001. In probands with cerebral events, however, parental stroke was not associated with sibling stroke. The overall frequency of ≥2 siblings with the same condition was also greater in probands with ACS than in those with cerebral events (5.43, 3.03-9.76; p<0.00001), despite similar overall incidence of MI and stroke in our study population. 142(15.7%) ACS occurred in families with ≥2 affected FDRs compared with 56(5.1%) TIA/strokes. All results were similar when analyses were confined to probands with MI only versus stroke only, and independent of smoking. Conclusions: -Heritability of coronary events was greater than that of cerebral events, such that MI was more likely to cluster in families than was stroke.

Circ Cardiovasc Genet: 27 Jul 2011; epub ahead of print

Autosomal Recessive Atrial Dilated Cardiomyopathy with Standstill Evolution Associated with Mutation of Natriuretic Peptide Precursor A.

Disertori M, Quintarelli S, Grasso M, Pilotto A, ... Specchia C, Arbustini E
Background: -Atrial dilatation and atrial standstill are etiologically heterogeneous phenotypes with poorly defined nosology. In 1983 we described 8-years follow-up of idiopathic atrial dilatation with standstill evolution in 8 patients from 3 families. We later identified 5 additional patients with identical phenotypes: 1 member of the largest original family and 4 unrelated to the 3 original families. All families are from a same geographic area in the North-East Italy. Methods and results: -We followed-up the 13 patients for up to 37 years, extended the clinical investigation and monitoring to living relatives and investigated the genetic basis of the disease. The disease was characterized by: 1) clinical onset in adulthood; 2) bi-atrial dilatation up to giant size; 3) early supraventricular arrhythmias with progressive loss of atrial electrical activity to atrial standstill; 4) thromboembolic complications; 5) stable, normal left ventricular function and NYHA functional class during the long-term course of the disease. By linkage analysis we mapped a locus at 1p36.22 containing the natriuretic precursor A (NPPA) gene. By sequencing NPPA we identified a homozygous missense mutation (p.Arg150Gln) in all living affected individuals of the 6 families. All patients showed low serum levels of Atrial Natriuretic Peptide (ANP). Heterozygous mutation carriers were healthy and demonstrated normal levels of ANP. Conclusions: -Autosomal recessive Atrial Dilated Cardiomyopathy is a rare disease associated with homozygous mutation of the NPPA gene and characterized by extreme atrial dilatation with standstill evolution, thromboembolic risk, preserved left ventricular function and severely decreased levels of ANP.

Circ Cardiovasc Genet: 30 Dec 2012; epub ahead of print

Admixture Mapping of Coronary Artery Calcified Plaque in African Americans with Type 2 Diabetes.

Divers J, Palmer ND, Lu L, Register TC, ... Langefeld CD, Freedman BI
Background: -The presence and severity of coronary artery calcified plaque (CAC) differs markedly between individuals of African and European descent, suggesting that admixture mapping (AM) may be informative for identifying genetic variants associated with subclinical cardiovascular disease (CVD). Methods and results: -AM of CAC was performed in 1,040 unrelated African Americans with type 2 diabetes mellitus from the African American-Diabetes Heart Study (AA-DHS), Multi-Ethnic Study of Atherosclerosis (MESA), and Family Heart Study (FamHS) using the Illumina custom ancestry informative marker (AIM) panel. All cohorts obtained computed tomography scanning of the coronary arteries using identical protocols. For each AIM, the probability of inheriting 0, 1, and 2 copies of a European-derived allele was determined. Linkage analysis was performed by testing for association between each AIM using these probabilities and CAC, accounting for global ancestry, age, gender and study. Markers on 1p32.3 in the GLIS1 gene (rs6663966, LOD=3.7), 1q32.1 near CHIT1 (rs7530895, LOD=3.1), 4q21.2 near PRKG2 (rs1212373, LOD=3.0) and 11q25 in the OPCML gene (rs6590705, LOD=3.4) had statistically significant LOD scores, while markers on 8q22.2 (rs6994682, LOD=2.7), 9p21.2 (rs439314, LOD=2.7), and 13p32.1 (rs7492028, LOD=2.8) manifested suggestive evidence of linkage. These regions were uniformly characterized by higher levels of European ancestry associating with higher levels or odds of CAC. Findings were replicated in 1,350 AAs without diabetes and 2,497 diabetic European Americans from MESA and the Diabetes Heart Study. Conclusions: -Fine mapping these regions will likely identify novel genetic variants that contribute to CAC and clarify racial differences in susceptibility to subclinical CVD.

Circ Cardiovasc Genet: 11 Dec 2012; epub ahead of print

Candidate Gene Association Resource (CARe): Design, Methods, and Proof of Concept.

Musunuru K, Lettre G, Young T, Farlow DN, ... Boerwinkle E, Gabriel SB
Background: -The National Heart, Lung, and Blood Institute\'s Candidate Gene Association Resource (CARe), a planned cross-cohort analysis of genetic variation in cardiovascular, pulmonary, hematological, and sleep-related traits, comprises more than 40,000 participants representing four ethnic groups in nine community-based cohorts. The goals of CARe include the discovery of new variants associated with traits using a candidate gene approach and the discovery of new variants using the genome-wide association mapping approach specifically in African Americans. Methods and results: -CARe has assembled DNA samples for more than 40,000 individuals self-identified as European-American, African-American, Hispanic, or Chinese-American, with accompanying data on hundreds of phenotypes that have been standardized and deposited in the CARe Phenotype Database. All participants were genotyped for seven single-nucleotide polymorphisms (SNPs) selected based on prior association evidence. We performed association analyses relating each of these SNPs to lipid traits, stratified by gender and ethnicity and adjusted for age and age2. In at least two of the ethnic groups, SNPs near CETP, LIPC, and LPL strongly replicated for association with high-density lipoprotein cholesterol concentrations, PCSK9 with low-density lipoprotein cholesterol levels, and LPL and APOA5 with serum triglycerides. Notably, some SNPs showed varying effect sizes and significance of association in different ethnic groups. Conclusions: -The CARe Pilot Study validates the operational framework for phenotype collection, SNP genotyping, and analytical pipeline of the CARe project and validates the planned candidate gene study of ~2,000 biologic candidate loci in all participants and genome-wide association study in ~8,000 African-American participants. CARe will serve as a valuable resource for the scientific community.

Circ Cardiovasc Genet: 19 Apr 2010; epub ahead of print

MicroRNA-150: A Novel Marker of Left Ventricular Remodeling After Acute Myocardial Infarction.

Devaux Y, Vausort M, McCann GP, Zangrando J, ... Wagner DR, Squire IB
Background: -Left ventricular (LV) remodeling after acute myocardial infarction (AMI) is associated with adverse prognosis. MicroRNAs (miRNAs) regulate the expression of several genes involved in LV remodeling. Our aim was to identify miRNAs associated with LV remodeling after AMI. Methods and results: -We studied 90 patients following first ST elevation AMI (STEMI). A derivation cohort consisted of 60 patients characterized by echocardiography pre-discharge and at 6 months follow-up. Thirty patients characterized by magnetic resonance imaging pre-discharge and at 4 months follow-up were the validation cohort. Remodeling was defined as increase in LV end-diastolic volume between discharge and follow-up (ΔEDV >0). Circulating miRNAs were measured by microarrays and PCR. Using a systems-based approach, we identified several miRNAs potentially involved in LV remodeling. In the derivation cohort, one of these miRNAs, miR-150, was down-regulated in patients with remodeling (ΔEDV >0) compared to patients without remodeling (ΔEDV ≤0). In the validation cohort, patients with remodeling had 2-fold lower levels of miR-150 than those without (P=0.03). miR-150 outperformed N terminal-pro-brain natriuretic peptide (Nt-pro-BNP) to predict remodeling (area under the receiver operating characteristic curve of 0.74 and 0.60, respectively). miR-150 reclassified 54% (95% confidence interval (CI), 5-102%; P=0.03) of patients misclassified by Nt-pro-BNP and 59% (95% CI, 9-108%; P=0.02) of patients misclassified by a multi-parameter clinical model including age, gender, and admission levels of troponin I, creatine kinase, and Nt-pro-BNP. Conclusions: -Low circulating levels of miR-150 are associated with LV remodeling after first STEMI. miR-150 has potential as a novel biomarker in this setting.

Circ Cardiovasc Genet: 01 Apr 2013; epub ahead of print

Brugada Syndrome Disease Phenotype Explained in Apparently Benign Sodium Channel Mutations.

Hoshi M, Du XX, Shinlapawittayatorn K, Liu H, ... Ficker E, Deschênes I
-Brugada syndrome (BrS) is an arrhythmogenic disorder that has been linked to mutations in SCN5A, the gene encoding for the pore-forming α-subunit of the cardiac sodium channel. Typically, BrS mutations in SCN5A result in a reduction of sodium current with some mutations even exhibiting a dominant-negative effect on wild-type (WT) channels thus leading to an even more prominent decrease in current amplitudes. However, there is also a category of apparently benign ("atypical") BrS SCN5A mutations that in vitro demonstrates only minor biophysical defects. It is therefore not clear how these mutations produce a BrS phenotype. We hypothesized that similar to dominant-negative mutations atypical mutations could lead to a reduction in sodium currents when co-expressed with WT to mimic the heterozygous patient genotype.

Circ Cardiovasc Genet: 26 Feb 2014; epub ahead of print

Integration of Genetics into a Systems Model of ECG Traits using HumanCVD BeadChip.

Gaunt TR, Shah S, Nelson CP, Drenos F, ... Kumari M, Day IN
Background: -Electrocardiographic (ECG) traits are important, substantially heritable, determinants of risk of arrhythmias and sudden cardiac death. Methods and results: -In this study, three population-based cohorts (n=10,526) genotyped with the Illumina HumanCVD Beadchip and four quantitative ECG traits (PR interval, QRS axis, QRS duration and QTc interval) were evaluated for single nucleotide polymorphism (SNP) associations. Six gene regions contained SNPs associated with these traits at p< 10(-6), including SCN5A (PR interval and QRS duration), CAV1-CAV2 locus (PR interval), CDKN1A (QRS duration), NOS1AP, KCNH2 and KCNQ1 (QTc interval). Expression QTL analyses of top associated SNPs were undertaken in human heart and aortic tissues. NOS1AP, SCN5A, IGFBP3, CYP2C9 and CAV1 showed evidence of differential allelic expression. We modelled the effects of ion channel activity on ECG parameters, estimating the change in gene expression that would account for our observed associations, thus relating epidemiological observations and eQTL data to a systems model of the ECG. Conclusions: -These association results replicate and refine the mapping of previous genome-wide association study (GWAS) findings for ECG traits, whilst the expression analysis and modelling approaches offer supporting evidence for a functional role of some of these loci in cardiac excitation/conduction.

Circ Cardiovasc Genet: 08 Nov 2012; epub ahead of print

Genome-Wide Association Study of Cardiac Structure and Systolic Function in African Americans: The Candidate Gene Association Resource (CARe) Study.

Fox ER, Musani SK, Barbalic M, Lin H, ... Cappola TP, Vasan RS
Background: -Using data from four community-based cohorts of African Americans (AA), we tested the association between genome-wide markers (SNPs) and cardiac phenotypes in the Candidate-gene Association REsource (CARe) study. Methods and results: -Among 6,765 AA, we related age, sex, height and weight-adjusted residuals for nine cardiac phenotypes (assessed by echocardiogram or MRI) to 2.5 million SNPs genotyped using Genome-Wide Affymetrix Human SNP Array 6.0 (Affy6.0) and the remainder imputed. Within cohort genome-wide association analysis was conducted followed by meta-analysis across cohorts using inverse variance weights (genome-wide significance threshold=4.0 x10(-07)). Supplementary pathway analysis was performed. We attempted replication in 3 smaller cohorts of African ancestry and tested look-ups in one consortium of European ancestry (EchoGEN). Across the 9 phenotypes, variants in 4 genetic loci reached genome-wide significance: rs4552931 in UBE2V2 (p=1.43x10(-07)) for left ventricular mass (LVM); rs7213314 in WIPI1 (p=1.68 x10(-07)) for LV internal diastolic diameter (LVIDD); rs1571099 in PPAPDC1A (p= 2.57 x10(-08)) for interventricular septal wall thickness (IVST); and rs9530176 in KLF5 (p=4.02 x10(-07)) for ejection fraction (EF). Associated variants were enriched in three signaling pathways involved in cardiac remodeling. None of the 4 loci replicated in cohorts of African ancestry were confirmed in look-ups in EchoGEN. Conclusions: -In the largest GWAS of cardiac structure and function to date in AA, we identified 4 genetic loci related to LVM, IVST, LVIDD and EF that reached genome-wide significance. Replication results suggest that these loci may represent unique to individuals of African ancestry. Additional large-scale studies are warranted for these complex phenotypes.

Circ Cardiovasc Genet: 30 Dec 2012; epub ahead of print

Genetic Architecture of Carotid Artery Intima-Media Thickness in Mexican Americans.

Melton PE, Carless MA, Curran JE, Dyer TD, ... Blangero J, Almasy L
Background: -Intima-media thickness (IMT) of the common and internal carotid arteries is an established surrogate for atherosclerosis and predicts risk of stroke and myocardial infarction. Often IMT is measured as the average of these two arteries, yet they are believed to result from separate biological mechanisms. The aim of this study was to conduct a family-based genome-wide association study (GWAS) for IMT to identify polymorphisms influencing IMT and to determine if distinct carotid artery segments are influenced by different genetic components. Methods and results: -IMT for the common and internal carotid arteries was determined through B-mode ultrasound in 772 Mexican Americans from the San Antonio Family Heart Study. A GWAS utilizing 931,219 single nucleotide polymorphisms (SNPs) was undertaken with six internal and common carotid artery IMT phenotypes utilizing an additive measured genotype model. The most robust association detected was for two SNPs (rs16983261, rs6113474, p=1.60e(-7)) in complete linkage disequilibrium on chromosome 20p11 for the internal carotid artery near wall, next to the gene PAX1. We also replicated previously reported GWAS regions on chromosomes 19q13 and 7q22. We found no overlapping associations between internal and common carotid artery phenotypes at p<5.0e0(-6). The genetic correlation between the two carotid IMT arterial segments was 0.51. Conclusions: -This study represents the first large scale GWAS of carotid IMT in a non-European population and identified several novel loci. We do not detect any shared GWAS signals between common and internal carotid arterial segments but the moderate genetic correlation implies both common and unique genetic components.

Circ Cardiovasc Genet: 13 Mar 2013; epub ahead of print

The Cardiac Phenotype in Patients with a CHD7 Mutation.

Corsten-Janssen N, Kerstjens-Frederikse WS, du Marchie Sarvaas GJ, Baardman ME, ... Van Ravenswaaij-Arts CM, Kapusta L
Background: -Loss-of-function mutations in CHD7 cause CHARGE syndrome, a variable combination of multiple congenital malformations including heart defects. Heart defects are reported in 66%-92% of patients with a CHD7 mutation, but most studies are small and do not provide a detailed classification of the defects. We present the first, detailed, descriptive study on the cardiac phenotype of 299 patients with a CHD7 mutation and discuss the role of CHD7 in cardiac development. Methods and results: -We have collected information on congenital heart defects in 299 patients with a pathogenic CHD7 mutation of whom 220 (74%) had a congenital heart defect. Detailed information on the heart defects was available for 202 of these patients. We classified the heart defects based on embryonic cardiac development and compared the distribution to 1,007 equally classified non-syndromic heart defects of patients registered by EUROCAT, a European Registry of Congenital Anomalies. Heart defects are highly variable in patients with CHD7 mutations, but atrioventricular septal defects (AVSD) and conotruncal heart defects are overrepresented. Gender did not have an effect on the presence of heart defects, but truncating CHD7 mutations resulted in a heart defect significantly more often than missense or splice-site mutations (chi-square, p<0.001). Conclusions: -CHD7 plays a very important role in cardiac development because we found a wide range of heart defects in 74% of a large cohort of patients with a CHD7 mutation. Conotruncal defects and AVSDs are overrepresented in patients with CHD7 mutations compared to patients with non-syndromic heart defects.

Circ Cardiovasc Genet: 15 May 2013; epub ahead of print

Prevalence and Potential Genetic Determinants of Sensorineural Deafness in KCNQ1 Homozygosity and Compound Heterozygosity.

Giudicessi JR, Ackerman MJ
Background: -Homozygous or compound heterozygous mutations in KCNQ1 cause Jervell and Lange-Nielsen syndrome (JLNS), a rare, autosomal recessive form of long QT syndrome (LQTS) characterized by deafness, marked QT prolongation, and a high risk of sudden death. However, it is not understood why some individuals with mutations on both KCNQ1 alleles present without deafness. Here, we sought to determine the prevalence and genetic determinants of this phenomenon in a large referral population of LQTS patients. Methods and results: -Retrospective analysis of all LQTS patients evaluated from July 1998 to April 2012 was used to identify those with ≥1 KCNQ1 mutation. Of the 249 KCNQ1-positive patients identified, 15 patients (6.0%) harbored a rare putative pathogenic mutation on both KCNQ1 alleles. Surprisingly, 11 (73%) of these patients presented without the sensorineural deafness associated with JLNS. The degree of QT interval prolongation and number of breakthrough cardiac events were similar between cases with and without deafness. Interestingly, truncating mutations were more prevalent in JLNS (79%) than non-deaf cases (36%, p<0.001) derived from this study and those in the literature. Conclusions: -Here, we provide evidence that the "recessive" inheritance of a severe LQT1 phenotype in the absence of an auditory phenotype may represent a more common pattern of LQTS inheritance than previously anticipated and that these cases should be treated as a higher-risk LQTS subset similar to their JLNS counterparts. Furthermore, mutation type may serve as a genetic determinant of deafness, but not cardiac expressivity, in individuals harboring ≥1 KCNQ1 mutation on each allele.

Circ Cardiovasc Genet: 07 Feb 2013; epub ahead of print

Mutations in the Sarcomere Protein Gene MYH7 in Ebstein\'s Anomaly.

Postma AV, van Engelen K, van de Meerakker J, Rahman T, ... Goodship J, Klaassen S
Background: -Ebstein\'s anomaly is a rare congenital heart malformation characterized by adherence of the septal and posterior leaflets of the tricuspid valve to the underlying myocardium. An association between Ebstein\'s anomaly with left ventricular noncompaction (LVNC) and mutations in MYH7 encoding β-myosin heavy chain has been reported; here we have screened for MYH7 mutations in a cohort of probands with Ebstein\'s anomaly in a large population-based study. Methods and results: -Mutational analysis in a cohort of 141 unrelated probands with Ebstein\'s anomaly was performed by next generation sequencing and direct DNA sequencing of MYH7. Heterozygous mutations were identified in 8 of 141 samples (6%). Seven distinct mutations were found, 5 were novel and 2 were known to cause hypertrophic cardiomyopathy (HCM). All mutations except for one 3-bp deletion were missense mutations, one was a de novo change. Mutation-positive probands and family members showed various congenital heart malformations as well as LVNC. Among 8 mutation-positive probands, 6 had LVNC, whereas among 133 mutation-negative probands, none had LVNC. The frequency of MYH7 mutations was significantly different between probands with and without LVNC accompanying Ebstein\'s anomaly (p<0.0001). LVNC segregated with the MYH7 mutation in the pedigrees of three of the probands, one of which also included another individual with Ebstein\'s anomaly. Conclusions: -Ebstein\'s anomaly is a congenital heart malformation that is associated with mutations in MYH7. MYH7 mutations are predominantly found in Ebstein\'s anomaly associated with LVNC and may warrant genetic testing and family evaluation in this subset of patients.

Circ Cardiovasc Genet: 03 Dec 2010; epub ahead of print

Genetic Loci for Coronary Calcification and Serum Lipids Relate to Aortic and Carotid Calcification.

Bos D, Ikram MA, Isaacs A, Verhaaren BF, ... van der Lugt A, Vernooij MW
Background: -Atherosclerosis in different vessel beds shares lifestyle and environmental risk factors. We investigated whether this also holds for genetic risk factors. Hence, we used genetic loci for coronary artery calcification (CAC) and serum lipid levels -one of the strongest risk factors for atherosclerosis- to assess their relationship with atherosclerosis in different vessel beds. Methods and results: -In 1,987 persons from the population-based Rotterdam Study we used three SNPs (single nucleotide polymorphisms) for CAC and 132 SNPs for total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides. To quantify atherosclerotic calcification, as marker of atherosclerosis, all participants underwent non-enhanced CT of the aortic arch and carotid arteries. We investigated associations between genetic risk scores of the joint effect of the SNPs and calcification. The joint effect of CAC-SNPs was associated with larger calcification volumes in all vessel beds [difference in calcification volume per SD increase in genetic risk score 0.15(95%CI:0.11;0.20) in aorta, 0.14(95%CI:0.10;0.18) in extracranial carotids, 0.11(95%CI:0.07;0.16) in intracranial carotids]. The joint effect of total cholesterol-SNPs, LDL-SNPs and of all lipid SNPs together was associated with larger calcification volumes in both the aortic arch and the carotid arteries, but attenuated after adjusting for the lipid fraction and lipid lowering medication. Conclusions: -The genetic basis for aortic arch and carotid artery calcification overlaps with the most important loci of coronary artery calcification. Furthermore, serum lipids share a genetic predisposition with both calcification in the aortic arch and the carotid arteries, providing novel insights into the etiology of atherosclerosis.

Circ Cardiovasc Genet: 17 Dec 2012; epub ahead of print

The Toronto HCM Genotype Score for Prediction of a Positive Genotype in Hypertrophic Cardiomyopathy.

Gruner C, Ivanov J, Care M, Williams L, ... Woo A, Rakowski H
Background: -Genotyping in hypertrophic cardiomyopathy (HCM) has gained increasing attention in the past decade. Its major role is for family screening, and only rarely influences decision-making processes in any individual patient. It is associated with substantial costs, and cost-effectiveness can only be achieved in the presence of high detection rates for disease-causing sarcomere protein gene mutations. Therefore, our aim was to develop a score based on clinical and echocardiographic variables that allows prediction of the probability of a positive genotype. Methods and results: -Clinical and echocardiographic variables were collected in 471 consecutive patients undergoing genetic testing at a tertiary referral center between July 2005 and November 2010. Logistic regression for a positive genotype was employed to construct integer risk weights for each independent predictor variable. These were summed for each patient to create the Toronto HCM genotype score. A positive genotype was found in 163/471 patients (35%). Independent predictors with associated risk weights in parentheses were as follows: age at diagnosis 20-29 (-1), 30-39 (-2), 40-49 (-3), 50-59 (-4), 60-69 (-5), 70-79 (-6), ≥80 (-7); female gender (4); arterial hypertension (-4); positive family history for HCM (6); morphology category (5); ratio of maximal wall thickness to posterior wall thickness <1.46 (0); 1.47-1.70 (1), 1.71-1.92 (2), 1.93-2.26 (3), ≥2.27 (4). The model had a receiver operator curve of 0.80 and Hosmer-Lemeshow goodness-of-fit p = 0.22. Conclusions: -The Toronto genotype score is an accurate tool to predict a positive genotype in an HCM cohort at a tertiary referral center.

Circ Cardiovasc Genet: 13 Dec 2012; epub ahead of print

The Impact of Ancestry and Common Genetic Variants on QT Interval in African Americans.

Smith JG, Avery CL, Evans DS, Nalls MA, ... Whitsel EA, Newton-Cheh C
Background: -Ethnic differences in cardiac arrhythmia incidence have been reported, with a particularly high incidence of sudden cardiac death (SCD) and low incidence of atrial fibrillation in individuals of African ancestry. We tested the hypotheses that African ancestry and common genetic variants are associated with prolonged duration of cardiac repolarization, a central pathophysiological determinant of arrhythmia, as measured by the electrocardiographic QT interval. Methods and results: -First, individual estimates of African and European ancestry were inferred from genome-wide single nucleotide polymorphism (SNP) data in seven population-based cohorts of African Americans (n=12 097) and regressed on measured QT interval from electrocardiograms. Second, imputation was performed for 2.8 million SNPs and a genome-wide association (GWA) study of QT interval performed in ten cohorts (n=13 105). There was no evidence of association between genetic ancestry and QT interval (p=0.94). Genome-wide significant associations (p<2.5x10(-8)) were identified with SNPs at two loci, upstream of the genes NOS1AP (rs12143842, p=2x10(-15)) and ATP1B1 (rs1320976, p=2x10(-10)). The most significant SNP in NOS1AP was the same as the strongest SNP previously associated with QT interval in individuals of European ancestry. Low p-values (p<10(-5)) were observed for SNPs at several other loci previously identified in GWA studies in individuals of European ancestry, including KCNQ1, KCNH2, LITAF and PLN. Conclusions: -We observed no difference in duration of cardiac repolarization with global genetic indices of African ancestry. In addition, our GWA study extends the association of polymorphisms at several loci associated with repolarization in individuals of European ancestry to include African Americans.

Circ Cardiovasc Genet: 19 Nov 2012; epub ahead of print

Sarcomere Protein Gene Mutations in Patients with Apical Hypertrophic Cardiomyopathy.

Gruner C, Care M, Siminovitch K, Moravsky G, ... Woo A, Rakowski H
Background: -Apical hypertrophic cardiomyopathy (HCM) is a unique form of HCM with left ventricular hypertrophy confined to the cardiac apex. The purpose of our study was to report genetic findings in a large series of unrelated patients with apical HCM and compare them to a non apical HCM cohort. Methods and results: -Overall, 429 patients with HCM underwent genetic testing. The panel included 8 sarcomere protein genes and 3 other genes (GLA, PRKAG2, and LAMP2). Sixty-one patients were diagnosed with apical HCM. A positive genotype was found in 8 patients with apical HCM. The genotype positive and genotype negative patients had similar maximal wall thicknesses (17.5±3.5mm versus 17.6±3.3mm, p=0.71) and similar frequency of HCM related events (2/8; 25% versus 13/53; 25%, p=0.98). Thirteen percent with apical HCM and 40% with non apical HCM had a positive genotype (p<0.001) most often involving the MYBPC3 and MYH7 genes. Conclusions: -In apical HCM a positive genotype was found less frequently than in non apical HCM, and it was most often involving MYBPC3 and MYH7 genes. Only 13% of patients with apical HCM were found to be genotype positive, indicating that genome-wide association studies and gene expression profiling are needed for better understanding of the genetic background of the disease. There was no significant genotype-phenotype correlation in our cohort with apical HCM.

Circ Cardiovasc Genet: 22 Apr 2011; epub ahead of print

A Blood Based Gene Expression Test for Obstructive Coronary Artery Disease Tested in Symptomatic Non-Diabetic Patients Referred for Myocardial Perfusion Imaging: The COMPASS Study.

Thomas GS, Voros S, McPherson JA, Lansky AJ, ... Douglas PS, Rosenberg S
Background: -Obstructive coronary artery disease (CAD) diagnosis in symptomatic patients often involves non-invasive testing before invasive coronary angiography (ICA). A blood-based gene expression score (GES) was previously validated in non-diabetic patients referred for ICA but not in symptomatic patients referred for myocardial perfusion imaging (MPI). Methods and results: -This prospective multi-center study obtained peripheral blood samples for GES before MPI in 537 consecutive patients. Patients with abnormal MPI usually underwent ICA; all others had research coronary CT-angiography (CTA), with core laboratories defining coronary anatomy. A total of 431 patients completed GES, coronary imaging (ICA or CTA), and MPI. Mean age was 56±10 (48% women). The pre-specified primary endpoint was GES receiver-operator characteristics (ROC) analysis to discriminate ≥50% stenosis (15% prevalence by core laboratory analysis). ROC curve area (AUC) for GES was 0.79 (95% CI 0.73-0.84, p<.001), with sensitivity, specificity, and negative predictive value (NPV) of 89%, 52%, and 96%, respectively, at a pre-specified threshold of ≤15 with 46% of patients below this score. The GES outperformed clinical factors by ROC and reclassification analysis and also showed significant correlation with maximum percent stenosis. Six-month follow-up on 97% of patients showed that 27/28 patients with adverse cardiovascular events or revascularization had GES >15. Site and core-lab MPI had AUCs of 0.59 and 0.63, respectively, significantly less than GES. Conclusions: -A GES has high sensitivity and NPV for obstructive CAD. In this population clinically referred for MPI, the GES outperformed clinical factors and MPI. Clinical Trial registration Information-www.clinicaltrials.gov; Identifier: NCT01117506.

Circ Cardiovasc Genet: 17 Feb 2013; epub ahead of print

The Effect of Survival Bias on Case-Control Genetic Association Studies of Highly Lethal Diseases.

Anderson CD, Nalls MA, Biffi A, Rost NS, ... Meschia JF, Rosand J
Background: -Survival bias is the phenomenon by which individuals are excluded from analysis of a trait because of mortality related to the expression of that trait. In genetic association studies, variants increasing risk for disease onset as well as risk of disease-related mortality (lethality) could be difficult to detect in genetic association case-control designs, possibly leading to underestimation of a variant\'s effect on disease risk. Methods and results: -We modeled cohorts for three diseases of high lethality (intracerebral hemorrhage, ischemic stroke, and myocardial infarction) using existing longitudinal data. Based on these models, we simulated case-control genetic association studies for genetic risk factors of varying effect sizes, lethality, and minor allele frequencies (MAF). For each disease, erosion of detected effect size was larger for case-control studies of individuals of advanced age (age > 75 years) and/or variants with very high event-associated lethality (Genotype Relative Risk for event-related death > 2.0). We found that survival bias results in no more than 20% effect size erosion for cohorts with mean age < 75 years, even for variants that double lethality risk. Furthermore, we found that increasing effect size erosion was accompanied by depletion of MAF in the case population, yielding a "signature" of the presence of survival bias. Conclusions: -Our simulation provides formulas to allow estimation of effect size erosion given a variant\'s odds-ratio (OR) of disease, OR of lethality, and MAF. These formulas will add precision to power calculation and replication efforts for case-control genetic studies. Our approach requires validation using prospective data.

Circ Cardiovasc Genet: 04 Feb 2011; epub ahead of print

Common Variants in Epithelial Sodium Channel Genes Contribute to Salt-Sensitivity of Blood Pressure: The GenSalt Study.

Zhao Q, Gu D, Hixson JE, Liu DP, ... Hamm LL, He J
Background: -Rare mutations of the epithelial sodium channel (ENaC) lead to Mendelian forms of salt-sensitive hypertension or salt-wasting hypotension. We aimed to examine the association between common variants in the ENaC genes and salt-sensitivity of blood pressure (BP). Methods and results: -A total of 1,906 Han Chinese participated in the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study, which includes a 7-day low-sodium intake (51.3 mmol sodium/day) followed by a 7-day high-sodium intake (307.8 mmol sodium/day). Nine BP measurements were obtained at baseline and each intervention period using a random-zero sphygmomanometer. Single nucleotide polymorphisms (SNPs), both tagging and functional, from the three ENaC subunits, α, β, and γ (SCNN1A, SCNN1B, and SCNN1G), were genotyped. Multiple common SNPs in SCNN1G were significantly associated with BP response to low-sodium intervention (rs4073930, p=1.7×10(-5); rs4073291, p=1.1×10(-5); rs7404408, p=1.9×10(-5); rs5735, p=3.0×10(-4); rs4299163, p=0.004; and rs4499238, p=0.002) even after correcting for multiple testing. For example, under an additive model, the minor allele G of SNP rs4073291 was associated with 1.33 mmHg lower systolic BP (SBP) reduction during low-sodium intervention. Conclusions: -This large dietary sodium intervention study indicates that common variants of ENaC subunits may contribute to the variation of BP response to dietary sodium intake. Future studies are warranted to confirm these findings in an independent population and to identify functional variants for salt-sensitivity. Clinical Trial registration Information-http://clinicaltrials.gov; Identifier: NCT00721721.

Circ Cardiovasc Genet: 12 May 2011; epub ahead of print

The S1103Y Cardiac Sodium Channel Variant Is Associated with ICD Events in African Americans with Heart Failure and Reduced Ejection Fraction.

Sun AY, Koontz JI, Shah SH, Piccini JP, ... Hranitzky PM, Pitt GS
Background: -Risk stratifying heart failure patients for primary prevention implantable cardioverter-defibrillators (ICDs) remains a challenge, especially for African Americans, who have an increased incidence of sudden cardiac death but have been underrepresented in clinical trials. We hypothesized that the S1103Y cardiac sodium channel SCN5A variant influences the propensity for ventricular arrhythmias in African American patients with heart failure and reduced ejection fraction. Methods and results: -112 African Americans with ejection fractions (EF) <35% receiving primary prevention ICDs were identified from the Duke Electrophysiology Genetic and Genomic Studies (EPGEN) biorepository and followed for appropriate ICD therapy (either antitachycardia pacing or shock) for documented sustained ventricular tachycardia or fibrillation. The S1103Y variant was over-represented in patients receiving appropriate ICD therapy compared to subjects who did not (35% vs 13%, p=0.03). Controlling for baseline characteristics, the adjusted hazard ratio using a Cox Proportional Hazard Model for ICD therapy in Y1103 allele carriers was 4.33 (95% CI 1.60-11.73, p=<0.01). There was no difference in mortality between carriers and non-carriers. Conclusions: -This is the first report that the S1103Y variant is associated with a higher incidence of ventricular arrhythmias in African Americans with heart failure and reduced ejection fraction.

Circ Cardiovasc Genet: 18 Apr 2011; epub ahead of print

Short Read (Next-gen) Sequencing: A Tutorial with Cardiomyopathy Diagnostics as an Exemplar.

Punetha J, Hoffman EP
Rapid advances in DNA sequencing technologies have made it increasingly cost effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel, or \'next generation\' [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein coding genes in the genome (~1.1% of genome) and an exome test for a single patient generates about 6 gigabases (10(9) bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care, but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ~27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation.

Circ Cardiovasc Genet: 14 Jul 2013; epub ahead of print

Putting Pleiotropy and Selection into Context Defines a New Paradigm for Interpreting Genetic Data.

Predazzi IM, Rokas A, Deinard A, Schnetz-Boutaud N, ... Sirugo G, Williams SM
Background: -Natural selection shapes many human genes, including some related to complex diseases. Understanding how selection affects genes, especially pleiotropic ones, may be important in evaluating disease associations and the role played by environmental variation. This may be of particular interest for genes with antagonistic roles that cause divergent patterns of selection. The lectin like low-density lipoprotein 1 receptor (LOX-1), encoded by OLR1, is exemplary. It has antagonistic functions in the cardiovascular and immune systems as the same protein domain binds oxidized LDL and bacterial cell wall proteins - the former contributing to atherosclerosis, the latter presumably protecting from infection. We studied patterns of selection in this gene, in humans and non-human primates, to determine whether variable selection can lead to conflicting results in CVD association studies. Methods and results: -We analyzed sequences from 11 non-human primate species as well as SNP and sequence data from multiple human populations. Results indicate that the derived allele is favored across primate lineages (probably due to recent positive selection). However, both the derived and ancestral alleles were maintained in human populations, especially European ones (possibly due to balancing selection derived from LOX-1\'s dual roles). Balancing selection likely reflects response to diverse environmental pressures among humans. Conclusions: -These data indicate that differential selection patterns, within and between species, in OLR1 render association studies difficult to replicate even if the gene is etiologically connected to CVD. Selection analyses can identify genes exhibiting gene-environment interactions critical for unraveling disease association.

Circ Cardiovasc Genet: 24 Apr 2013; epub ahead of print

Association of Genome-Wide Variation with Highly Sensitive Cardiac Troponin-T (hs-cTnT) Levels in European- and African-Americans: A Meta-Analysis from the Atherosclerosis Risk in Communities and the Cardiovascular Health Studies.

Yu B, Barbalic M, Brautbar A, Nambi V, ... Psaty BM, Boerwinkle E
Background: -High levels of cardiac troponin T measured by a highly sensitive assay (hs-cTnT) are strongly associated with incident coronary heart disease (CHD) and heart failure (HF). No large-scale genome-wide association study (GWAS) of hs-cTnT has been reported to date. We sought to identify novel genetic variants that are associated with hs-cTnT levels. Methods and results: -We performed a GWAS in 9,491 European-Americans and 2,053 African-Americans free of CHD and HF from 2 prospective cohorts: the Atherosclerosis Risk in Communities Study (ARIC) and the Cardiovascular Health Study (CHS). GWASs were conducted in each study and race stratum. Fixed-effect meta-analyses combined the results of linear regression from 2 cohorts within each race stratum, and then across race strata to produce overall estimates and p-values. The meta-analysis identified a significant association at chromosome 8q13 (rs10091374, p = 9.06×10(-9)) near the nuclear receptor coactivator 2 (NCOA2) gene. Over-expression of NCOA2 can be detected in myoblasts. An additional analysis using logistic regression and the clinically motivated 99(th) percentile cut-point detected a significant association at 1q32 (rs12564445, p = 4.73×10(-8)) in the gene TNNT2, which encodes the cardiac troponin T protein itself. The hs-cTnT-associated SNPs were not associated with CHD in a large case-control study, but rs12564445 was significantly associated with incident HF in ARIC European-Americans (HR = 1.16, p-value = 0.004). Conclusions: -We identified 2 loci, near NCOA2 and in the TNNT2 gene, at which variation was significantly associated with hs-cTnT levels. Further use of the new assay should enable replication of these results.

Circ Cardiovasc Genet: 17 Dec 2012; epub ahead of print

Differential MicroRNA Expression Profiles in Peripheral Arterial Disease.

Stather PW, Sylvius N, Wild JB, Choke E, Sayers RD, Bown MJ
Background- Peripheral arterial disease (PAD) is a clinical condition caused by an atherosclerotic process affecting the arteries of the limbs. Despite major improvements in surgical endovascular techniques, PAD is still associated with high mortality and morbidity. Recently, microRNAs (miRNAs), a class of short noncoding RNA controlling gene expression, have emerged as major regulators of multiple biological processes. Methods and results- A whole-miRNA transcriptome profiling was performed in peripheral blood from an initial sample set of patients and controls. A 12-miRNA PAD-specific signature, which includes let 7e, miR-15b, -16, -20b, -25, -26b, -27b, -28-5p, -126, -195, -335, and -363, was further investigated and validated in 2 additional sample sets. Each of these 12 miRNAs exhibited good diagnostic value as evidenced by receiver operating characteristic curve analyses. Pathway enrichment analysis using predicted and validated targets identified several signaling pathways relevant to vascular disorders. Several of these pathways, including cell adhesion molecules, were confirmed by quantifying the expression level of several candidate genes regulating the initial stages of the inflammatory atherosclerotic process. The expression level of 7 of these candidate genes exhibits striking inverse correlation with that of several, if not all, of the miRNAs of the PAD-specific miRNA signature. Conclusions- These results demonstrate the potential of miRNAs for the diagnosis of PAD and provide further insight into the molecular mechanisms leading to the development of PAD, with the potential for future therapeutic targets.

Circ Cardiovasc Genet: 15 Oct 2013; 6:490-7

Novel Loci Associated with PR Interval in a Genome-Wide Association Study of Ten African American Cohorts.

Butler AM, Yin X, Evans DS, Nalls MA, ... Magnani JW, Avery CL
Background: -The PR interval (PR) as measured by the resting, standard 12-lead electrocardiogram (ECG) reflects the duration of atrial/atrioventricular nodal depolarization. Substantial evidence exists for a genetic contribution to PR, including genome-wide association studies that have identified common genetic variants at nine loci influencing PR in populations of European and Asian descent. However, few studies have examined loci associated with PR in African Americans. Methods and results: -We present results from the largest genome-wide association study to date of PR in 13,415 adults of African descent from ten cohorts. We tested for association between PR (ms) and approximately 2.8 million genotyped and imputed single nucleotide polymorphisms. Imputation was performed using HapMap 2 YRI and CEU panels. Study-specific results, adjusted for global ancestry and clinical correlates of PR, were meta-analyzed using the inverse variance method. Variation in genome-wide test statistic distributions was noted within studies (lambda range: 0.9-1.1), although not after genomic control correction was applied to the overall meta-analysis (lambda: 1.008). In addition to generalizing previously reported associations with MEIS1, SCN5A, ARHGAP24, CAV1, and TBX5 to African American populations at the genome-wide significance level (P<5.0x10(-8)), we also identified a novel locus: ITGA9, located in a region previously implicated in SCN5A expression. The 3p21 region harboring SCN5A also contained two additional independent secondary signals influencing PR (P<5.0x10(-8)). Conclusions: -This study demonstrates the ability to map novel loci in African Americans as well as the generalizability of loci associated with PR across populations of African, European and Asian descent.

Circ Cardiovasc Genet: 08 Nov 2012; epub ahead of print

Genome-Wide Association Study Identifies Novel Loci Associated With Concentrations of Four Plasma Phospholipid Fatty Acids in the De Novo Lipogenesis Pathway: Results from the CHARGE Consortium.

Wu JH, Lemaitre RN, Manichaikul A, Guan W, ... Fornage M, Mozaffarian D
Background: -Palmitic acid(16:0), stearic acid(18:0), palmitoleic acid(16:1n-7), and oleic acid(18:1n-9) are major saturated and mono-unsaturated fatty acids that affect cellular signaling and metabolic pathways. They are synthesized via de novo lipogenesis (DNL) and are the main saturated and mono-unsaturated fatty acids in the diet. Levels of these fatty acids have been linked to diseases including type 2 diabetes and coronary heart disease. Methods and results: -Genome-wide association studies were conducted in 5 population-based cohorts comprising 8,961 participants of European ancestry to investigate the association of common genetic variation with plasma levels of these four fatty acids. We identified polymorphisms in 7 novel loci associated with circulating levels of one or more of these fatty acids. ALG14 (asparagine-linked glycosylation 14 homolog) polymorphisms were associated with higher 16:0(P=2.7x10(-11)) and lower 18:0(P=2.2x10(-18)). FADS1 and FADS2 (desaturases) polymorphisms were associated with higher 16:1n-7(P=6.6x10(-13)) and 18:1n-9(P=2.2x10(-32)), and lower 18:0(P =1.3x10(-20)). LPGAT1 (lysophosphatidylglycerol acyltransferase) polymorphisms were associated with lower 18:0(P=2.8x10(-9)). GCKR (glucokinase regulator, P=9.8x10(-10)) and HIF1AN (factor inhibiting hypoxia-inducible factor-1, P=5.7x10(-9)) polymorphisms were associated with higher 16:1n-7, whereas PKD2L1 (polycystic kidney disease 2-like 1, P=5.7x10(-15)) and a locus on chromosome 2(not near known genes) were associated with lower 16:1n-7(P=4.1x10(-8)). Conclusions: -Our findings provide novel evidence that common variations in genes with diverse functions, including protein-glycosylation, polyunsaturated fatty acid metabolism, phospholipid modeling, and glucose- and oxygen-sensing pathways, are associated with circulating levels of four fatty acids in the DNL pathway. These results expand our knowledge of genetic factors relevant to DNL and fatty acid biology.

Circ Cardiovasc Genet: 29 Jan 2013; epub ahead of print

The Impact of Partial and Complete Loss of Function Mutations in Endothelial Lipase on HDL Levels and Functionality in Humans.

Singaraja RR, Sivapalaratnam S, Hovingh K, Dubé MP, ... Kastelein JJ, Hayden MR
Background: -Endothelial lipase is a phospholipase with activity against high density lipoprotein. Although a small number of mutations in LIPG have been described, the role of LIPG in protection against atherosclerosis is unclear. Methods and results: -We identified eight loss-of-function (LOF) mutations in LIPG in individuals with high HDL-C. Functional analysis confirmed that most rare mutations abolish lipase activity in vitro, indicating complete LOF (CLOF), while two more common mutations N396S and R476W reduce activity by ~50%, indicating partial LOF (PLOF), and implying ~50% and ~75% remaining EL function in heterozygous CLOF and PLOF mutation carriers respectively. CLOF mutation carriers had significantly higher plasma HDL-C levels compared to PLOF mutation carriers. Apo-B depleted serum from CLOF carriers showed significantly enhanced cholesterol efflux acceptor capacity, whereas only trends were observed in PLOF carriers. Carriers of LIPG mutations exhibited trends toward reduced CAD in four independent cohorts (meta-analysis OR=0.7, p=0.04). Conclusions: -Our data suggest that the impact of LIPG mutations is directly related to their effect on EL function, and support that antagonism of EL function improves cardioprotection.

Circ Cardiovasc Genet: 16 Dec 2012; epub ahead of print

Proteomic Analysis Yields an Unexpected Trans-Acting Point in Control of the Human Sympathochromaffin Phenotype.

Chiron S, Wei Z, Chen Y, Zhang K, ... Mahata SK, O\'Connor DT
Background: -The secretory protein chromogranin A (CHGA) plays a necessary role in formation of catecholamine storage vesicles and also gives rise to a catecholamine release-inhibitory fragment. Since genetic variation in the proximal human CHGA promoter predicts autonomic function and blood pressure, here we explored how a common genetic variant alters transcription of the gene. Methods and results: -Bioinformatic analysis suggested that the common G-462A promoter variant (rs9658634) may disrupt as many as 3 transcriptional control motifs: LEF1, COUP-TF, and PPARγ-RXRα. During electrophoretic mobility shifts, chromaffin cell nuclear proteins bound specifically to the A (though not G) allele of CHGA promoter G-462A. Upon oligonucleotide affinity chromatography followed by LC-MS/MS analysis of A-allele eluates, the transcription factor LEF1 (Lymphoid Enhancer-binding Factor-1) was identified. Interaction of LEF1 with the A-allele at G-462A was confirmed by super-shift. Upon co-transfection, LEF1 discriminated between the allelic variants, especially in chromaffin cells. Allele specificity of trans-activation by LEF1 was transferable to an isolated G-462A element fused to a heterologous (SV40) promoter. Since beta-catenin (CTNNB1) can hetero-dimerize with LEF1, we tested the effect of co-transfection of this factor, and again found A-allele-specific perturbation of CHGA transcription. Conclusions: -Common genetic variation within the human CHGA promoter alters the interaction of specific factors in trans with the promoter, with LEF1 identified by proteomic analysis and confirmed by super-shift. Co-expression experiments show functional effects of LEF1 and CTNNB1 on CHGA promoter. The findings document a novel role for components of the immune and WNT pathways in control of human sympathochromaffin phenotypes.

Circ Cardiovasc Genet: 09 May 2011; epub ahead of print

Growth-Differentiation Factor-15 for Long-Term Risk Prediction in Patients Stabilized After an Episode of Non-ST-Segment-Elevation Acute Coronary Syndrome.

Eggers KM, Kempf T, Lagerqvist B, Lindahl B, ... Wollert KC, Wallentin L
Background: Growth-differentiation factor-15 (GDF-15) has emerged as a prognostic biomarker in patients with non-ST-segment-elevation acute coronary syndrome. This study assessed the time course and the long-term prognostic relevance of GDF-15 levels measured repetitively in patients with non-ST-segment-elevation acute coronary syndrome during 6 months after the acute event. Methods and results: GDF-15 and other biomarkers were measured at randomization, after 6 weeks, and after 3 and 6 months in 950 patients with non-ST-segment-elevation acute coronary syndrome included in the FRagmin and Fast Revascularization during InStability in Coronary artery disease II study. Study end points were death, recurrent myocardial infarction, and their composite during 5-year follow-up. Median GDF-15 levels decreased slightly from 1357 ng/L at randomization to 1302 ng/L at 6 months (P<0.001). GDF-15 was consistently related to cardiovascular risk factors and biochemical markers of hemodynamic stress, renal dysfunction, and inflammation. Moreover, GDF-15 was independently related to the 5-year risk of the composite end point when measured at both 3 months (adjusted hazard ratio, 1.8 [1.0 to 3.0]) and 6 months (adjusted hazard ratio, 2.3 [1.3 to 4.1]). Serial measurements of GDF-15 at randomization and 6 months helped to identify patient cohorts at different levels of risk, with patients with persistently elevated GDF-15 levels >1800 ng/L having the highest rate of the composite end point. Conclusions: GDF-15 is independently related to adverse events in non-ST-segment-elevation acute coronary syndrome both in the acute setting and for at least 6 months after clinical stabilization. Therefore, continued research on GDF-15 should be focused on the usefulness of GDF-15 for support of clinical management in acute and chronic ischemic heart disease.

Circ Cardiovasc Genet: 17 Feb 2010; 3:88-96

Sarcomere Gene Mutations in Isolated Left Ventricular Noncompaction Cardiomyopathy Do Not Predict Clinical Phenotype.

Probst S, Oechslin E, Schuler P, Greutmann M, ... Jenni R, Klaassen S
Background: -Left ventricular noncompaction of the myocardium (LVNC) has been recognized as a cardiomyopathy with a genetic etiology. Mutations in genes encoding sarcomere proteins were shown to be associated with LVNC. We have evaluated the potential clinical impact of genetic analysis of sarcomere genes in patients with LVNC. Methods and results: -In this report, we identified 5 mutations in cardiac myosin-binding protein C (MYBPC3) and 2 mutations in α-tropomyosin (TPM1) in a cohort of unrelated adult probands with isolated LVNC. The mutations in MYBPC3 and TPM1 and in 6 other previously reported sarcomere genes in this cohort resulted in a total of 18 heterozygous mutations in 63 probands (29%). β-myosin heavy chain (MYH7) was the most prevalent disease gene and accounts for 13% of cases followed by MYBPC3 (8%). Comparing sarcomere mutation-positive and mutation-negative LVNC probands showed no significant differences in terms of average age, myocardial function, presence of heart failure or tachyarrhythmias at initial presentation or at follow-up. Familial disease was found in 16 probands of which 8 were sarcomere mutation-positive. Non-penetrance was detected in 2 of 8 mutation-positive families with LVNC. Conclusions: -Mutations in sarcomere genes account for a significant proportion of cases of isolated LVNC in this cohort (29%). The distribution of disease genes confirms genetic heterogeneity and opens new perspectives in genetic testing in patients with LVNC and their relatives at high risk of inheriting the cardiomyopathy. The presence or absence of a sarcomere gene mutation in LVNC cannot be related to the clinical phenotype.

Circ Cardiovasc Genet: 09 May 2011; epub ahead of print

Genomic Variation Associated with Mortality among Adults of European and African Ancestry with Heart Failure: The CHARGE Consortium.

Morrison AC, Felix JF, Cupples LA, Glazer NL, ... Vasan RS, Smith NL
Background: -Prognosis and survival are significant concerns for individuals with heart failure (HF). In order to better understand the pathophysiology of HF prognosis, the association between 2,366,858 single nucleotide polymorphisms (SNPs) and all-cause mortality was evaluated among individuals with incident HF from four community-based prospective cohorts: the Atherosclerosis Risk in Communities Study, the Cardiovascular Health Study, the Framingham Heart Study, and the Rotterdam Study. Methods and results: -Participants were 2,526 individuals of European ancestry and 466 individuals of African ancestry who suffered an incident HF event during follow-up in the respective cohorts. Within each study, the association between genetic variants and time to mortality among individuals with HF was assessed by Cox proportional hazards models that included adjustment for sex and age at the time of the HF event. Prospective fixed-effect meta-analyses were conducted for the four study populations of European ancestry (N=1,645 deaths) and for the two populations of African ancestry (N=281 deaths). Genome-wide significance was set at P=5.0x10(-7). Meta-analytic findings among individuals of European ancestry revealed one genome-wide significant locus on chromosome 3p22 in an intron of CKLF-like MARVEL transmembrane domain containing 7 (CMTM7, p = 3.2x10(-7)). Eight additional loci in individuals of European ancestry and four loci in individuals of African ancestry were identified by high-signal SNPs (p < 1.0x10(-5)), but did not meet genome-wide significance. Conclusions: -This study identified a novel locus associated with all-cause mortality among individuals of European ancestry with HF. This finding warrants additional investigation, including replication, in other studies of HF.

Circ Cardiovasc Genet: 19 Apr 2010; epub ahead of print

TGFβRIIb Mutations Trigger Aortic Aneurysm Pathogenesis by Altering TGFβ2 Signal Transduction.

Bee KJ, Wilkes DC, Devereux RB, Basson CT, Hatcher CJ
Background: -Thoracic aortic aneurysm (TAA) is a common progressive disorder involving gradual dilation of the ascending and/or descending thoracic aorta that eventually leads to dissection or rupture. Nonsydromic TAA can occur as a genetically triggered, familial disorder that is usually transmitted in a monogenic autosomal dominant fashion and is known as familial TAA (FTAA). Genetic analyses of families affected with TAA have identified several chromosomal loci and further mapping of FTAA genes has highlighted disease-causing mutations in at least four genes: myosin heavy chain 11 (MYH11), a-smooth muscle actin (ACTA2), transforming growth factor beta receptors I and II (TGFβRI and TGFβRII). Methods and results: -We evaluated 100 probands to determine the mutation frequency in MYH11, ACTA2, TGFbRI and TGFbRII in an unbiased population of individuals with genetically mediated TAA. In this study, 9% of patients had a mutation in one of the genes analyzed. 3% of patients had mutations in ACTA2, 3% in MYH11, 1% in TGFβRII and no mutations were found in TGFβRI. Additionally, we identified mutations in a 75 base pair alternatively spliced TGFbRII exon, exon 1a that produces the TGFβRIIb isoform and accounted for 2% of patients with mutations. Our in vitro analyses indicate that the TGFβRIIb activating mutations alter receptor function upon TGFb2 signaling. Conclusions: -We propose that TGFbRIIb expression is a regulatory mechanism for TGFb2 signal transduction. Dysregulation of the TGFb2 signaling pathway, as a consequence of TGFbRIIb mutations, results in aortic aneurysm pathogenesis.

Circ Cardiovasc Genet: 25 Oct 2012; epub ahead of print

Familial History of Stroke Is Associated with Acute Coronary Syndromes in Women.

Banerjee A, Lim CC, Silver LE, Welch SJ, Banning AP, Rothwell PM
Background: -Stroke in female first-degree relatives (FDRs) is a powerful risk factor for ischemic stroke in women, but its association with acute coronary syndromes (ACS) is unknown. Family history (FH) of stroke is omitted from existing myocardial infarction risk prediction tools, which perform less well in women than in men. Our objective was to study the sex-of-parent and sex-of-proband interactions for FH of stroke in ACS patients. Methods and results: -In a prospective, population-based study (Oxford Vascular Study) of all patients with ACS or stroke/transient ischemic attack, FH data for stroke and myocardial infarction were analyzed by sex of proband and FDRs, and coronary angiograms were reviewed, where available; 942 of 1058 ACS probands and 1015 of 1152 stroke/transient ischemic attack probands had complete FH data; 24.1% of ACS probands and 24.3% of stroke/transient ischemic attack probands had history of stroke in ≥1 FDR. Maternal stroke was more common than paternal stroke in female ACS probands (odds ration [OR], 2.53; 1.39 to 4.61) but not in male probands (OR, 0.92; 0.64 to 1.32) (difference-P = 0.004). Overall, female ACS probands were more likely to have female than male FDRs with stroke (OR, 2.09; 1.29 to 3.37), whereas the opposite trend was seen in male ACS probands (OR, 0.69; 0.50 to 0.97) (difference-P = 0.0002). However, there was no association between parental history of stroke and disease localization or presence of multivessel disease on coronary angiography. Conclusions: -FH of stroke is as common in ACS patients as in stroke/transient ischemic attack patients and sex-of-parent/sex-of-proband interactions are similar. Stroke in female FDRs may help to identify women at increased risk of ACS as well as ischemic stroke.

Circ Cardiovasc Genet: 03 Feb 2011; epub ahead of print

Genome-Wide Association Study Evaluating Lp-PLA2 Mass and Activity at Baseline and Following Rosuvastatin Therapy.

Chu AY, Guilianini F, Grallert H, Dupuis J, ... Chasman DI, Ridker PM
Background: -Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a pro-inflammatory enzyme bound to LDL-C and other circulating lipoproteins. Two measures of Lp-PLA(2), mass and activity, are associated with increased cardiovascular risk. Data are sparse regarding genetic determinants of Lp-PLA(2) mass and activity, and no prior data are available addressing genetic determinants of statin-induced changes for this pro-inflammatory biomarker. Methods and results: -We performed a genome-wide association study of Lp-PLA(2) mass and activity at baseline and after 12 months of rosuvastatin therapy (20 mg/day) among 6,851 participants of European ancestry from the JUPITER trial, and performed replication in a meta-analysis of 13,664 participants from the CHARGE Consortium. Novel associations were identified and replicated at MS4A4E and TMEM49 for baseline Lp-PLA(2) activity with genome-wide significant joint p-values (P=2.0x10(-11) and P=2.9x10(-9), respectively). Additionally, genome-wide associations (P<5x10(-8)) were identified and replicated for baseline Lp-PLA(2) mass at CETP, and for Lp-PLA(2) activity at the APOC1-APOE and PLA2G7 loci. Among 2,673 statin-allocated participants Lp-PLA(2) mass and activity were both reduced by more than 30% and LDL-C by 50% after 12 months of statin therapy (P<0.001 for both). Variants in ABCG2 and LPA were associated with change in statin-induced Lp-PLA(2) activity at genome-wide significance, but were substantially attenuated after adjustment for statin-induced changes in lipid levels. Conclusions: -Genome-wide significant associations at MS4A4E and TMEM49 may reflect novel influences on circulating levels of Lp-PLA(2) activity. Additionally, genome-wide significant associations with rosuvastatin-induced change in Lp-PLA(2) activity were observed in ABCG2 and LPA, likely due to their impact on statin-induced LDL-C lowering.

Circ Cardiovasc Genet: 01 Nov 2012; epub ahead of print

High Resolution Identity by Descent Mapping Uncovers the Genetic Basis for Blood Pressure Differences Between SHR Lines.

Bell R, Herring SM, Gokul N, Monita M, ... Boerwinkle E, Doris PA
Background: -The recent development of a large panel of genome-wide single nucleotide polymorphisms (SNPs) provides the opportunity to examine genetic relationships between distinct SHR lines that share hypertension, but differ in their susceptibility to hypertensive end-organ disease. Methods and results: -We compared genotypes at nearly 10,000 SNPs obtained for the hypertension end-organ injury-susceptible SHR-A3 (SHRSP, SHR-stroke prone) line and the injury-resistant SHR-B2 line. This revealed that that the two lines were genetically identical by descent (IBD) across 86.6% of the genome. Areas of the genome that were not IBD were distributed across 19 of the 20 autosomes and the X chromosome. A block structure of non-IBD comprising a total of 121 haplotype blocks was formed by clustering of SNPs inherited from different ancestors. To test the null hypothesis that distinct SHR lines share a common set of hypertension susceptibility alleles we compared blood pressure in adult SHR animals from both lines and their F1 and F2 progeny using telemetry. In 16-18wk old animals fed a normal diet, systolic blood pressure (SBP, mm Hg) in SHR-A3 was 205.7 ± 3.86 (mean ± SEM, n = 26), while in similar SHR-B2 animals SBP was 186.7 ± 2.53 (n = 20). In F1 and F2 animals, SBP was 188.2 ± 4.23, (n = 19) and 185.6 ± 1.1 (n = 211) respectively (p<10(-6), ANOVA). In order to identify non-IBD haplotype blocks contributing to blood pressure differences between these SHR lines we developed a high throughput SNP genotyping system to genotype SNPs marking non-IBD blocks. We mapped a single non-IBD block on chromosome 17 extending over less than 10Mb at which SHR-A3 alleles significantly elevate blood pressure compared with SHR-B2. Conclusions: -Thus hypertension in SHR-A3 and -B2 appears to arise from an overlapping set of susceptibility alleles, with SHR-A3 possessing an additional hypertension locus that contributes to further increase blood pressure.

Circ Cardiovasc Genet: 16 Mar 2011; epub ahead of print

Clinical and Genetic Correlates of Circulating Angiopoietin-2 and Soluble Tie-2 in the Community.

Lieb W, Zachariah J, Xanthakis V, Safa R, ... Sawyer DB, Vasan RS
Background: -Experimental studies suggest that endothelial growth factors play an important role in angiogenesis and vascular remodeling. The clinical and genetic correlates of circulating angiopoietin-2 (Ang-2) and its soluble receptor/regulator Tie-2 (sTie-2) have not been determined in a community-based sample. Methods and results: -Serum Ang-2 and sTie-2 were assayed in 3778 Framingham Third Generation cohort participants (mean age 40+/-9 years, 53% women). Clinical correlates and heritability of both biomarkers were assessed using generalized estimating equations and variance-component analyses. Ang-2 levels were higher and sTie-2 levels lower in women as compared to men. Ang-2 was positively related to age, smoking, systolic blood pressure (BP), hypertension treatment and diabetes (p<0.05 for all) but was inversely associated with total cholesterol and diastolic BP (p<0.0001 for both). Soluble Tie-2 was positively associated with body mass index, diabetes and triglycerides, but inversely related to age, alcohol consumption and glomerular filtration rate (p<0.05 for all). Both Ang-2 and sTie-2 were higher in participants with metabolic syndrome (p<0.005), with stronger associations of Ang-2 with BP traits and of sTie-2 with obesity-dyslipidemia components. Heritability estimates for Ang-2 and sTie-2 were 27% and 56%, respectively (p<0.0001). A region on chromosome 9 was significantly linked to circulating sTie-2 levels (LOD score 8.31). Conclusions: -Circulating levels of Ang-2 and sTie-2 are heritable traits that are associated with cardiovascular (CVD) risk factors including the metabolic syndrome. These observations are consistent with the notion that angiogenesis and vascular remodeling are in part determined by genetic influences, and associated with metabolic risk factors.

Circ Cardiovasc Genet: 29 Mar 2010; epub ahead of print

Functional Characterization of a Novel Mutation in NKX2-5 Associated with Congenital Heart Disease and Adult-Onset Cardiomyopathy.

Costa MW, Guo G, Wolstein O, Vale M, ... Fatkin D, Harvey RP
Background: -The transcription factor NKX2-5 is crucial for heart development and mutations in this gene have been implicated in diverse congenital heart diseases (CHD) and conduction defects (CD) in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown. Methods and results: -Mutation screening was performed in 220 probands with adult-onset dilated cardiomypathy (DCM). Six NKX2-5 coding sequence variants were identified, including 3 non-synonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain (HD), was identified in one family. A subset of family members had CHD, but there was an unexpectedly high prevalence of DCM. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes, due to reduced degradation by the ubiquitin-proteasome system (UPS). In functional assays, DNA binding activity of I184M was reduced, resulting in impaired activation of target genes, despite increased expression levels of mutant protein. Conclusions: -Certain NKX2-5 HD mutations show abnormal protein degradation via the UPS and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function, and contribute to NKX2-5-related cardiomyopathies with graded severity.

Circ Cardiovasc Genet: 09 May 2013; epub ahead of print

A Novel Strategy Using Cardiac Sodium Channel Polymorphic Fragments to Rescue Trafficking Deficient SCN5A Mutations.

Shinlapawittayatorn K, Dudash LA, Du XX, Heller L, ... Ficker E, Deschênes I
Background: -Brugada Syndrome (BrS) is associated with mutations in the cardiac sodium channel (Na(v)1.5). We previously reported that the function of a trafficking-deficient BrS Na(v)1.5 mutation, R282H, could be restored by co-expression with the sodium channel polymorphism H558R. Here, we tested the hypothesis that peptide fragments from Na(v)1.5, spanning the H558R-polymorphism, can be used to restore trafficking of trafficking-deficient BrS sodium channel mutations. Methods and results: -Whole-cell patch-clamping revealed that co-transfection in HEK293 cells of the R282H channel with either the 40 or 20 amino acid cDNA fragments of Na(v)1.5 containing the H558R polymorphism restored trafficking of this mutant channel. Fluorescence Resonance Energy Transfer (FRET) suggested that the trafficking-deficient R282H channel was misfolded and this was corrected upon co-expression with R558-containing peptides which restored trafficking of the R282H channel. Importantly, we also expressed the peptide spanning the H558R-polymorphism with 8 additional BrS Na(v)1.5 mutations with reduced currents, and demonstrated that the peptide was able to restore significant sodium currents in 4 of them. Conclusions: -In the present study, we demonstrated that small peptides, spanning the H558R-polymorphism, are sufficient to restore trafficking defect of BrS-associated Na(v)1.5 mutations. Our findings suggest that it might be possible to use short-cDNA constructs as a novel strategy that is tailored to specific disease-causing mutants of BrS.

Circ Cardiovasc Genet: 15 Aug 2011; epub ahead of print

Gene-Centric Analysis Identifies Variants Associated with Interleukin-6 Levels and Shared Pathways with Other Inflammation Markers.

Shah T, Zabaneh D, Gaunt T, Swerdlow DI, ... Hingorani AD, Casas JP
Background: -The inflammatory cytokine interleukin-6 (IL-6), a possible risk factor for coronary heart disease, has an estimated heritability of over 60%, but to date few genetic variants influencing IL-6 levels are known. Methods and results: -We used the IBC HumanCVD BeadChip in the Whitehall II (WHII, N=4,911) and British Women\'s Heart and Health Studies (BWHHS, N=3,445) to identify single nucleotide polymorphisms (SNPs) associated with circulating IL-6 levels. Twenty-two SNPs from seven loci (IL6R/TDRD10, HLA-DRB1, BUD13, SEZ6L, IL1RN, TRIB3 and ABO) were associated with IL-6 (p<10(-5)), though none were in the IL6 gene itself. With the exception of TRIB3, all loci have been previously reported in genome-wide association studies for autoimmune and cardiovascular diseases. Fourteen SNPs in the IL6R region in high linkage disequilibrium (r(2)>0.9) with a non-synonymous variant, rs2228145, were also associated with IL-6 and CRP concentration (p<10(-5)). An IL-6 specific weighted allele score explained 2% of the variance of log IL-6 levels (p = 2.44 x 10(-22)) in WHII and 1% (p = 1.9 x 10(-8)) in BWHHS. Conclusions: -Multiple common genetic variants of modest effect influence IL-6 concentration. Several loci contain SNPs exhibiting overlapping associations with autoimmune and cardiovascular disorders and other circulating biomarkers. Genetic variants associated with IL-6 provide important tools for probing the causal relevance of IL-6 signalling in a range of cardiometabolic diseases.

Circ Cardiovasc Genet: 17 Mar 2013; epub ahead of print

Towards Breaking the Histone Code - Bayesian Graphical Models for Histone Modifications.

Mitra R, Müller P, Liang S, Xu Y, Ji Y
Background: -Histones are proteins that wrap DNA around in small spherical structures called nucleosomes. Histone modifications (HMs) refer to the post-translational modifications to the histone tails. At a particular genomic locus, each of these HMs can either be present or absent, and the combinatory patterns of the presence or absence of multiple HMs, or the \'histone codes\', are believed to co-regulate important biological processes. We aim to use raw data on HM markers at different genomic loci to (1) decode the complex biological network of HMs in a single region and (2) demonstrate how the HM networks differ in different regulatory regions. We suggest that these differences in network attributes form a significant link between histones and genomic functions. Methods and results: -We develop a powerful graphical model under Bayesian paradigm. Posterior inference is fully probabilistic, allowing us to compute the probabilities of distinct dependence patterns of the HMs using graphs. Furthermore, our model-based framework allows for easy but important extensions for inference on differential networks under various conditions, such as the different annotations of the genomic locations (e.g., promoters versus insulators). We applied these models to ChIP-Seq data based on CD4+ T lymphocytes. The results confirmed many existing findings and provided a unified tool to generate various promising hypotheses. Differential network analyses revealed new insights on co-regulation of HMs of transcriptional activities in different genomic regions. Conclusions: -The use of Bayesian graphical models and borrowing strength across different conditions provide high power to infer histone networks and their differences.

Circ Cardiovasc Genet: 09 Jun 2013; epub ahead of print

Comparison of Echocardiographic and Cardiac Magnetic Resonance Imaging in Hypertrophic Cardiomyopathy Sarcomere Mutation Carriers without Left Ventricular Hypertrophy.

Valente AM, Lakdawala NK, Powell AJ, Evans SP, ... Colan SD, Ho CY
Background: -Left ventricular hypertrophy (LVH) typically manifests during or after adolescence in sarcomere mutation carriers at risk for developing hypertrophic cardiomyopathy (HCM). Guidelines recommend serial imaging of mutation carriers without LVH (G+/LVH-) to monitor for phenotypic evolution, but the optimal strategy is undefined. Compared with echocardiography (echo), cardiac magnetic resonance imaging (CMR) offers improved endocardial visualization and potential to assess scar. However the incremental advantage offered by CMR for early diagnosis of HCM is unclear. Therefore, we systematically compared echo and CMR in G+/LVH- subjects. Methods and results: -Forty sarcomere mutation carriers with normal echo wall thickness (< 12 mm or z-score < 2.5 in children) underwent concurrent CMR. Mean age was 21.7 ± 11.1 years, 55% were female). If LV wall thickness appeared non-uniform, the size and location of relatively thickened segments were noted. Late gadolinium enhancement (LGE) was assessed with CMR. Diagnostic agreement between echo and CMR was good (90%), although CMR measurements of LV wall thickness were ~19% lower than echo. Four subjects had mild hypertrophy (12.6-14 mm, ≤2 segments) appreciated by CMR but not echo. No subjects had LGE. During median 35-month follow up, 2 subjects developed overt HCM, including 1 with mild LVH by CMR at baseline. Conclusions: -Echo is unlikely to miss substantial LVH; however CMR identified mild hypertrophy in ~10% of mutation carriers with normal echo wall thickness. CMR may be a useful adjunct in HCM family screening, particularly in higher risk situations, or if echocardiographic images are suboptimal or suggest borderline LVH.

Circ Cardiovasc Genet: 20 May 2013; epub ahead of print

Identification of the BCAR1-CFDP1-TMEM170A Locus as a Determinant of Carotid Intima-Media Thickness and Coronary Artery Disease Risk.

Gertow K, Sennblad B, Strawbridge RJ, Ohrvik J, ... Humphries SE, Hamsten A
Background: -Carotid intima-media thickness (cIMT) is a widely accepted marker of subclinical atherosclerosis. To date, large-scale investigations of genetic determinants of cIMT are sparse. Methods and results: -In order to identify cIMT-associated genes and genetic variants, a discovery analysis using the Illumina 200K CardioMetabochip was conducted in 3,430 subjects with detailed ultrasonographic determinations of cIMT from the IMPROVE study. Segment-specific IMT measurements of common carotid (CC), bifurcation, and internal carotid arteries, and composite IMT variables considering the whole carotid tree (IMT(mean), IMT(max), and IMT(mean-max)), were analysed. A replication stage investigating 42 single nucleotide polymorphisms (SNPs) for association with CC-IMT was undertaken in five independent European cohorts (total n=11,590). A locus on chromosome 16 (lead SNP rs4888378, intronic in CFDP1) was associated with cIMT at significance levels passing multiple-testing correction at both stages (array-wide significant discovery P=6.75x10(-7) for IMT(max); replication P=7.24x10(-6) for CC-IMT; adjustments for sex, age and population substructure where applicable; minor allele frequency 0.43 and 0.41, respectively). The protective minor allele was associated with lower carotid plaque score in a replication cohort (P=0.04, n=2120), and lower coronary artery disease (CAD) risk in two case-control studies of subjects with European ancestry (odds ratio [95%CI] 0.83 [0.77-0.90], P=6.53x10(-6); n=13,591, and 0.95 [0.92-0.98], P=1.83x10(-4), n=82,297, respectively). Queries of human biobank datasets (n=126-138) revealed associations of rs4888378 with nearby gene expression in vascular tissues. Conclusions: -This study identified rs4888378 in the BCAR1-CFDP1-TMEM170A locus as a novel genetic determinant of cIMT and CAD risk in individuals of European descent.

Circ Cardiovasc Genet: 14 Nov 2012; epub ahead of print

Genetic Determinants of Major Blood Lipids in Pakistan Is Compared with Europeans.

Saleheen D, Soranzo N, Rasheed A, Scharnagl H, ... Danesh J, Deloukas P
Background: -Evidence is sparse about the genetic determinants of major lipids in Pakistanis. Methods and results: -45,000 variants across 2000 genes were assessed in 3200 Pakistanis, and compared with 2450 Germans using the same gene array and similar lipid assays. We also did a meta-analysis of selected lipid-related variants in Europeans. Pakistani genetic architecture was distinct from that of several ethnic groups represented in international reference samples. 41 variants at 14 loci were significantly associated with levels of HDL-C, triglyceride or LDL-C. The most significant lipid-related variants identified among Pakistanis corresponded to genes previously shown to be relevant to Europeans, such as CETP associated with HDL-C levels (rs711752; P<10(-13)); APOA5/ZNF259 (rs651821; P<10(-13)) and GCKR (rs1260326; P<10(-13)) with triglyceride levels; and CELSR2 variants with LDL-C levels (rs646776; P<10(-9)). For Pakistanis, these 41 variants explained 6.2%, 7.1%, and 0.9% of the variation in HDL-C, triglyceride, and LDL-C, respectively. Compared with Europeans, the allele frequency of rs662799 in APOA5 among Pakistanis was higher and its impact on triglyceride concentration was greater (P<10(-4)). Conclusions: -Several lipid-related genetic variants are common to Pakistanis and Europeans, though they explain only a modest portion of population variation in lipid concentration. Allelic frequencies and effect sizes of lipid-related variants can differ between Pakistanis and Europeans.

Circ Cardiovasc Genet: 23 Jun 2010; epub ahead of print

Exome Sequencing and Genome-Wide Linkage Analysis in 17 Families Illustrates the Complex Contribution of TTN Truncating Variants to Dilated Cardiomyopathy.

Norton N, Li D, Rampersaud E, Morales A, ... Nickerson DA, Hershberger RE
Background: -Familial dilated cardiomyopathy is a genetically heterogeneous disease with >30 known genes. TTN truncating variants were recently implicated in a candidate gene study to cause 25% of familial and 18% of sporadic dilated cardiomyopathy (DCM) cases. Methods and results: -We used an unbiased genome-wide approach employing both linkage analysis and variant filtering across the exome sequences of 48 individuals affected with DCM from 17 families to identify genetic cause. Linkage analysis ranked the TTN region as falling under the second highest genome-wide multipoint linkage peak, MLOD 1.59. We identified six TTN truncating variants carried by affected with DCM in 7 of 17 DCM families (LOD 2.99); 2 of these 7 families also had novel missense variants segregated with disease. Two additional novel truncating TTN variants did not segregate with DCM. Nucleotide diversity at the TTN locus, including missense variants, was comparable to five other known DCM genes. The average number of missense variants in the exome sequences from the DCM cases or the ~5,400 cases from the Exome Sequencing Project was ~23 per individual. The average number of TTN truncating variants in the Exome Sequencing Project was 0.014 per individual. We also identified a region (chr9q21.11-q22.31) with no known DCM genes with a maximum heterogeneity LOD score of 1.74. Conclusions: -These data suggest that TTN truncating variants contribute to DCM cause. However, the lack of segregation of all identified TTN truncating variants illustrates the challenge of determining variant pathogenicity even with full exome sequencing.

Circ Cardiovasc Genet: 17 Feb 2013; epub ahead of print

Coronary Heart Disease in Systemic Lupus Erythematosus Is Associated with Interferon Regulatory Factor 8 Gene Variants.

Leonard D, Svenungsson E, Sandling JK, Berggren O, ... Syvänen AC, Rönnblom L
Background: -Patients with Systemic Lupus Erythematosus (SLE) have increased morbidity and mortality in coronary heart disease (CHD). We asked if there was a genetic influence on CHD in SLE. Methods and results: -The association between single nucleotide polymorphisms (SNPs) and CHD in two populations of SLE patients was assessed. Patients were genotyped on a custom 12k Illumina Array. The allele frequencies were compared between patients with (n=66) and without (n=509) CHD. We found 61 SNPs with an association (p<0.01) to CHD with the strongest association for three SNPs located in the Interferon Regulatory Factor 8 gene (IRF8). Comparison of the allele frequencies of these 61 SNPs in patients with (n=27) and without CHD (n=212) in the second study population revealed that 2 SNPs, rs925994 and rs10514610 in IRF8, (LD, r(2) = 0.84) were associated with CHD in both study populations. Meta-analysis of the SNP rs925994 gave an odds ratio of 3.6 (2.1-6.3), p-value 1.9 x 10(-6). The identified IRF8 allele remained as a risk factor for CHD after adjustment for traditional CHD risk factors. The IRF8 risk allele was associated with presence of carotid plaques (p<0.001) and increased intima-media thickness (p=0.01). By electrophoretic mobility shift assays we show weaker binding of protein to the risk allele of the highly linked SNP rs11117415 and by flow cytometry a reduced frequency of circulating B cells was detected in patients with the IRF8 risk allele. Conclusions: -There is a considerable genetic component for CHD in SLE with IRF8 as a strong susceptibility locus.

Circ Cardiovasc Genet: 09 May 2013; epub ahead of print

Exome Sequencing and Systems Biology Converge to Identify Novel Mutations in the L-Type Calcium Channel, CACNA1C, Linked to Autosomal Dominant Long QT Syndrome.

Boczek NJ, Best JM, Tester DJ, Giudicessi JR, ... Kamp TJ, Ackerman MJ
Background: -Long QT syndrome (LQTS) is the most common cardiac channelopathy with 15 elucidated LQTS-susceptibility genes. Approximately 20% of LQTS cases remain genetically elusive. Methods and results: -We combined whole exome sequencing (WES) and bioinformatic/systems biology to identify the pathogenic substrate responsible for non-syndromic, genotype-negative, autosomal dominant LQTS in a multigenerational pedigree and established the spectrum and prevalence of variants in the elucidated gene among a cohort of 102 unrelated patients with "genotype-negative/phenotype-positive" LQTS. WES was utilized on three members within a genotype-negative/phenotype-positive family. Genomic triangulation combined with bioinformatic tools and ranking algorithms led to the identification of a CACNA1C mutation. This mutation, Pro857Arg-CACNA1C, co-segregated with the disease within the pedigree, was ranked by three disease-network algorithms as the most probable LQTS-susceptibility gene, and involves a conserved residue localizing to the PEST domain in the II-III linker. Functional studies reveal that Pro857Arg-CACNA1C leads to a gain-of-function with increased ICa,L and increased surface membrane expression of the channel compared to wildtype. Subsequent mutational analysis identified 3 additional variants within CACNA1C in our cohort of 102 unrelated cases of genotype-negative/phenotype-positive LQTS. Two of these variants also involve conserved residues within Cav1.2\'s PEST domain. Conclusions: -This study provides evidence that coupling WES and bioinformatic/systems biology is an effective strategy for the identification of potential disease causing genes/mutations. The identification of a functional CACNA1C mutation co-segregating with disease in a single pedigree suggests that CACNA1C perturbations may underlie autosomal dominant LQTS in the absence of Timothy syndrome.

Circ Cardiovasc Genet: 15 May 2013; epub ahead of print

Copy Number Variation in Glutathione S-transferases M1 and T1 and Ischemic Vascular Disease: Four Studies and Meta-analyses.

Nørskov MS, Frikke-Schmidt R, Loft S, Sillesen H, ... Nordestgaard BG, Tybjærg-Hansen A
Background: -Glutathione S-transferases (GSTs) M1 and T1 detoxify products of oxidative stress, and may protect against atherosclerosis and ischemic vascular disease (IVD). We tested the hypothesis that copy number variation (CNV) in GSTM1 and -T1 genes, associated with stepwise decreases in catalytic activity, predicted risk of IVD. Methods and results: -We included 23,059 Danes from two general population studies and two case-control studies, of whom 4,930 had ischemic heart disease (IHD) and 2,086 had ischemic cerebrovascular disease (ICVD). A real-time PCR method genotyped for the exact number of GSTM1 and -T1 gene copies. We also performed meta-analyses, including own and former studies totalling 13,196 IHD cases and 33,228 controls. CNV in GSTM1 or GSTT1, or genotype combinations were not associated with an increased risk of IHD, myocardial infarction, ICVD, ischemic stroke, or any ischemic vascular event in studies individually or combined, or in meta-analyses. Furthermore, genotypes did not interact with smoking on risk of disease endpoints. Finally, GST genotypes did not associate with markers of inflammation and oxidation, or interact with smoking on markers of inflammation in the general population. In contrast, we observed the well-established association between CNV in GSTM1 and risk of bladder cancer. Conclusions: -In studies including 6,557 IVD cases and 16,502 controls and in meta-analyses of 13,196 cases and 33,228 controls, CNV in GSTM1 and -T1 genes did not associate with risk of IVD or with markers of inflammation. These observations were independent of smoking exposure.

Circ Cardiovasc Genet: 12 May 2011; epub ahead of print

Natriuretic Peptide Receptor-3 Gene (NPR3): Nonsynonymous Polymorphism Results in Significant Reduction in Protein Expression Due to Accelerated Degradation.

Pereira NL, Lin D, Pelleymounter L, Moon I, ... Yee VC, Weinshilboum RM
Background: -The primary role of natriuretic peptide receptor 3 (NPR3) or NPR-C is in the clearance of natriuretic peptides that play an important role in modulating intravascular volume and vascular tone. Genetic variation in NPR3 has been associated with variation in blood pressure and obesity. Despite the importance of NPR3, sequence variation in the gene has not been addressed using DNA from different ethnic populations. We set out to identify and functionally characterize genetic variation in NPR3 in 3 ethnic groups. Methods and results: -DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence NPR3 exons, splice junctions and flanking regions. We identified 105 polymorphisms, 50 of which were novel, including 8 nonsynonymous (ns) SNPs, 7 were novel. Expression constructs were created for the nsSNP. HEK-293 cells were transfected with constructs for wild type (WT) and variant allozymes; and recombinant proteins were measured by quantitative Western blot analysis. The most significant change in NPR3 protein was observed for the Arg146 variant allozyme, with 20% of WT protein, due primarily to autophagy-dependent degradation. NPR3 structural modeling confirmed that the Arg146 variant protein was not compatible with WT conformation and could result in protein misfolding or instability. Conclusions: -Multiple novel NPR3 genetic polymorphisms were identified in 3 ethnic groups. The Arg146 allozyme displayed a significant decrease in protein quantity, due to degradation mediated predominantly by autophagy. This genetic variation could have a significant effect on the metabolism of natriuretic peptides with potential clinical implications.

Circ Cardiovasc Genet: 14 Mar 2013; epub ahead of print

Two Chromosome 9p21 Haplotype Blocks Distinguish Between Coronary Artery Disease and Myocardial Infarction Risk.

Fan M, Dandona S, McPherson R, Allayee H, ... Roberts R, Stewart AF
Background: -Variants at the 9p21 locus associate with the risk of coronary artery disease (CAD) or myocardial infarction (MI). However, atherosclerotic plaque deposition is distinct from MI (plaque rupture and thrombosis) and recent studies showed no association between these variants and MI in patients with preexisting CAD. We performed haplotype analysis at the 9p21 locus to test whether haplotypes at distinct linkage disequilibrium (LD) blocks predict these phenotypes. Methods and results: -Using 24 single-nucleotide polymorphisms genotyped in Caucasians without diabetes, we reconstructed haplotypes at the 9p21 locus. Angiographic CAD/MI patients had at least 1 epicardial stenosis > 50% (n=2352) whereas controls were asymptomatic and over age 60 (n=2116). For CAD patients, regression models examined association of haplotypes with initial age of symptomatic CAD, number of diseased vessels, and history of MI. In the case-control study, only haplotypes at one block tagged by rs1333049 associated with CAD more so than MI. These haplotypes also associated with early onset of CAD (β=-0.13 p=1.37*10(-4)) and disease severity (β=0.1823, p=0.006), but not with prevalent MI among CAD patients. In contrast, haplotypes at another block tagged by rs518394 associated with prevalent MI (β=0.239, p= 2.05*10(-4)), but remarkably these are inversely associated with disease severity (β=-0.196, p=0.003). This MI association was replicated in the Cleveland Clinic GeneBank premature CAD cohort (n=1385, β=0.207, p= 0.019). Conclusions: -Variants/haplotypes at two blocks are distinguished at 9p21, those at one block predispose to atherosclerosis whereas those at the other predispose to MI among individuals with preexisting CAD.

Circ Cardiovasc Genet: 02 Jun 2013; epub ahead of print

Subtle Abnormalities in Contractile Function Are an Early Manifestation of Sarcomere Mutations in Dilated Cardiomyopathy.

Lakdawala NK, Thune JJ, Colan SD, Cirino AL, ... Seidman CE, Ho CY
Background: -Sarcomere mutations cause both dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), however the steps leading from mutation to disease are not well described. By studying mutation carriers before a clinical diagnosis develops, we characterize the early manifestations of sarcomere mutations in DCM, and investigate how these manifestations differ from sarcomere mutations associated with HCM. Methods and results: -Sixty-two genotyped individuals in families with sarcomeric DCM underwent clinical evaluation including strain echocardiography. The group included 12 subclinical DCM mutation carriers with normal cardiac dimensions and ejection fraction (LVEF≥55%), 21 overt DCM subjects, and 29 related mutation (-) normal controls. Results were compared to a previously characterized cohort of 60 subclinical HCM subjects (sarcomere mutation carriers without LV hypertrophy). Systolic myocardial tissue velocity, longitudinal, circumferential, and radial strain, and longitudinal and radial strain rate were reduced by 10-23% in subclinical DCM mutation carriers compared to controls (p<0.001 for all comparisons), after adjusting for age and family relations. No significant differences in diastolic parameters were identified comparing the subclinical and control cohorts. The opposite pattern of contractile abnormalities with reduced diastolic but preserved systolic function was seen in subclinical HCM. Conclusions: -Subtle abnormalities in systolic function are present in subclinical DCM mutation carriers, despite normal LV size and EF. In contrast, impaired relaxation and preserved systolic function appear to be the predominant early manifestations of sarcomere mutations that lead to HCM. These findings support the theory that the mutation\'s intrinsic impact on sarcomere function influences whether a dilated or hypertrophic phenotype develops.

Circ Cardiovasc Genet: 04 Sep 2012; epub ahead of print

Common Genetic Variants of MGP Are Associated with Calcification in the Arterial Wall But Not Calcification Present in the Atherosclerotic Plaques.

Wang Y, Chen J, Zhang Y, Yu W, ... Chen X, Hui R
Background: -Two endophenotypes of arterial calcification, calcification on arterial wall and calcification in atherosclerotic plaques, are associated with different types of cardiovascular events. Mgp-deficient mice showed MGP is strongly associated with calcification on arterial wall without atherosclerotic plaques, and MGP variants were not significantly associated with myocardial infarction. MGP may play different roles in the two endophenotypes. Methods and results: -We analyzed the associations of MGP variants rs4236, rs1800801, and rs1800802 with the two endophenotypes determined by multi-detector computed tomography angiography. Total 585 with calcification on coronary artery wall, 675 with calcification in coronary atherosclerotic plaques, 454 with calcification on aortic wall, and 725 controls were enrolled. After Bonferroni correction, rs4236 and rs1800801 were still associated with calcification on arterial wall, the odds ratios were 0.708(95%CI, 0.540-0.928) for rs4236 and 0.652(95%CI, 0.479-0.888) for rs1800801 in coronary artery wall calcification, and 0.699(95%CI, 0.525-0.931) for rs4236 and 0.650(95%CI, 0.467-0.905) for rs1800801 in aortic wall calcification, respectively. The variants were correlated with calcification severity by ln(CAC Agatston score+1) in coronary artery wall calcification, but not in atherosclerotic plaque calcification. In accordance with their associations with calcification on arterial wall, rs4236C and rs1800801A were associated with higher MGP plasma levels while rs1800802C with lower MGP levels in normal controls. Due to the role of calcification in plaque vulnerability, their associations with acute myocardial infarction were also determined in 771 controls and 752 patients, no association was found. Conclusions: -MGP genetic variants showed association with calcification on arterial wall but not calcification in atherosclerotic plaques.

Circ Cardiovasc Genet: 15 May 2013; epub ahead of print

The Impact of Inherited Genetic Variants Associated with Lipid Profile, Hypertension, and Coronary Artery Disease on the Risk of Intracranial and Abdominal Aortic Aneurysms.

van \'t Hof FN, Ruigrok YM, Baas AF, Kiemeney LA, ... Rinkel GJ, de Bakker PI
Background: -Epidemiological studies show that an unfavourable lipid profile and coronary artery disease (CAD) are risk traits for abdominal aortic aneurysms (AAA) but not for intracranial aneurysms (IA), and that hypertension is a main risk trait for IA but not for AAA. To evaluate these observations, we investigated single nucleotide polymorphisms (SNPs) associated with serum lipid levels, hypertension, and CAD, and tested their contribution to AAA and IA risk. Methods and results: -We defined sets of SNPs previously reported to be associated with serum lipid levels, CAD and blood pressure (BP). From previously collected genome-wide data, we extracted genotypes for these SNP sets in 709 IA cases and 2692 controls, and 807 AAA cases and 1905 controls (all of Dutch origin). We computed genetic scores for each individual by summing the observed number of risk alleles weighted by their previously published effect size. Using logistic regression we tested the genetic scores for association to IA and AAA, and found significant associations for genetic scores of total cholesterol (p=3.6x10(-6)), low-density lipoprotein cholesterol (p=5.7x10(-7)) and CAD (p=0.0014) with AAA, and for the BP score with IA (p=0.0030). Conclusions: -We demonstrate that genetic risk profiles of lipid factors and CAD are associated with AAA but not with IA, and the genetic risk profile of BP is associated with IA but not with AAA. These findings are consistent with epidemiological observations.

Circ Cardiovasc Genet: 09 May 2013; epub ahead of print

Hypertension Susceptibility Loci and Blood Pressure Response to Antihypertensives - Results from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study.

Gong Y, McDonough CW, Wang Z, Hou W, ... Turner ST, Johnson JA
Background: -To date, 39 SNPs have been associated with blood pressure (BP) or hypertension (HTN) in genome-wide association studies (GWAS) in Caucasians. Our hypothesis is that the loci/SNPs associated with BP/HTN are also associated with BP response to antihypertensive drugs. Methods and results: -We assessed the association of these loci with BP response to atenolol or hydrochlorothiazide monotherapy in 768 hypertensive participants in the Pharmacogenomics Responses of Antihypertensive Responses (PEAR) study. Linear regression analysis was performed in Caucasians for each SNP in an additive model adjusting for baseline BP, age, gender and principal components for ancestry. Genetic scores were constructed to include SNPs with nominal associations and empirical p values were determined by permutation test. Genotypes of 37 loci were obtained from Illumina 50K cardiovascular or Omni1M GWAS chips. In Caucasians, no SNPs reached Bonferroni-corrected alpha of 0.0014, six reached nominal significance (p<0.05) and 3 were associated with atenolol BP response at p < 0.01. The genetic score of the atenolol BP lowering alleles was associated with response to atenolol (p =3.3*10(-6) for SBP; p=1.6*10(-6) for DBP). The genetic score of the HCTZ BP lowering alleles was associated with response to HCTZ (p = 0.0006 for SBP; p = 0.0003 for DBP). Both risk score p values were < 0.01 based on the empirical distribution from the permutation test. Conclusions: -These findings suggest selected signals from hypertension GWAS may predict BP response to atenolol and HCTZ when assessed through risk scoring. Clinical Trial registration Information-clinicaltrials.gov; Identifier: NCT00246519.

Circ Cardiovasc Genet: 21 Oct 2012; epub ahead of print

The Association of Genome-Wide Variation with the Risk of Incident Heart Failure in Adults of European and African Ancestry: A Prospective Meta-Analysis from the CHARGE Consortium.

Smith NL, Felix JF, Morrison AC, Demissie S, ... Boerwinkle E, Vasan RS
Background: -Although genetic factors contribute to the onset of heart failure (HF), no large-scale genome-wide investigation of HF risk has been published to date. We investigated the association of 2,478,304 single nucleotide polymorphisms (SNPs) with incident HF by meta-analyzing data from 4 community-based prospective cohorts: the Atherosclerosis Risk in Communities Study, the Cardiovascular Health Study, the Framingham Heart Study, and the Rotterdam Study. Methods and results: -Eligible participants for these analyses were of European or African ancestry and free of clinical HF at baseline. Each study independently conducted genome-wide scans and imputed data to the ~2.5 million SNPs in HapMap. Within each study, Cox proportional hazards regression models provided age- and sex-adjusted estimates of the association between each variant and time to incident HF. Fixed-effect meta-analyses combined results for each SNP from the 4 cohorts to produce an overall association estimate and p-value. A genome-wide significance p-value threshold was set a priori at 5.0x10(-7). During a mean follow-up of 11.5 years, 2,526 incident HF events (12%) occurred in 20,926 European-ancestry participants. The meta-analysis identified a genome-wide significant locus at chromosomal position 15q22 (1.4x10(-8)), which was 58.8 kb from USP3. Among 2,895 African-ancestry participants, 466 incident HF events (16%) occurred during a mean follow-up of 13.7 years. One genome-wide significant locus was identified at 12q14 (6.7x10(-8)), which was 6.3 kb from LRIG3. Conclusions: -We identified 2 loci that were associated with incident HF and exceeded genome-wide significance. The findings merit replication in other community-based settings of incident HF.

Circ Cardiovasc Genet: 06 May 2010; epub ahead of print

Apolipoprotein E Polymorphisms and Postprandial Triglyceridemia before and after Fenofibrate Treatment in the GOLDN Study.

Irvin MR, Kabagambe EK, Tiwari HK, Parnell LD, ... Ordavas JM, Arnett DK
Background: -While much is known about the effect of APOE alleles on fasting lipid concentrations, less is known about the effect of APOE alleles on postprandial triglyceridemia or the triglyceride response to fenofibrate. Methods and results: -We evaluated the effects of the APOE locus on fasting and postprandial triglyceride concentrations as part of the Genetics of Lipid Lowering and Diet Network (GOLDN) study. Participants were evaluated following a high-fat meal challenge before (N=1072) and after 3 weeks of daily treatment with 160 mg of fenofibrate (N=738). Mixed models adjusted for gender, age, waist circumference and family relationship were used to examine the association of the epsilon4 carrier and epsilon2 carrier status versus epsilon3 homozygotes with fasting triglycerides and the area under the curve (AUC) for triglycerides during the high-fat meal challenge. Compared to the epsilon3/epsilon3 genotype, epsilon2 carriers had on average higher fasting triglyceride concentrations (130.5 mg/dL vs. 109.3 mg/dL, P<0.001). After fenofibrate treatment, the APOE genotype differences persisted in the fasting state (epsilon2 carriers: 85.1 mg/dL vs. epsilon3/epsilon3: 75.9 mg/dL, P<0.05). Carriers of the epsilon4 allele had significantly higher fasting triglyceride concentrations only pre-fenofibrate (120.9 mg/dL vs. 109.3 md/dL, P=0.008). APOE alleles did not have an effect on response to fenofibrate. Postprandial triglycerides were significantly higher for epsilon2 carriers versus epsilon3 homozygotes (but not epsilon4 carriers) both before and after fenofibrate treatment (P=0.01 and P=0.005, respectively). Conclusions: -APOE polymorphisms are important determinants of triglyceride concentrations, especially in the fasting state.

Circ Cardiovasc Genet: 23 Aug 2010; epub ahead of print

Large-Scale Candidate Gene Analysis in Whites and African-Americans Identifies IL6R Polymorphism in Relation to Atrial Fibrillation: The NHLBI CARe Project.

Schnabel RB, Kerr KF, Lubitz SA, Alkylbekova EL, ... Benjamin EJ, Heckbert SR
Background: -The genetic background of atrial fibrillation (AF) in whites and African Americans is largely unknown. Genes in cardiovascular pathways have not been systematically investigated. Methods and results: -We examined a panel of approximately 50,000 common single nucleotide polymorphisms (SNPs) in 2,095 cardiovascular candidate genes and AF in three cohorts with participants of European (n=18,524; 2,260 cases) or African American descent (n=3,662; 263 cases) in the National Heart Lung and Blood Institute\'s Candidate Gene Association Resource. Results in whites were followed up in the German Competence Network for AF (n=906, 468 cases). The top result was assessed in relation to incident ischemic stroke in the Cohorts for Heart and Aging Research in Genomic Epidemiology Stroke Consortium (n= 19,602 whites, 1544 incident strokes). SNP rs4845625 in the IL6R gene was associated with AF (relative risk (RR) C allele, 0.90; 95% confidence interval (CI), 0.85-0.95; P=0.0005) in whites, but did not reach statistical significance in African Americans (RR, 0.86; 95% CI, 0.72-1.03; P=0.09). The results were comparable in the German AF Network replication, (RR, 0.71; 95% CI, 0.57-0.89; P=0.003). No association between rs4845625 and stroke was observed in whites. The known chromosome 4 locus near PITX2 in whites also was associated with AF in African Americans (rs4611994, hazard ratio, 1.40; 95% CI, 1.16-1.69; P=0.0005). Conclusions: -In a community-based cohort meta-analysis, we identified genetic association in IL6R with AF in whites. Additionally, we demonstrated that the chromosome 4 locus known from recent genome-wide association studies in whites is associated with AF in African Americans.

Circ Cardiovasc Genet: 17 Aug 2011; epub ahead of print

Venous Thromboembolism Does Not Share Strong Familial Susceptibility with Ischemic Stroke: A Nationwide Family Study in Sweden.

Zöller B, Li X, Ohlsson H, Sundquist J, Sundquist K
Background: -Coagulation allelic variants associated with venous thromboembolism (VTE) have been suggested to be involved in the pathogenesis of ischemic stroke. This nationwide study aimed at determining whether VTE shares familial susceptibility with ischemic stroke. Method and Results-The Swedish Multigeneration Register of 0-75-year-old subjects was linked to the Swedish Hospital Discharge Register and the Cause of Death Register for the period 1987-2007. Odds ratios (OR), for VTE, and ischemic stroke were determined in two ways: odds of ischemic stroke in offspring whose parents had been diagnosed with VTE, and odds of VTE in offspring whose parents had been diagnosed with ischemic stroke. The analyses were repeated for siblings and spouses. Offspring of parents with VTE (n=25 929) were at increased risk for ischemic stroke (n=5595): OR 1.10 (95% CI 1.06-1.14). Siblings of probands with VTE (n=45 132) had no increased risk of ischemic stroke (n=1716): OR 1.05 (95% CI 1.00-1.11). Spouses of probands with VTE (n=24 106) were at increased risk for ischemic stroke (n=940): OR 1.18 (95% CI 1.10-1.27). The risks for VTE in relatives of probands with ischemic stroke were: OR 1.15, 95% CI 1.10-1.21 (offspring), OR 1.07, 95% CI 1.02-1.12 (siblings), and OR 1.21, 95% CI 1.11-1.32 (spouses). Conclusions: -Venous thromboembolism does not share strong familial susceptibility with ischemic stroke in the Swedish population. Moreover, familial non-genetic factors contribute to the observed weak familial associations. The present study suggests that it is unlikely that strong shared disease-causing mutations exist to a large extent in the Swedish population.

Circ Cardiovasc Genet: 01 Sep 2011; epub ahead of print

Cardiac Structural and Sarcomere Genes Associated with Cardiomyopathy Exhibit Marked Intolerance of Genetic Variation.

Pan S, Caleshu CA, Dunn KE, Foti MJ, ... Soyinka O, Ashley EA
Background: -The clinical significance of variants in genes associated with inherited cardiomyopathies can be difficult to determine due to uncertainty regarding population genetic variation and a surprising amount of tolerance of the genome even to loss of function variants. We hypothesized that genes associated with cardiomyopathy might be particularly resistant to the accumulation of genetic variation. Methods and results: -We analyzed the rates of single nucleotide genetic variation in all known genes from the exomes of >5,000 individuals from the National Heart, Lung, and Blood Institute\'s Exome Sequencing Project (ESP), as well as the rates of structural variation from the Database of Genomic Variants. Most variants were rare, with over half unique to one individual. Cardiomyopathy associated genes exhibited a rate of nonsense variants 96.1% lower than other Mendelian disease genes. We tested the ability of in-silico algorithms to distinguish between a set of variants in MYBPC3, MYH7, and TNNT2 with strong evidence for pathogenicity and variants from the ESP data. Algorithms based on conservation at the nucleotide level (GERP, PhastCons) did not perform as well as amino acid level prediction algorithms (Polyphen-2, SIFT). Variants with strong evidence for disease causality were found in the ESP data at prevalence higher than expected. Conclusions: -Genes associated with cardiomyopathy carry very low rates of population variation. The existence in population data of variants with strong evidence for pathogenicity suggests that even for Mendelian disease genetics, a probabilistic weighting of multiple variants may be preferred over the \'single gene\' causality model.

Circ Cardiovasc Genet: 16 Oct 2012; epub ahead of print

Gene-Targeted Analysis of Copy Number Variants Identifies Three Novel Associations with Coronary Heart Disease Traits.

Costelloe SJ, El-Sayed Moustafa JS, Drenos F, Palmen J, ... Coin LJ, Talmud PJ
Background: -Copy number variants (CNVs) are a major form of genomic variation which may be implicated in complex disease phenotypes. However, investigation of the role of CNVs in coronary heart disease (CHD) traits has been limited. Methods and results: -We examined the utility of the cnvHap algorithm for CNV detection, using data for 2500 men from the Northwick Park Heart Study II (NPHS-II). An Illumina custom chip including 722 single nucleotide polymorphisms covering 76 CHD-trait genes was employed. Common CNVs were significantly associated (at P < 0.05, after correction) with CHD phenotypes in five genes. Novel associations of CNVs in TLR4 with apolipoprotein AI (apoAI) were replicated (P < 0.05) in the Whitehall II (WHII) cohort (4887 subjects), while newly described associations of CNVs in SREBP1 with apolipoprotein AI and associations of IL-6ST with apolipoprotein B were replicated in data from 3546 subjects from the North Finnish Birth Cohort 1996 (NFBC1966), (P < 0.05) while was replicated in NFBC1966. Conclusions: -This study supports the use of CNV detection algorithms such as cnvHap as potential tools for the identification of novel CNVs, some of which show significant association and replication with CHD risk phenotypes. However, the functional basis for these associations requires further substantiation.

Circ Cardiovasc Genet: 12 Sep 2012; epub ahead of print

Common variants in HSPB7 and FRMD4B associated with advanced heart failure.

Cappola TP, Li M, He J, Ky B, ... Cresci S, Dorn GW
Background: -Heart failure results from abnormalities in multiple biological processes that contribute to cardiac dysfunction. We tested the hypothesis that inherited variation in genes of known importance to cardiovascular biology would thus contribute to heart failure risk. Methods and results: -We utilized the ITMAT/Broad/CARe (IBC) cardiovascular SNP-array to screen referral populations of advanced heart failure patients for variants in ~2,000 genes of predicted importance to cardiovascular biology. Our design was a two-stage case-control study. In Stage 1, genotypes in Caucasian heart failure patients (n=1,590; ejection fraction 32+/-16%) were compared to those in unaffected controls (n=577; ejection fraction 67+/-8%) recruited from the same referral centers. Associations were tested for independent replication in Stage 2 (n=308 cases, 2,314 controls). Two intronic SNPs showed replicated associations with all-cause heart failure: rs1739843 in HSPB7 (combined P=3.09x10(-6)) and rs6787362 in FRMD4B (P=6.09x10(-6)). For both SNPs the minor allele was protective. In subgroup analyses, rs1739843 associated with both ischemic and nonischemic heart failure, whereas rs6787362 associated principally with ischemic heart failure. Linkage disequilibrium surrounding rs1739843 suggested that the causal variant resides in a region containing HSPB7 and a neighboring gene, CLCNKA, whereas the causal variant near rs6787362 is probably within FRMD4B. Allele frequencies for these SNPs were substantially different in African Americans (n=635 cases, 714 controls) and showed no association with heart failure in this population. Conclusions: -Our findings identify regions containing HSPB7 and FRMD4B as novel susceptibility loci for advanced heart failure. More broadly, in an era of genome-wide association studies, we demonstrate how knowledge of candidate genes can be leveraged as a complementary strategy to discern the genetics of complex disorders.

Circ Cardiovasc Genet: 03 Feb 2010; epub ahead of print

Phylogenetic and Physicochemical Analyses Enhance the Classification of Rare Non-Synonymous Single Nucleotide Variants in Type 1 and 2 Long QT Syndrome.

Giudicessi JR, Kapplinger JD, Tester DJ, Alders M, ... Wilde AA, Ackerman MJ
Background: -Hundreds of non-synonymous single nucleotide variants (nsSNVs) have been identified in the two most common LQTS-susceptibility genes (KCNQ1 and KCNH2). Unfortunately, a ~3% Background: and KCNH2 nsSNVs amongst healthy individuals complicates the ability to distinguish rare pathogenic mutations from similarly rare yet presumably innocuous variants. Methods and results: -In this study, 4 tools [1) conservation across species, 2) Grantham values, 3) SIFT, and 4) PolyPhen] were used to predict "pathogenic" or "benign" status for nsSNVs identified across 388 clinically "definite" LQTS cases and 1344 ostensibly healthy controls. From these data, estimated predictive values (EPVs) were determined for each tool independently, in concert with previously published protein topology-derived EPVs, and synergistically when ≥ 3 tools were in agreement. Overall, all 4 tools displayed a statistically significant ability to distinguish between case-derived and control-derived nsSNVs in KCNQ1, whereas each tool, except Grantham values, displayed a similar ability to differentiate KCNH2 nsSNVs. Collectively, when at least 3 of the 4 tools agreed on the "pathogenic" status of C-terminal nsSNVs located outside the KCNH2/Kv11.1 cyclic nucleotide binding domain, the topology-specific EPV improved from 56% to 91%. Conclusions: -While in silico prediction tools should not be used to predict independently the pathogenicity of a novel, rare nSNV, our results support the potential clinical utility of the synergistic use of these tools to enhance the classification of nsSNVs, particularly for Kv11.1\'s difficult to interpret C-terminal region.

Circ Cardiovasc Genet: 04 Sep 2012; epub ahead of print

LAMP2 Microdeletions in Patients with Danon Disease.

Yang Z, Funke BH, Cripe LH, Vick GW, ... Towbin JA, Vatta M
Background: -Danon disease is an X-linked dominant disorder characterized by the clinical triad of hypertrophic cardiomyopathy (HCM), skeletal myopathy and variable mental retardation. Pathologically, autophagic vacuoles are noted in both skeletal and cardiac muscle. It exhibits an X-linked dominant mode of inheritance and males are severely affected, while females develop milder and later-onset cardiac symptoms. Danon disease has been associated with mutations in the LAMP2 gene located at Xq24, typically resulting in splicing defects or protein truncation affecting the lysosome-associated membrane glycoprotein 2 (LAMP2). Because of its rarity, the full spectrum of genetic mutation resulting in Danon disease has not been elucidated. Methods and results: -We analyzed three males with clinical and pathological findings consistent with Danon disease. Comprehensive mutational analysis failed to yield detectable products for selected LAMP2 exons and genomic DNA deletion was suspected. Genomic junction fragment PCR analysis in Case-1 identified a novel Alu-mediated 34kb microdeletion encompassing the entire 5\'UTR and exon-1 of LAMP2. In Case-2 and -3, junctional PCR and Southern Blot analyses mapped the breakpoint to a MIRb and (TA)(n) simple repeats present in intron-3, which determined a 64kb and a 58Kb deletion, respectively, thereby ablating exons-4-10. Western blot analysis confirmed the absence of LAMP2 in protein extract from lymphocytes of index Case-2. Conclusions: -This is the first report of Danon disease caused by microdeletions at Xq24, which functionally ablate LAMP2. The microdeletion mechanism appears to involve one Alu-mediated unequal recombination and two chromosomal breakage points involving TA-rich repeat sequences.

Circ Cardiovasc Genet: 22 Feb 2010; epub ahead of print

BMPR2 Mutations Influence Phenotype More Obviously in Male Patients with Pulmonary Arterial Hypertension.

Liu D, Wu WH, Mao YM, Yuan P, ... Ju FL, Jing ZC
Background: -BMPR2 mutations predispose to idiopathic and heritable pulmonary arterial hypertension (IPAH and HPAH). The influence of BMPR2 mutations on clinical outcome is not concordant in different ethnic groups. Although the BMPR2 mutation spectrum and mutation rate in Chinese PAH patients has been reported previously, the influence of genotype on phenotype and whether this influence is associated with sex has not been investigated. Methods and results: -We analyzed data from 305 PAH patients considered as either idiopathic or heritable who underwent genetic counseling in Shanghai Pulmonary Hospital. The clinical, functional, and hemodynamic characteristics of BMPR2 mutation carriers and noncarriers were compared. The more severe hemodynamic compromise at diagnosis in BMPR2 mutation carriers versus noncarriers is concordant with other ethnic groups. In the Chinese PAH cohort, BMPR2 mutations were associated with a higher risk of mortality after adjustment for age and sex [HR=1.971; 95% CI 1.121-3.466; P = 0.018]. The overall survival difference between mutation carriers and noncarriers was more obvious in male patients, which was reflected by a higher mortality risk of male mutation carriers than that of male noncarriers after adjustment for age at diagnosis [HR = 3.702; 95% CI 1.416-9.679; P = 0.008]. In females, this trend did not reach statistical significance. Conclusions: -BMPR2 mutations influence phenotype more obviously in male PAH patients. The etiology of female PAH patients is more complicated and the influence of BMPR2 mutations may be modified by other unknown factors, making disparities in the prognosis between female mutation carriers and noncarriers less evident.

Circ Cardiovasc Genet: 26 Aug 2012; epub ahead of print

Matrix Metalloproteinase-9 Genotype as a Potential Genetic Marker for Abdominal Aortic Aneurysm.

Duellman T, Warren CL, Peissig P, Wynn M, Yang J
Background: -Degradation of extracellular matrix support in the large abdominal arteries contributes to the abnormal dilation of the aorta leading to abdominal aortic aneurysms (AAA) and matrix metalloproteinase-9 (MMP-9) is the predominant enzyme targeting elastin and collagen present in the walls of the abdominal aorta. Previous studies have suggested a potential association between MMP-9 genotype and AAA but these studies have been limited only to the p-1562 and (CA) dinucleotide repeat microsatellite polymorphisms in the promoter region of the MMP-9 gene. We determined the functional alterations caused by fifteen MMP-9 single nucleotide polymorphisms (SNPs) reported to be relatively abundant in the human genome through Western blots, gelatinase, and promoter-reporter assays and incorporated this information to perform a logistic-regression analysis of MMP-9 SNPs in 336 human AAA cases and controls. Methods and results: -Significant functional alterations were observed for 6 exon SNPs and 4 promoter SNPs. Genotype analysis of frequency-matched (age, sex, history of hypertension, hypercholesterolemia, and smoking) cases and controls revealed significant genetic heterogeneity exceeding 20% observed for six SNPs in our population of mostly Caucasian subjects from Northern Wisconsin. A step-wise logistic regression analysis with 6 functional SNPs, where weakly contributing confounds were eliminated using Akaike information criteria, gave a final 2 SNP (D165N and p-2502) model with an overall odds ratio of 2.45 (95% confidence interval 1.06, 5.70). Conclusions: -The combined approach of direct experimental confirmation of the functional alterations of MMP-9 SNPs and logistic regression analysis revealed significant association between MMP-9 genotype and AAA.

Circ Cardiovasc Genet: 02 Sep 2012; epub ahead of print

CREB1 Is a Strong Genetic Predictor of the Variation in Exercise Heart Rate Response to Regular Exercise: The HERITAGE Family Study.

Rankinen T, Argyropoulos G, Rice T, Rao DC, Bouchard C
Background: -A genome-wide linkage scan identified a quantitative trait locus (QTL) for exercise training-induced changes in submaximal exercise (50W) heart rate (DeltaHR50) on chromosome 2q33.3-q34 in the HERITAGE Family Study (N=472). Methods and results: -To fine map the region, 1,450 tagSNPs were genotyped between 205 and 215 Mb on chromosome 2. The strongest evidence of association with DeltaHR50 was observed with two SNPs located in the 5\' region of the cAMP responsive element binding protein 1 (CREB1) gene (rs2253206: p=1.6x10(-5) and rs2360969: p=4.3x10(-5)). The associations remained significant (p=0.01 and p=0.023, respectively) after accounting for multiple testing. Regression modeling of the 39 most significant SNPs in the single-SNP analyses identified nine SNPs that collectively explained 20% of the DeltaHR50 variance. CREB1 SNP rs2253206 had the strongest effect (5.45% of variance), followed by SNPs in the FASTKD2 (3.1%), MAP2 (2.6%), SPAG16 (2.1%), ERBB4 (3 SNPs ~1.4% each), IKZF2 (1.4%), and PARD3B (1.0%) loci. In conditional linkage analysis, six SNPs from the final regression model (CREB1, FASTKD2, MAP2, ERBB4, IKZF2, and PARD3B) accounted for the original linkage signal: the LOD score dropped from 2.10 to 0.41 after adjusting for all six SNPs. Functional studies revealed that the common allele of rs2253206 exhibits significantly (p<0.05) lower promoter activity than the minor allele. Conclusions: -Our data suggest that functional DNA sequence variation in the CREB1 locus is strongly associated with DeltaHR50 and explains considerable proportion of the QTL variance. However, at least five additional SNPs seem to be required to fully account for the original linkage signal.

Circ Cardiovasc Genet: 21 Apr 2010; epub ahead of print

Identification of functional modules by integration of multiple data sources using a bayesian network classifier.

Wang J, Zuo Y, Liu L, Man Y, Tadesse MG, Ressom HW
Background- Prediction of functional modules is indispensable for detecting protein deregulation in human complex diseases such as cancer. Bayesian network is one of the most commonly used models to integrate heterogeneous data from multiple sources such as protein domain, interactome, functional annotation, genome-wide gene expression, and the literature. Methods and results- In this article, we present a Bayesian network classifier that is customized to (1) increase the ability to integrate diverse information from different sources, (2) effectively predict protein-protein interactions, (3) infer aberrant networks with scale-free and small-world properties, and (4) group molecules into functional modules or pathways based on the primary function and biological features. Application of this model in discovering protein biomarkers of hepatocellular carcinoma leads to the identification of functional modules that provide insights into the mechanism of the development and progression of hepatocellular carcinoma. These functional modules include cell cycle deregulation, increased angiogenesis (eg, vascular endothelial growth factor, blood vessel morphogenesis), oxidative metabolic alterations, and aberrant activation of signaling pathways involved in cellular proliferation, survival, and differentiation. Conclusions- The discoveries and conclusions derived from our customized Bayesian network classifier are consistent with previously published results. The proposed approach for determining Bayesian network structure facilitates the integration of heterogeneous data from multiple sources to elucidate the mechanisms of complex diseases.

Circ Cardiovasc Genet: 15 Apr 2014; 7:206-17