Kerri L. Wiggins

Genetic loci for retinal arteriolar microcirculation

Narrow arterioles in the retina have been shown to predict hypertension as well as other vascular diseases, likely through an increase in the peripheral resistance of the microcirculatory flow. In this study, we performed a genome-wide association study in 18,722 unrelated individuals of European ancestry from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium and the Blue Mountain Eye Study, to identify genetic determinants associated with variations in retinal arteriolar caliber. Retinal vascular calibers were measured on digitized retinal photographs using a standardized protocol. One variant (rs2194025 on chromosome 5q14 near the myocyte enhancer factor 2C MEF2C gene) was associated with retinal arteriolar caliber in the meta-analysis of the discovery cohorts at genome-wide significance of P-value <5×10−8. This variant was replicated in an additional 3,939 individuals of European ancestry from the Australian Twins Study and Multi-Ethnic Study of Atherosclerosis (rs2194025, P-value = 2.11×10−12 in combined meta-analysis of discovery and replication cohorts). In independent studies of modest sample sizes, no significant association was found between this variant and clinical outcomes including coronary artery disease, stroke, myocardial infarction or hypertension. In conclusion, we found one novel loci which underlie genetic variation in microvasculature which may be relevant to vascular disease. The relevance of these findings to clinical outcomes remains to be determined.

Genetic risk factors for ischaemic stroke and its subtypes (the METASTROKE collaboration): a meta-analysis of genome-wide association studies.

Summary Background Various genome-wide association studies (GWAS) have been done in ischaemic stroke, identifying a few loci associated with the disease, but sample sizes have been 3500 cases or less. We established the METASTROKE collaboration with the aim of validating associations from previous GWAS and identifying novel genetic associations through meta-analysis of GWAS datasets for ischaemic stroke and its subtypes. Methods We meta-analysed data from 15 ischaemic stroke cohorts with a total of 12 389 individuals with ischaemic stroke and 62 004 controls, all of European ancestry. For the associations reaching genome-wide significance in METASTROKE, we did a further analysis, conditioning on the lead single nucleotide polymorphism in every associated region. Replication of novel suggestive signals was done in 13 347 cases and 29 083 controls. Findings We verified previous associations for cardioembolic stroke near PITX2 (p=2·8×10−16) and ZFHX3 (p=2·28×10−8), and for large-vessel stroke at a 9p21 locus (p=3·32×10−5) and HDAC9 (p=2·03×10−12). Additionally, we verified that all associations were subtype specific. Conditional analysis in the three regions for which the associations reached genome-wide significance (PITX2, ZFHX3, and HDAC9) indicated that all the signal in each region could be attributed to one risk haplotype. We also identified 12 potentially novel loci at p<5×10−6. However, we were unable to replicate any of these novel associations in the replication cohort. Interpretation Our results show that, although genetic variants can be detected in patients with ischaemic stroke when compared with controls, all associations we were able to confirm are specific to a stroke subtype. This finding has two implications. First, to maximise success of genetic studies in ischaemic stroke, detailed stroke subtyping is required. Second, different genetic pathophysiological mechanisms seem to be associated with different stroke subtypes. Funding Wellcome Trust, UK Medical Research Council (MRC), Australian National and Medical Health Research Council, National Institutes of Health (NIH) including National Heart, Lung and Blood Institute (NHLBI), the National Institute on Aging (NIA), the National Human Genome Research Institute (NHGRI), and the National Institute of Neurological Disorders and Stroke (NINDS).

Novel Associations of Multiple Genetic Loci With Plasma Levels of Factor VII, Factor VIII, and von Willebrand Factor The CHARGE (Cohorts for Heart and Aging Research in Genome Epidemiology) Consortium

A complex cascade of coagulation factors underlies hemostasis and prevents life-threatening blood loss from damaged blood vessels. The hemostatic factors VII and VIII, both produced in the liver, play central roles in the initiation and propagation, respectively, of fibrin formation. In the tissue-factor pathway, blood coagulation factor VII (FVII), once activated, serves as a catalyst for factor X (FX) activation, which converts prothrombin to thrombin. During propagation, activated factor VIII (FVIII) activates FX in the presence of activated factor IX. Von Willebrand factor (vWF), produced by endothelial cells and megakaryocytes, has multiple roles in hemostasis. Its primary role is to serve as an adhesion molecule that anchors platelets to exposed collagen after endothelial cell damage. The factor also acts as a carrier protein of FVIII, thereby prolonging the half-life of FVIII. Elevated circulating levels of FVIII and vWF are risk factors for venous thrombosis but the data supporting an association of FVII levels with arterial thrombosis are less consistent.1-5 Hemorrhagic complications are associated with deficiency in FVII and vWF (von Willebrand disease), as well as X-linked deficiency in FVIII (Hemophilia A).6-9 Plasma levels of these proteins are affected by environmental factors but they also are genetically influenced.10-13 Heritability estimates range from 0.53-0.63 for FVII, 0.40-0.61 for FVIII, and 0.31-0.75 for vWF.12, 13 To date, our understanding of genetic variation influencing plasma levels has been focused primarily on cis-acting variation in the genes encoding each protein product (F7, F8, and VWF, respectively). A large-scale genome-wide investigation of the genomic correlates of plasma levels has not been previously published. Using data from 23,608 adults, we investigated genome-wide associations between common genetic variation and plasma levels of FVII, FVIII, and vWF.Background— Plasma levels of coagulation factors VII (FVII), VIII (FVIII), and von Willebrand factor (vWF) influence risk of hemorrhage and thrombosis. We conducted genome-wide association studies to identify new loci associated with plasma levels. Methods and Results— The setting of the study included 5 community-based studies for discovery comprising 23 608 European-ancestry participants: Atherosclerosis Risk In Communities Study, Cardiovascular Health Study, British 1958 Birth Cohort, Framingham Heart Study, and Rotterdam Study. All subjects had genome-wide single-nucleotide polymorphism (SNP) scans and at least 1 phenotype measured: FVII activity/antigen, FVIII activity, and vWF antigen. Each study used its genotype data to impute to HapMap SNPs and independently conducted association analyses of hemostasis measures using an additive genetic model. Study findings were combined by meta-analysis. Replication was conducted in 7604 participants not in the discovery cohort. For FVII, 305 SNPs exceeded the genome-wide significance threshold of 5.0×10−8 and comprised 5 loci on 5 chromosomes: 2p23 (smallest P value 6.2×10−24), 4q25 (3.6×10−12), 11q12 (2.0×10−10), 13q34 (9.0×10−259), and 20q11.2 (5.7×10−37). Loci were within or near genes, including 4 new candidate genes and F7 (13q34). For vWF, 400 SNPs exceeded the threshold and marked 8 loci on 6 chromosomes: 6q24 (1.2×10−22), 8p21 (1.3×10−16), 9q34 (<5.0×10−324), 12p13 (1.7×10−32), 12q23 (7.3×10−10), 12q24.3 (3.8×10−11), 14q32 (2.3×10−10), and 19p13.2 (1.3×10−9). All loci were within genes, including 6 new candidate genes, as well as ABO (9q34) and VWF (12p13). For FVIII, 5 loci were identified and overlapped vWF findings. Nine of the 10 new findings were replicated. Conclusions— New genetic associations were discovered outside previously known biological pathways and may point to novel prevention and treatment targets of hemostasis disorders.

Genome-Wide Association Study of Retinopathy in Individuals without Diabetes

Background Mild retinopathy (microaneurysms or dot-blot hemorrhages) is observed in persons without diabetes or hypertension and may reflect microvascular disease in other organs. We conducted a genome-wide association study (GWAS) of mild retinopathy in persons without diabetes. Methods A working group agreed on phenotype harmonization, covariate selection and analytic plans for within-cohort GWAS. An inverse-variance weighted fixed effects meta-analysis was performed with GWAS results from six cohorts of 19,411 Caucasians. The primary analysis included individuals without diabetes and secondary analyses were stratified by hypertension status. We also singled out the results from single nucleotide polymorphisms (SNPs) previously shown to be associated with diabetes and hypertension, the two most common causes of retinopathy. Results No SNPs reached genome-wide significance in the primary analysis or the secondary analysis of participants with hypertension. SNP, rs12155400, in the histone deacetylase 9 gene (HDAC9) on chromosome 7, was associated with retinopathy in analysis of participants without hypertension, −1.3±0.23 (beta ± standard error), p = 6.6×10−9. Evidence suggests this was a false positive finding. The minor allele frequency was low (∼2%), the quality of the imputation was moderate (r2 ∼0.7), and no other common variants in the HDAC9 gene were associated with the outcome. SNPs found to be associated with diabetes and hypertension in other GWAS were not associated with retinopathy in persons without diabetes or in subgroups with or without hypertension. Conclusions This GWAS of retinopathy in individuals without diabetes showed little evidence of genetic associations. Further studies are needed to identify genes associated with these signs in order to help unravel novel pathways and determinants of microvascular diseases.

Diabetes Mellitus, Glycemic Control, and Risk of Atrial Fibrillation

BACKGROUNDDiabetes may be an independent risk factor for atrial fibrillation. However, results from prior studies are in conflict, and no study has examined diabetes duration or glycemic control.OBJECTIVETo examine the association of diabetes with risk of atrial fibrillation and to describe risk according to diabetes duration and glycemic control.DESIGNA population-based case-control study.PARTICIPANTSWithin a large, integrated healthcare delivery system, we identified 1,410 people with newly-recognized atrial fibrillation from ICD-9 codes and validated cases by review of medical records. 2,203 controls without atrial fibrillation were selected from enrollment lists, stratified on age, sex, hypertension, and calendar year.MAIN MEASURESInformation on atrial fibrillation, diabetes and other characteristics came from medical records. Diabetes was defined based on physician diagnoses recorded in the medical record, and pharmacologically treated diabetes was defined as receiving antihyperglycemic medications. Information about hemoglobin A1c levels came from computerized laboratory data.KEY RESULTSAmong people with atrial fibrillation, 252/1410 (17.9%) had pharmacologically treated diabetes compared to 311/2203 (14.1%) of controls. The adjusted OR for atrial fibrillation was 1.40 (95% CI 1.15-1.71) for people with treated diabetes compared to those without diabetes. Among those with treated diabetes, the risk of developing atrial fibrillation was 3% higher for each additional year of diabetes duration (95% CI 1-6%). Compared to people without diabetes, the adjusted OR for people with treated diabetes with average hemoglobin A1c ≤7 was 1.06 (95% CI 0.74-1.51); for A1c >7 but ≤8, 1.48 (1.09-2.01); for A1c >8 but ≤9, 1.46 (1.02-2.08); and for A1c >9, 1.96 (1.22–3.14).CONCLUSIONSDiabetes was associated with higher risk of developing atrial fibrillation, and risk was higher with longer duration of treated diabetes and worse glycemic control. Future research should identify and test approaches to reduce the risk of atrial fibrillation in people with diabetes.

Four Novel Loci (19q13, 6q24, 12q24, and 5q14) Influence the Microcirculation In Vivo

There is increasing evidence that the microcirculation plays an important role in the pathogenesis of cardiovascular diseases. Changes in retinal vascular caliber reflect early microvascular disease and predict incident cardiovascular events. We performed a genome-wide association study to identify genetic variants associated with retinal vascular caliber. We analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n = 6,652). All participants had retinal photography and retinal arteriolar and venular caliber measured from computer software. In the discovery cohorts, 179 single nucleotide polymorphisms (SNP) spread across five loci were significantly associated (p<5.0×10−8) with retinal venular caliber, but none showed association with arteriolar caliber. Collectively, these five loci explain 1.0%–3.2% of the variation in retinal venular caliber. Four out of these five loci were confirmed in independent replication samples. In the combined analyses, the top SNPs at each locus were: rs2287921 (19q13; p = 1.61×10−25, within the RASIP1 locus), rs225717 (6q24; p = 1.25×10−16, adjacent to the VTA1 and NMBR loci), rs10774625 (12q24; p = 2.15×10−13, in the region of ATXN2,SH2B3 and PTPN11 loci), and rs17421627 (5q14; p = 7.32×10−16, adjacent to the MEF2C locus). In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. Our population-based genome-wide association study demonstrates four novel loci associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. These data provide further insights into the contribution and biological mechanisms of microcirculatory changes that underlie cardiovascular disease.

Common variants at 6p21.1 are associated with large artery atherosclerotic stroke

Genome-wide association studies (GWAS) have not consistently detected replicable genetic risk factors for ischemic stroke, potentially due to etiological heterogeneity of this trait. We performed GWAS of ischemic stroke and a major ischemic stroke subtype (large artery atherosclerosis, LAA) using 1,162 ischemic stroke cases (including 421 LAA cases) and 1,244 population controls from Australia. Evidence for a genetic influence on ischemic stroke risk was detected, but this influence was higher and more significant for the LAA subtype. We identified a new LAA susceptibility locus on chromosome 6p21.1 (rs556621: odds ratio (OR) = 1.62, P = 3.9 × 10−8) and replicated this association in 1,715 LAA cases and 52,695 population controls from 10 independent population cohorts (meta-analysis replication OR = 1.15, P = 3.9 × 10−4; discovery and replication combined OR = 1.21, P = 4.7 × 10−8). This study identifies a genetic risk locus for LAA and shows how analyzing etiological subtypes may better identify genetic risk alleles for ischemic stroke.

Multiple Loci Are Associated with White Blood Cell Phenotypes

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count—6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count—17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count—6p21, 19p13 at EPS15L1; monocyte count—2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.

ABO genotype and risk of thrombotic events and hemorrhagic stroke

Summary.  Background: The non‐O alleles of the ABO genotype have been associated with an increased risk of thrombosis. Risk associated with the specific A1, A2 or B alleles is not well defined. Objectives: To examine the association of the ABO genotype with myocardial infarction (MI), ischemic stroke, hemorrhagic stroke, and venous thrombosis (VT). Patients and methods: We used data from two ongoing population‐based case–control studies of MI, stroke, and VT. Cases included hypertensive adults and postmenopausal women with incident non‐fatal MI (n = 1063), ischemic stroke (n = 469), and hemorrhagic stroke (n = 91), and postmenopausal women with incident non‐fatal VT (n = 504). Controls were frequency matched to cases on age, sex, hypertension status, and year of identification. ABO genotypes were determined using single‐nucleotide polymorphisms, and subjects were grouped by diplotype according to the presence of O1, O2, A11, A2 and B alleles. Logistic regression was used to test the association of diplotypes with risk of each outcome. Results: As compared with the O1O1 group, the A11 allele was associated with an increased risk of VT [odds ratio (OR) 1.79; 95% confidence interval (CI) 1.41–2.26] and MI (OR 1.23; 95% CI 1.05–1.44). The B allele was associated with an increased risk of VT (OR 1.82; 95% CI 1.29–2.57) and ischemic stroke (OR 1.59; 95% CI 1.17–2.17). The AB diplotype category was associated with a 2.7‐fold risk of VT (OR 2.70; 95% CI 1.73–4.21). No other associations reached significance. Conclusions: The VT and MI findings are confirmatory, and the ischemic stroke finding with the B allele is a novel finding and needs replication.

Variation in eicosanoid genes, non-fatal myocardial infarction and ischemic stroke

OBJECTIVES Eicosanoids are lipid mediators that may play a role in atherosclerosis. We investigated the association of common genetic variation in prostaglandin H synthase 1 (PTGS1), prostaglandin H synthase 2 (PTGS2), thromboxane A2 synthase (TBXAS1), prostacyclin synthase (PTGIS), prostaglandin E synthase (PTGES), 5-lipoxygenase activating protein (ALOX5AP), 12-lipoxygenase (ALOX12) and 15-lipoxygenase (ALOX15) with the risks of myocardial infarction (MI) and ischemic stroke. A secondary aim was to replicate the interaction of PTGS2 rs20417 (-765G to C) with aspirin use on coronary heart disease risk observed in the Atherosclerosis Risk in Communities Study (ARIC). METHODS We conducted a case-control study in a large Health Maintenance Organization. Cases were men and women, aged 30-79 years with incident non-fatal myocardial infarction (n=1063) or ischemic stroke (n=469) between January 1995 and December 2004. Controls (n=3462) were randomly selected and frequency matched to cases on age, sex, hypertension and calendar year. RESULTS Common variation in TBXAS1 and PTGIS was associated with MI risk (p-value for global Chi-square test, 0.01 and 0.03, respectively). Common variation in ALOX5AP, ALOX12, ALOX15, PTGS1, PTGS2 and PTGES was not associated with risks of MI and ischemic stroke. We replicated the observation of the Atherosclerosis Risk in Communities Study and observed an interaction of rs20417 with aspirin use on myocardial infarction risk (p for interaction=0.03). CONCLUSIONS Study results suggest that variation in TBXAS1 and PTGIS may influence MI risk, and carriers of rs20417C allele might derive greater benefits from aspirin use in primary prevention.