Andrade, Mariza de

Human genetic studies support an inverse causal relationship between leukocyte telomere length (LTL) and coronary artery disease (CAD), but directionally mixed effects for LTL and diverse malignancies. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by expansion of hematopoietic cells bearing leukemogenic mutations, predisposes both hematologic malignancy and CAD. TERT (which encodes telomerase reverse transcriptase) is the most significantly associated germline locus for CHIP in genome-wide association studies. Here, we investigated the relationship between CHIP, LTL, and CAD in the Trans-Omics for Precision Medicine (TOPMed) program ( n = 63,302) and UK Biobank ( n = 47,080). Bidirectional Mendelian randomization studies were consistent with longer genetically imputed LTL increasing propensity to develop CHIP, but CHIP then, in turn, hastens to shorten measured LTL (mLTL). We also demonstrated evidence of modest mediation between CHIP and CAD by mLTL. Our data promote an understanding of potential causal relationships across CHIP and LTL toward prevention of CAD.

Background: Peripheral artery disease (PAD) affects >200 million people worldwide and is associated with high mortality and morbidity. We sought to identify genomic variants associated with PAD overall and in the contexts of diabetes and smoking status. Methods: We identified genetic variants associated with PAD and then meta-analyzed with published summary statistics from the Million Veterans Program and UK Biobank to replicate their findings. Next, we ran stratified genome-wide association analysis in ever smokers, never smokers, individuals with diabetes, and individuals with no history of diabetes and corresponding interaction analyses, to identify variants that modify the risk of PAD by diabetic or smoking status. Results: We identified 5 genome-wide significant ( P association ≤5×10 −8 ) associations with PAD in 449 548 (N cases =12 086) individuals of European ancestry near LPA (lipoprotein [a]), CDKN2BAS1 (CDKN2B antisense RNA 1), SH2B3 (SH2B adaptor protein 3) - PTPN11 (protein tyrosine phosphatase non-receptor type 11), HDAC9 (histone deacetylase 9), and CHRNA3 (cholinergic receptor nicotinic alpha 3 subunit ) loci (which overlapped previously reported associations). Meta-analysis with variants previously associated with PAD showed that 18 of 19 published variants remained genome-wide significant. In individuals with diabetes, rs116405693 at the CCSER1 (coiled-coil serine rich protein 1 ) locus was associated with PAD (odds ratio [95% CI], 1.51 [1.32–1.74], P diabetes =2.5×10 −9 , P interactionwithdiabetes =5.3×10 −7 ). Furthermore, in smokers, rs12910984 at the CHRNA3 locus was associated with PAD (odds ratio [95% CI], 1.15 [1.11–1.19], P smokers =9.3×10 −10 , P interactionwithsmoking =3.9×10 −5 ). Conclusions: Our analyses confirm the published genetic associations with PAD and identify novel variants that may influence susceptibility to PAD in the context of diabetes or smoking status.

Background: Lp(a) (lipoprotein [a]) levels are higher in individuals of African ancestry (AA) than in individuals of European ancestry (EA). We examined associations of genetically predicted Lp(a) levels with (1) atherosclerotic cardiovascular disease subtypes: coronary heart disease, cerebrovascular disease, peripheral artery disease, and abdominal aortic aneurysm and (2) nonatherosclerotic cardiovascular disease phenotypes, stratified by ancestry. Methods: We performed (1) Mendelian randomization analyses for previously reported cardiovascular associations and (2) Mendelian randomization–phenome-wide association analyses for novel associations. Analyses were stratified by ancestry in electronic Medical Records and Genomics, United Kingdom Biobank, and Million Veteran Program cohorts separately and in a combined cohort of 804 507 EA and 103 580 AA participants. Results: In Mendelian randomization analyses using the combined cohort, a 1-SD genetic increase in Lp(a) level was associated with atherosclerotic cardiovascular disease subtypes in EA—odds ratio and 95% CI for coronary heart disease 1.28 (1.16–1.41); cerebrovascular disease 1.14 (1.07–1.21); peripheral artery disease 1.22 (1.11–1.34); abdominal aortic aneurysm 1.28 (1.17–1.40); in AA, the effect estimate was lower than in EA and nonsignificant for coronary heart disease 1.11 (0.99–1.24) and cerebrovascular disease 1.06 (0.99–1.14) but similar for peripheral artery disease 1.16 (1.01–1.33) and abdominal aortic aneurysm 1.34 (1.11–1.62). In EA, a 1-SD genetic increase in Lp(a) level was associated with aortic valve disorders 1.34 (1.10–1.62), mitral valve disorders 1.18 (1.09–1.27), congestive heart failure 1.12 (1.05–1.19), and chronic kidney disease 1.07 (1.01–1.14). In AA, no significant associations were noted for aortic valve disorders 1.08 (0.94–1.25), mitral valve disorders 1.02 (0.89–1.16), congestive heart failure 1.02 (0.95–1.10), or chronic kidney disease 1.05 (0.99–1.12). Mendelian randomization–phenome-wide association analyses identified novel associations in EA with arterial thromboembolic disease, nonaortic aneurysmal disease, atrial fibrillation, cardiac conduction disorders, and hypertension. Conclusions: Many cardiovascular associations of genetically increased Lp(a) that were significant in EA were not significant in AA. Lp(a) was associated with atherosclerotic cardiovascular disease in four major arterial beds in EA but only with peripheral artery disease and abdominal aortic aneurysm in AA. Additionally, novel cardiovascular associations were detected in EA.

P-selectin is a cellular adhesion molecule that enhances procoagulant activity and activation of leukocyte integrins. P-selectin has been shown to be crucial in the development of atherosclerosis. Using a stratified random sample of 2406 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) at Exam 2, we investigated the association of soluble P-selectin and cardiovascular disease. The Tobit model was used to investigate the association of P-selectin levels and coronary artery calcium (CAC) and Cox proportional hazards regression was used to assess the association between protein level and incident cardiovascular disease (CVD) events (myocardial infarction, stroke, angina, or other atherosclerotic death). Mean levels of soluble P-selectin differed significantly by ethnicity; 46±18 ng/mL African Americans, 58±20 ng/mL Caucasians, 43±15 ng/mL Chinese, and 61±20 ng/mL Hispanics (p<0.0001). CAC was present in 46% of African Americans, 61% of Caucasians, 52% of Chinese, and 53% of Hispanics. P-selectin levels were significantly associated with CAC in Caucasians (p < 0.001) and African Americans (p = 0.025), see Table. This association was robust to adjustment for traditional risk factors and a significant interaction of P-selectin and ethnicity was observed (p= 0.02). Over an average of 6.5 years of follow-up there were 111 incident CVD events after MESA exam 2, 26 in African Americans, 33 in Caucasians, 16 in Chinese, and 36 in Hispanics. The risk of CVD events increased 34% per standard deviation increase in P-selectin levels only in Caucasians (p = 0.007) and this finding was consistent after adjustment for CVD risk factors (HR = 1.30, p = 0.017). We have shown in this large cohort that mean circulating P-selectin levels differed by ethnicity. Despite similar percentages of subjects with CAC in each ethnic group, P-selectin levels were significantly associated with CAC only in African Americans and Caucasians. Furthermore, in Caucasians only, P-selectin levels were associated with incident CVD events. Association of P-selectin, per SD (20 ng/mL) for the population and cardiovascular disease Outcome African Americans n = 579 Caucasians n = 620 Chinese n = 602 Hispanics n = 605 CAC, Agatston Score Beta (S.E.) P-value Beta (S.E.) P-value Beta (S.E.) P-value Beta (S.E.) P-value Model 1 85.9 (38.4) 0.025 99.4 (28.7) <0.001 8.2 (33.3) 0.81 32.1 (27.3) 0.24 Model 2 79.3 (39.8) 0.046 94.7 (29.1) 0.001 −19.5 (33.6) 0.56 27.8 (27.6) 0.31 CVD Events HR (CI) HR (CI) HR (CI) HR (CI) Model 1 1.08 (0.75, 1.57) 0.67 1.34 (1.08, 1.65) 0.007 1.09 (0.57, 2.08) 0.80 0.99 (0.71, 1.39) 0.97 Model 2 1.10 (0.76, 1.58) 0.61 1.30 (1.05, 1.62) 0.017 0.82 (0.38, 1.78) 0.62 1.00 (0.71, 1.43) 0.98 Model 1 = age and sex ; Model 2 = age, sex, BMI, smoking status, LDL, HDL, triglycerides, hypertension, diabetes

Background: Bicuspid aortic valve disease (BAV), the most common congenital cardiovascular malformation, has an incidence of 0.5–1.0% of live births. While most cases of BAV appear to be sporadic, familial inheritance patterns have been observed consistent with autosomal dominant inheritance with variable penetrance. However, little is known about specific genetic loci responsible for familial BAV. Here, we performed linkage analysis on a large multi-generational pedigree affected with BAV. Methods: We identified a large, five-generation pedigree (136 family members) with 10 individuals having BAV. Two-dimensional echocardiography was used to assign aortic valve phenotype. Genome-wide linkage analysis using 430 microsatellite markers (Marshfield Clinic) and fine mapping using 100 single nucleotide polymorphisms (Affymetrix) on chromosome 9 was performed on genomic DNA from all available family members. Logarithm of odds (LOD) scores of >2.0 were considered suggestive of linkage. Comprehensive splice site/open reading frame mutational analysis of candidate genes residing in the putative locus is underway using PCR, DHPLC, and DNA sequencing. A candidate gene, KLF9, Krüppel-like factor 9 was analyzed for mutations because of its role in cardiogenesis. Results: Multi-point genome-wide linkage analysis demonstrated a 7 cM region on chromosome 9q21 that was suggestive of linkage for familial BAV with a maximum multipoint LOD score of 2.8 flanked by the microsatellite markers GATA7D12 and D9S1834. This region contains several candidate genes with biological plausibility for BAV phenotype. KLF9- encoded Krüppel-like factor 9, localized to chromosome 9q21, was targeted as a prime candidate gene for familial BAV. However, no mutations involving the translated exons of KLF9 were detected. Further fine mapping studies and candidate gene analysis are currently underway. Conclusions: We report a novel susceptibility locus on chromosome 9q21 for BAV in a large multi-generational family. Although coding region mutations in KLF9 are not responsible for BAV in this pedigree, several candidate genes with biological plausibility for the development of congenital BAV lie within this region and warrant further scrutiny.

Incident hypertension varies across races/ethnicities. Mechanisms implicated in the pathogenesis are endothelial dysfunction with reduction of nitric oxide and alterations of the Renin-Angiotensin-Aldosterone System (RAAS). Stromal Derived Factor 1 (SDF1) and Transforming Growth Factor β1 (TGFβ1) are essential in these processes. SDF1 is a chemotactic protein that regulates migration of endothelial progenitor cells from the bone marrow to the arterial wall to repair endothelial damage; TGFβ1 promotes extra-cellular matrix deposition within arterial vessels, inhibits nitric oxide production and stimulates renin release. In order to investigate the association of circulating levels of these proteins with incident hypertension across different ethnicities, an ethnic stratified random sample of 2,880 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) was used. Of those, 988 participants had available plasma samples and were without hypertension, defined as blood pressure ≥ 140/90 mmHg, or current use of antihypertensive medications, at baseline. During a median follow up of 8.5 years, 388 new cases of hypertension (101 in non-Hispanic whites, 94 in Chinese, 101 in African Americans and 92 in Hispanics) were observed. Time to hypertension incidence was estimated using Cox proportional hazards regression. Hazard Ratios were adjusted for age, sex, diabetes, LDL, HDL, triglycerides, BMI, smoking status and current alcohol use. The race/ethnicity by protein interaction p-values were 0.055 and 0.066 for TGFβ1 and SDF1 respectively, suggesting heterogeneity across races/ethnicities. In Chinese, a difference of one standard deviation of plasma TGFβ1 was associated with a 22% increase in incident hypertension (HR (95% CI) 1.22 (1.02, 1.47), p-value=0.03); and a difference of one standard deviation of plasma SDF1 was associated with a 31% decrease in incident hypertension (HR (95% CI) 0.69 (0.52, 0.91), p-value 0.008). No significant association was found between these proteins and incident hypertension among non-Hispanic whites, African or Hispanic Americans. In conclusion, soluble markers of endothelial integrity and of RAAS activation were associated with the incidence of hypertension among Chinese Americans. These results suggest heterogeneity in the pathophysiology of hypertension according to race/ethnicity.

L-selectin is an adhesion molecule constitutively expressed on leukocytes and mediating their interaction with the endothelium. As a member of the selectin family, it plays a crucial role in leukocyte adhesion to and migration through the endothelial wall during inflammation. A protective role of L-selectin in early atherosclerosis was demonstrated in animal models. Previous studies assessing the relationship of this protein with cardiovascular disease were inconsistent. Lower serum L-selectin levels were found in patients with coronary artery disease (CHD) and peripheral artery disease (PAD) compared to controls. In contrast, plasma levels of L-selectin were increased in patients with CHD and PAD. These inconsistencies might be due to an interaction of soluble L-selectin with clotting factors or anticoagulants present in plasma but not in serum. Also, the small number and Caucasian predominance of patients included in previous studies represent limitations that need to be addressed using a rigorous, population-based approach. Herein, we seek to investigate the association of serum L-selectin with subclinical outcomes in a large, ethnically heterogeneous population. In a randomly selected subset of 720 subjects for each of the four ethnicities included in the Multi-Ethnic Study of Atherosclerosis (MESA), serum samples were available for 2403 individuals (579 African Americans, 620 Caucasians, 600 Chinese and 604 Hispanics). Serum L-selectin levels and intima-media thickness (IMT) were measured at exam 2 and CAC was measured at exam 2 or 3 during the cohort follow up. Regression analysis and the Tobit model were used to assess the association of L-selectin levels with IMT and CAC respectively. The mean age was 63±10 years and 47% were men. The mean±SD protein levels were significantly different across ethnicities (867±192 ng/mL in African Americans, 956±215 ng/mL in Caucasians, 834±174 ng/mL in Chinese and 904±195 ng/mL in Hispanics; p<0.0001). In the univariate analysis, age was inversely correlated to the protein levels; in addition, levels were lower in males, regardless of ethnicity and in smokers, except among Hispanics. L-selectin was associated with modestly lower CAC only among Chinese (p=0.030), the association persisted after adjustment for traditional risk factors (p=0.046). No significant association was demontrated between the protein levels and IMT. In conclusion, in this large diverse population, we found that serum L-selectin was modestly associated to measures of subclinical atherosclerosis in Chinese but not in African, European or Hispanic Americans.

Pulse pressure (PP), defined as the difference between systolic and diastolic blood pressure, is considered a marker of large artery stiffness and has been shown to independently predict cardiovascular disease. Differences among ethnicities with regard to PP have been previously described, yet the underlying biological mechanisms are poorly understood. In addition, biomarkers of inflammation and matrix remodeling have been shown to be involved in the development of arterial stiffness. The aim of this study was to investigate the role of adhesion pathway components in the development of arterial stiffness measured by PP among four ethnicities. E-cadherin, HGF, L-selectin, P-selectin, 6-Ckine, MMP1, MMP2, RANTES, SDF1, SLPI, TGFβ1, TIMP2 and VCAM were measured in a random sample of 2402 participants (579 African Americans, 619 Caucasians, 600 Chinese and 604 Hispanics) in the Multi-Ethnic Study of Atherosclerosis (MESA) study at Exam 2. PP was measured concurrently and its association with each adhesion protein level was assessed via linear regression in each ethnicity. All analyses were adjusted for age, sex, and traditional cardiovascular risk factors. Mean PP was significantly different among ethnicities: 55.9±18.0 mmHg in African Americans, 52.1± 16.1 mmHg in Caucasians, 51.4±16.4 mmHg in Chinese, and 55.0±17.5 in Hispanics, p<0.001. MMP2 was positively associated with PP among African Americans (β=2.3 (0.6), p<0.001) and Caucasians (β=1.7 (0.5), p=0.001); TIMP2 among Caucasians (β=1.5 (0.5), p=0.002) and to a lesser extent among African Americans (β=1.2 (0.6) p=0.050) and Chinese (β=1.3 (0.6), p=0.034). SDF1 was associated to PP in African Americans (β=1.4 (0.6), p=0.017) and in Chinese (β=1.1 (0.5), p=0.042); finally an association existed between TGFβ1 and PP in C (β=1.1 (0.5), p=0.025). None of the studied proteins was associated with PP in Hispanics. In conclusion, components of the adhesion pathway were correlated to PP in this large multi-ethnic cohort, possibly giving new insight on the pathophysiology of arterial stiffness. In addition, heterogeneity by ethnicity suggests different mechanisms in the development of arterial stiffness.

Background: The Trans-Omics for Precision Medicine (TOPMed) program is performing whole-genome sequencing across multiple studies, including longitudinal cohorts, case-control, and case-only sample sets. Detecting the effects of low frequency variants requires large sample sizes, which can only be achieved by combining data across diverse study designs, including matching of case-only sample sets with controls from other studies. Here we present a strategy for combined analysis of venous thromboembolism (VTE) case status in 3 longitudinal cohorts and 3 case-only sample sets, in the context of whole-genome association studies. Methods: For each cohort study, we sampled ‘pure’ controls (without replacement) from the risk set of each incident case, within strata defined by sex, ancestry and birth-cohort. Pure controls had no event throughout their period of observation. A case’s risk set was defined as controls with no prior VTE history and under observation through an age at least as old as the case. For case-only sample sets, controls for each case were sampled from a cohort study, using the same risk set definition. Because of limited overlap in birth years between the cohort studies and the case-only sample sets, this matching was done within strata defined only by sex and ancestry group. Mixed model logistic regression will be used to account for relatedness as a random effect. Although conditional logistic regression is not practical for whole-genome association studies, case-control matching is implicitly recognized by a fixed effect for age-at-event for each matched set (1 case and >=1 matched controls). Additional fixed effects will include sample set and sex. We will also adjust for variations in case-control ratio among the matched sets. Results: In two cohort studies, we matched 1,231 cases to 4,820 controls (overall ratio = 1:3.9); only 500 controls and 5 cases could not be matched. For the case-only sample sets we matched 2,141 cases to 2,780 controls from one cohort study (overall ratio = 1:1.3); zero controls and only 14 cases could not be matched. Conclusions: We were successfully able to match nearly all cases to controls, and more than 90% of controls were also matched. By matching controls to cases based on age at event, we can account for the different risk of VTE by age clusters. Although this strategy does not provide an asymptotically unbiased estimated of the hazard ratio, compared to classical risk set sampling, it uses a large portion of the available data, it provides odds ratio estimates yielding the correct sign of association, and it reduces the potential influence of resampling subjects with rare variants. This strategy also enables combined analysis of multiple studies with different designs.

Menopausal hormone treatments (MHT) may limit progression of CIMT, a measure of atherosclerosis, but pose a risk for ischemic stroke. To test candidate gene variation for an association with CIMT, DNA from 714 women participating in KEEPS, a clinical trial of MHT to prevent progression of atherosclerosis, was genotyped using custom Illumina Infinium iSelect chemistry. Single nucleotide polymorphisms (SNPs; 13,229) were genotyped within 769 genes within the anticoagulant, procoagulant, fibrinolytic or innate immunity pathways. Of these SNPs, 75 were removed due to call rate<0.95, relatedness, duplication, and misidentification; 1275 were removed for quality control. There were 504 ancestry informative markers (AIM). Percent of European ancestry using AIM, age at enrollment and pulse pressure correlated positively with CIMT. Using logistic regression to adjust for these, one SNP (rs2236935 of MAP4K4 on chromosome 2) associated positively with CIMT (p‐value = 5.771e‐06). This gene encodes a protein of the serine/threonine protein kinase family involved in cytokine signaling pathways related to innate immunity, development of type II diabetes, and response to environmental stress. Whether this SNP remains associated with changes in CIMT in women randomized to hormonal treatment in KEEPS remains to be determined. (Supported by grants from the Aurora Foundation, NIH HL90639 and the Mayo Foundation)