Dov Shiffman

Cardiovascular Disease Risk Prediction With and Without Knowledge of Genetic Variation at Chromosome 9p21.3

Context Although genetic variation at chromosome 9p21.3 is associated with cardiovascular disease, it is not known whether evaluating this polymorphism adds in a clinically meaningful way to the evaluation of cardiovascular risk using more conventional risk factors, such as family history of early cardiovascular disease, smoking, blood pressure, cholesterol level, and C-reactive protein level. Contribution This study evaluated 9p21.3 polymorphism and more conventional cardiovascular risk factors in 22129 white, female health professionals followed for a median of 10 years and found that addition of the genetic information did not improve clinical classification of a womans risk for cardiovascular disease. Caution The findings may not apply to men or to nonwhite women. The Editors The success of risk prediction models for cardiovascular disease reflects an increasing understanding of the molecular basis of atherothrombosis. Aside from age, which integrates many biological activities and environmental exposures at once, other important components of risk prediction include plasma biomarkers for lipid metabolism, inflammation, thrombosis, and metabolic status (1). Current prediction models, however, cannot anticipate many cases of incident cardiovascular disease, motivating both the identification of new risk factors and the optimization of analytic methods for their use. Traditionally aggregated by the notion of family history, individual genetic variants may represent a class of risk factors with new prognostic information; they may be more removed from underlying disease processes than plasma risk factors or other clinical variables but may also be more general. For example, a recent exploration of the influence of lipid-associated variation on prediction of incident cardiovascular disease found residual predictive value for the genetic variation after adjustment for plasma lipid levels (2). Genetic variation at the chromosome 9p21.3 region is a good candidate for adding more information to risk prediction. Variation at this locus has consistently been associated with coronary artery disease (35) and diabetes (69). In addition, the risk allele is carried by almost 75% of the white population and the lack of correlation between any of the disease-associated 9p21.3 genetic variants and major cardiovascular risk factors suggests novel influences on disease progression. To assess whether knowledge of variation at 9p21.3 improves global risk prediction, we examined the effect of adding genetic information from a single nucleotide polymorphism (SNP) in the 9p21.3 region to previously published prediction models for the WHS (Womens Health Study), which has a large, prospective cohort of initially healthy U.S. women who were followed over 10 years for incident cardiovascular events. Methods Study Population Study participants were members of the WGHS (Womens Genome Health Study) (10), an ongoing prospective genetic evaluation study being conducted among initially healthy U.S. women who enrolled in the WHS, a trial of aspirin and vitamin E for the primary prevention of cardiovascular disease and cancer in women 45 years or older (11). Beginning in 1992, the WHS recruited female health professionals in the United States who had no major chronic disease at baseline, including cancer and cardiovascular disease, and followed them prospectively for incident myocardial infarction, stroke, coronary revascularization, and cardiovascular death. The institutional review board of the Brigham and Womens Hospital, Boston, Massachusetts, approved the study. Among the WHS participants, 28345 provided blood samples that were stored in liquid nitrogen until the time of analysis, as well as consent for ongoing analyses linking blood-derived observations with baseline risk factor profiles and incident disease events. Of these women, 23226 had standard risk factor information available and were genotyped for the rs10757274 polymorphism. To reduce the potential for population stratification to affect our results, we included only the 22129 (95.3%) white women. Risk Factor Ascertainment Baseline information on age, diabetes, smoking status, parental history of myocardial infarction before 60 years of age, blood pressure, and hypertension treatment were collected at study initiation. Plasma biomarkers were analyzed in a core laboratory facility, certified by the National Heart, Lung, and Blood Institute/Centers for Disease Control and Prevention Lipid Standardization Program, for total cholesterol, high-density lipoprotein cholesterol, apolipoprotein B-100, apolipoprotein A-I, high-sensitivity C-reactive protein, lipoprotein(a), and hemoglobin A1c. We determined genotypes for rs10757274 in the WGHS participants by using an oligonucleotide ligation procedure that combined polymerase chain reaction amplification of target sequences from 3 ng of genomic DNA with subsequent allele-specific oligonucleotide ligation (12). The ligation products of the 2 alleles were separated by hybridization to product-specific oligonucleotides, each coupled to spectrally distinct Luminex100 xMAP microspheres (Luminex, Austin, Texas). The captured products were fluorescently labeled with streptavidin Rphycoerythrin (Prozyme, San Leandro, California), sorted on the basis of microsphere spectrum, and detected by a Luminex100 instrument (12). Outcome Ascertainment Study participants were followed through March 2004 for total cardiovascular disease, which comprised incident myocardial infarction, ischemic stroke, coronary revascularization, and cardiovascular deaths. An end-points committee adjudicated events by using medical record review. Morbidity data were available on nearly all the women through 8 years of follow-up. Risk Prediction Models To assess the effect of variation at rs10757274 on global cardiovascular disease risk prediction, we considered covariates from 2 nongenetic risk prediction models. The first included the covariates from the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (ATP III) risk score, as well as history of diabetes (noted as a high-risk equivalent) (13). The second used the covariates from the Reynolds Risk Score (1), a model that includes additional biomarker information, as well as data on family history. To provide direct comparability and the highest level of internal validity, we elected on an a priori basis to model all covariates, including rs10757274, in the same base population rather than use -coefficients for the nongenetic covariates derived from previously published models. In so doing, we avoided the potential for bias that might occur by modeling the effect of the genetic data within the test cohort but using estimates of effect for the other covariates from a second, unrelated cohort. Statistical Analysis We used Cox proportional hazard models to generate crude and adjusted hazard ratios across genotypes and to test for trend. We used models with separate effects for each genotype in all analyses and generated adjusted survival curves by stratifying Cox proportional hazard models by genotype. We also used Cox models to generate estimates of predicted risk with and without genotype information, which we then assessed for accuracy. We saw no evidence of departures from proportionality in any of the models used. Our primary measure of discrimination was the Harrell c-index (14), a generalization of the area under the receiver-operating characteristic curve that allows for censored data. The c-index assesses the ability of the risk score to rank women who develop incident cardiovascular disease higher than women who do not. We assessed general calibration across deciles of predicted risk by using the HosmerLemeshow goodness-of-fit test (15) to compare the average predicted risk with the KaplanMeier risk estimate within each decile and considered a chi-square value of 20 or higher (P< 0.01) to be poor calibration (16). We assessed risk reclassification (1, 17) by sorting the predicted 10-year risk for each model into 4 categories (<5%, 5% to <10%, 10% to <20%, and 20%). We then compared the assigned categories for a pair of models. For each pair, we calculated the proportion of participants who were reclassified by the comparison model versus the reference model; we considered reclassification to be correct if the KaplanMeier risk estimate for the reclassified group was closer to the comparison category than the reference. We computed the HosmerLemeshow statistic for the reclassification tables (18), which assesses agreement between the KaplanMeier risk estimate and predicted risk within the reclassified categories. We also computed the Net Reclassification Improvement (19), which compares the shifts in reclassified categories by observed outcome, and the Integrated Discrimination Improvement (19), which directly compares the average difference in predicted risk for women who go on to develop cardiovascular disease with women who do not for the 2 models, on the women who were not censored before 8 years. Role of the Funding Source This study was supported by grants from the National Heart, Lung, and Blood Institute and National Cancer Institue, National Institutes of Health; the Donald W. Reynolds Foundation; and the Leducq Foundation. Additional support for DNA extraction, reagents, and data analysis was provided by Roche Diagnostics and Amgen. Genotyping of the 9p21.3 variant was performed by Celera. The funding sources had no role in the design, conduct, or reporting of this study or the decision to submit the manuscript for publication. Results Of the 22129 white women genotyped for this analysis, 5793 (26.2%) had no risk (G) alleles at rs10757374, 10952 (49.5%) had 1 risk allele, and 5384 (24.3%) had 2 risk alleles. The number of risk alleles had a significant association with family history of premature myocardial

Association of the Trp719Arg Polymorphism in Kinesin-Like Protein 6 With Myocardial Infarction and Coronary Heart Disease in 2 Prospective Trials The CARE and WOSCOPS Trials

OBJECTIVES We asked whether 35 genetic polymorphisms, previously found to be associated with cardiovascular disease, were associated with myocardial infarction (MI) in the CARE (Cholesterol and Recurrent Events) trial and with coronary heart disease (CHD) in the WOSCOPS (West of Scotland Coronary Prevention Study) trial and whether the risk associated with these polymorphisms could be reduced by pravastatin treatment. BACKGROUND Identification of genetic polymorphisms associated with CHD may improve assessment of CHD risk and understanding of disease pathophysiology. METHODS We tested the association between genotype and recurrent MI in the CARE study and between genotype and primary CHD in the WOSCOPS trial using regression models that adjusted for conventional risk factors: Cox proportional hazards models for the CARE study and conditional logistic regression models for a nested case-control study of the WOSCOPS trial. RESULTS We found that Trp719Arg (rs20455) in KIF6 was associated with coronary events. KIF6 encodes kinesin-like protein 6, a member of the molecular motor superfamily. In placebo-treated patients, carriers of the KIF6 719Arg allele (59.4% of the CARE trial cohort) had a hazard ratio of 1.50 (95% confidence interval [CI] 1.05 to 2.15) in the CARE trial and an odds ratio of 1.55 (95% CI 1.14 to 2.09) in the WOSCOPS trial. Among carriers, the absolute risk reduction by pravastatin was 4.89% (95% CI 1.81% to 7.97%) in the CARE trial and 5.49% (95% CI 3.52% to 7.46%) in the WOSCOPS trial. CONCLUSIONS In both the CARE and the WOSCOPS trials, carriers of the KIF6 719Arg allele had an increased risk of coronary events, and pravastatin treatment substantially reduced that risk.

Identification of Four Gene Variants Associated with Myocardial Infarction

Family history is a major risk factor for myocardial infarction (MI). However, known gene variants associated with MI cannot fully explain the genetic component of MI risk. We hypothesized that a gene-centric association study that was not limited to candidate genes could identify novel genetic associations with MI. We studied 11,053 single-nucleotide polymorphisms (SNPs) in 6,891 genes, focusing on SNPs that could influence gene function to increase the likelihood of identifying disease-causing gene variants. To minimize false-positive associations generated by multiple testing, two studies were used to identify a limited number of nominally associated SNPs; a third study tested the hypotheses that these SNPs are associated with MI. In the initial study (of 340 cases and 346 controls), 637 SNPs were associated with MI (P<.05); these were evaluated in a second study (of 445 cases and 606 controls), and 31 of the 637 SNPs were associated with MI (P<.05) and had the same risk allele as in the first study. For each of these 31 SNPs, we tested the hypothesis that it is associated with MI, using a third study (of 560 cases and 891 controls). We found that four of these gene variants were associated with MI (P<.05; false-discovery rate <10%) and had the same risk allele as in the first two studies. These gene variants encode the cytoskeletal protein palladin (KIAA0992 [odds ratio (OR) 1.40]), a tyrosine kinase (ROS1 [OR 1.75]), and two G protein-coupled receptors (TAS2R50 [OR 1.58] and OR13G1 [OR 1.40]); all ORs are for carriers of two versus zero risk alleles. These findings could lead to a better understanding of MI pathophysiology and improved patient risk assessment.

Association of Gene Variants With Incident Myocardial Infarction in the Cardiovascular Health Study

Objective—We asked whether single nucleotide polymorphisms (SNPs) that had been nominally associated with cardiovascular disease in antecedent studies were also associated with cardiovascular disease in a population-based prospective study of 4522 individuals aged 65 or older. Methods and Results—Based on antecedent studies, we prespecified a risk allele and an inheritance model for each of 74 SNPs. We then tested the association of these SNPs with myocardial infarction (MI) in the Cardiovascular Health Study (CHS). The prespecified risk alleles of 8 SNPs were nominally associated (1-sided P<0.05) with increased risk of MI in White CHS participants. The false discovery rate for these 8 was 0.43, suggesting that about 4 of these 8 are likely to be true positives. The 4 of these 8 SNPs that had the strongest evidence for association with cardiovascular disease before testing in CHS (association in 3 antecedent studies) were in KIF6 (CHS HR=1.29; 90%CI 1.1 to 1.52), VAMP8 (HR=1.2; 90%CI 1.02 to 1.41), TAS2R50 (HR=1.13; 90%CI 1 to 1.27), and LPA (HR=1.62; 90%CI 1.09 to 2.42). Conclusions—Although most of the SNPs investigated were not associated with MI in CHS, evidence from this investigation combined with previous studies suggests that 4 of these SNPs are likely associated with MI.

A Polymorphism in the Protease-Like Domain of Apolipoprotein(a) Is Associated With Severe Coronary Artery Disease

Objectives—The purpose of this study was to identify genetic variants associated with severe coronary artery disease (CAD). Methods and Results—We used 3 case-control studies of white subjects whose severity of CAD was assessed by angiography. The first 2 studies were used to generate hypotheses that were then tested in the third study. We tested 12 077 putative functional single nucleotide polymorphisms (SNPs) in Study 1 (781 cases, 603 controls) and identified 302 SNPs nominally associated with severe CAD. Testing these 302 SNPs in Study 2 (471 cases, 298 controls), we found 5 (in LPA, CALM1, HAP1, AP3B1, and ABCG2) were nominally associated with severe CAD and had the same risk alleles in both studies. We then tested these 5 SNPs in Study 3 (554 cases, 373 controls). We found 1 SNP that was associated with severe CAD: LPA I4399M (rs3798220). LPA encodes apolipoprotein(a), a component of lipoprotein(a). I4399M is located in the protease-like domain of apolipoprotein(a). Compared with noncarriers, carriers of the 4399M risk allele (2.7% of controls) had an adjusted odds ratio for severe CAD of 3.14 (confidence interval 1.51 to 6.56), and had 5-fold higher median plasma lipoprotein(a) levels (P=0.003). Conclusions—The LPA I4399M SNP is associated with severe CAD and plasma lipoprotein(a) levels.

Polymorphism in the apolipoprotein(a) gene, plasma lipoprotein(a), cardiovascular disease, and low-dose aspirin therapy.

OBJECTIVE A minor allele variant (rs3798220) of apolipoprotein(a) has been reported to be associated with elevated plasma lipoprotein(a) [Lp(a)] and increased cardiovascular risk. We investigated whether this allele was associated with elevated Lp(a) and cardiovascular risk in the Womens Health Study, a randomized trial of low-dose aspirin, and whether aspirin reduced cardiovascular risk in minor allele carriers. METHODS AND RESULTS Genotypes of rs3798220 were determined for 25,131 initially healthy Caucasian participants. Median Lp(a) levels at baseline were 10.0, 79.5, and 153.9mg/dL for major allele homozygotes, heterozygotes, and minor allele homozygotes, respectively (P<0.0001). During the 9.9 years of follow-up, minor allele carriers (3.7%) in the placebo group had twofold higher risk of major cardiovascular events than non-carriers (age-adjusted hazard ratio (HR)=2.21, 95% CI: 1.39-3.52). Among carriers, risk was reduced more than twofold by aspirin: for aspirin compared with placebo the age-adjusted HR was 0.44 (95% CI: 0.20-0.94); risk was not significantly reduced among non-carriers (age-adjusted HR=0.91, 95% CI: 0.77-1.08). This interaction between carrier status and aspirin allocation was significant (P=0.048). CONCLUSIONS In the Womens Health Study, carriers of an apolipoprotein(a) variant had elevated Lp(a), doubled cardiovascular risk, and appeared to benefit more from aspirin than non-carriers.

A Kinesin Family Member 6 Variant Is Associated With Coronary Heart Disease in the Women’s Health Study

OBJECTIVES We asked if carriers of the 719Arg allele of kinesin family member 6 (KIF6) have increased risk of coronary heart disease (CHD) in a cohort of initially healthy Caucasian American women. BACKGROUND The 719Arg allele of KIF6 (rs20455) has been reported to be associated with increased risk of CHD in a large population-based prospective study, ARIC (Atherosclerosis Risk in Communities), and in the placebo arms of 2 statin trials, CARE (Cholesterol and Recurrent Events) and WOSCOPS (West of Scotland Coronary Prevention Study). However, this KIF6 variant was not specifically investigated in the female subgroup in the ARIC study, and the CARE and WOSCOPS trials included only a small number of female patients. METHODS Genotypes of the rs20455 single nucleotide polymorphism (SNP) were determined among 25,283 initially healthy Caucasian women, age 45 years and older, participating in the WHS (Womens Health Study) who were prospectively followed over a 12-year period for incident cardiovascular events. The risk associated with the 719Arg allele of KIF6 was estimated using Cox proportional hazards models that adjusted for age and traditional risk factors. RESULTS During follow-up, 953 women suffered a first-ever CHD event (myocardial infarction, coronary revascularization, or cardiovascular death) or first-ever ischemic stroke. Compared with noncarriers, carriers of the 719Arg allele had an increased risk of CHD (hazard ratio [HR] = 1.24 [95% confidence interval (CI) 1.04 to 1.46, p = 0.013]) and myocardial infarction (HR = 1.34 [95% CI 1.02 to 1.75, p = 0.034]) but not ischemic stroke. CONCLUSIONS Confirming and extending previous reports, carriers of the 719Arg allele of KIF6 have 34% higher risk of myocardial infarction and 24% higher risk of CHD compared with noncarriers among 25,283 women from the WHS.

Five common gene variants identify elevated genetic risk for coronary heart disease

Purpose: Because multiple genetic variants influence risk for coronary heart disease, we combined multiple variants that had been associated with coronary heart disease in several studies into a genetic risk score and asked whether a high genetic risk score would be significantly associated with coronary heart disease after accounting for traditional risk factors.Methods: We considered five variants that were associated with coronary heart disease in two studies and confirmed in the Atherosclerosis Risk in Communities study: rs20455 (KIF6), rs3900940 (MYH15), rs7439293 (PALLD), rs2298566 (SNX19), and rs1010 (VAMP8). We calculated a genetic risk score for each Atherosclerosis Risk in Communities study participant and estimated the hazard ratio for incident coronary heart disease of a high genetic risk score (compared with not-high) in Cox models that adjusted for traditional risk factors during a median of 13 years of follow-up.Results: For white participants with a high genetic risk score (4% of the 9129 whites), compared with those without a high genetic risk score, the hazard ratio for incident coronary heart disease was 1.57 (95% confidence interval 1.21–2.04; P = 0.001). Internal validation using bootstrap samples estimated that a hazard ratio of 1.43 could be expected in external populations.Conclusions: After adjusting for traditional risk factors, those with a high genetic risk score had a 57% increased risk of incident coronary heart disease in the Atherosclerosis Risk in Communities study.

Gene Variants of VAMP8 and HNRPUL1 Are Associated With Early-Onset Myocardial Infarction

Objectives—Identify gene variants associated with early-onset myocardial infarction (MI). Methods and Results—We tested 11 647 single-nucleotide polymorphisms (SNPs) for association with early-onset MI in a case-control study (study 1 200 cases, 262 controls). To reduce the number of false positives among the 666 SNPs that were nominally associated with early-onset MI (P<0.05) in study 1, we tested these SNPs in study 2 (434 cases, 504 controls). We found that 8 of the 666 SNPs were associated with early-onset MI in study 2 (P<0.05) and had the same risk alleles as in study 1. These 8 SNPs were then tested for association with early-onset MI in study 3 (187 cases, 434 controls). We found that a VAMP8 variant (P=0.025; odds ratio [OR], 1.75; CI, 1.17 to 2.62) and an HNRPUL1 variant (P=0.0043; OR, 1.92; CI, 1.28 to 2.86) were associated with early-onset MI (nominal P<0.05; false discovery rate <10%) and had the same risk alleles in all 3 studies. Conclusions—Variants in 2 genes were associated with early-onset MI: VAMP8, which is involved in platelet degranulation, and HNRPUL1, which encodes a ribonuclear protein. The identification of these variants could improve understanding of disease mechanisms and suggest novel drug targets.

Risk prediction by genetic risk scores for coronary heart disease is independent of self-reported family history.

Abstract Aims Genetic risk scores (GRSs) have been associated with coronary heart disease (CHD) in large studies. We asked whether expanding an established 27-variant GRS (GRS27) to a 50-variant GRS (GRS50) improved CHD prediction and whether GRSs are independent of self-reported family history of CHD. Methods and results The association between GRSs and incident CHD was assessed in Cox models adjusting for established risk factors in 23 595 participants of the Malmö Diet and Cancer study—a prospective, population-based study. During a median follow-up of 14.4 years, 2213 participants experienced a first CHD event. After adjustment for established risk factors, both GRS27 and GRS50 were associated with incident CHD [hazard ratio (HR) = 1.70 for high (top quintile) vs. low (bottom quintile) of GRS27; 95% confidence interval (CI): 1.48–1.94; Ptrend = 1.6 × 10−15 and HR = 1.92 for GRS50; 95% CI: 1.67–2.20; Ptrend = 6.2 × 10−22]. Adding 23 single nucleotide polymorphisms (SNPs) to GRS27 improved risk prediction (P = 3 × 10−6). Further adjustment for self-reported family history did not appreciably change the risk estimates of either GRS27 (HR = 1.65; 95% CI: 1.45–1.89) or GRS50 (HR = 1.87; 95% CI: 1.63–2.14). The addition of GRS50 to established risk factors, including self-reported family history, improved discrimination (P < 0.0001) and reclassification (continuous net reclassification improvement index = 0.17, P < 0.0001). In young participants (below median age), those with high GRS50 had 2.4-fold greater risk (95% CI: 1.85–3.12) than those with low GRS50. Conclusion The addition of 23 SNPs to an existing GRS27 improved CHD risk prediction and was independent of self-reported family history. Coronary heart disease risk assessment by GRS could be particularly useful in young individuals.