Joshua P. Lewis

Association Analysis in African Americans of European-Derived Type 2 Diabetes Single Nucleotide Polymorphisms From Whole-Genome Association Studies

OBJECTIVE— Several whole-genome association studies have reported identification of type 2 diabetes susceptibility genes in various European-derived study populations. Little investigation of these loci has been reported in other ethnic groups, specifically African Americans. Striking differences exist between these populations, suggesting they may not share identical genetic risk factors. Our objective was to examine the influence of type 2 diabetes genes identified in whole-genome association studies in a large African American case-control population. RESEARCH DESIGN AND METHODS— Single nucleotide polymorphisms (SNPs) in 12 loci (e.g., TCF7L2, IDE/KIF11/HHEX, SLC30A8, CDKAL1, PKN2, IGF2BP2, FLJ39370, and EXT2/ALX4) associated with type 2 diabetes in European-derived populations were genotyped in 993 African American type 2 diabetic and 1,054 African American control subjects. Additionally, 68 ancestry-informative markers were genotyped to account for the impact of admixture on association results. RESULTS— Little evidence of association was observed between SNPs, with the exception of those in TCF7L2, and type 2 diabetes in African Americans. One TCF7L2 SNP (rs7903146) showed compelling evidence of association with type 2 diabetes (admixture-adjusted additive P [Pa] = 1.59 × 10−6). Only the intragenic SNP on 11p12 (rs9300039, dominant P [Pd] = 0.029) was also associated with type 2 diabetes after admixture adjustments. Interestingly, four of the SNPs are monomorphic in the Yoruba population of the HAPMAP project, with only the risk allele from the populations of European descent present. CONCLUSIONS— Results suggest that these variants do not significantly contribute to interindividual susceptibility to type 2 diabetes in African Americans. Consequently, genes contributing to type 2 diabetes in African Americans may, in part, be different from those in European-derived study populations. High frequency of risk alleles in several of these genes may, however, contribute to the increased prevalence of type 2 diabetes in African Americans.

A GENOME WIDE ASSOCIATION STUDY FOR DIABETIC NEPHROPATHY GENES IN AFRICAN AMERICANS

A genome-wide association study was performed using the Affymetrix 6.0 chip to identify genes associated with diabetic nephropathy in African Americans. Association analysis was performed adjusting for admixture in 965 type 2 diabetic African American patients with end-stage renal disease (ESRD) and in 1029 African Americans without type 2 diabetes or kidney disease as controls. The top 724 single nucleotide polymorphisms (SNPs) with evidence of association to diabetic nephropathy were then genotyped in a replication sample of an additional 709 type 2 diabetes-ESRD patients and 690 controls. SNPs with evidence of association in both the original and replication studies were tested in additional African American cohorts consisting of 1246 patients with type 2 diabetes without kidney disease and 1216 with non-diabetic ESRD to differentiate candidate loci for type 2 diabetes-ESRD, type 2 diabetes, and/or all-cause ESRD. Twenty-five SNPs were significantly associated with type 2 diabetes-ESRD in the genome-wide association and initial replication. Although genome-wide significance with type 2 diabetes was not found for any of these 25 SNPs, several genes, including RPS12, LIMK2, and SFI1 are strong candidates for diabetic nephropathy. A combined analysis of all 2890 patients with ESRD showed significant association SNPs in LIMK2 and SFI1 suggesting that they also contribute to all-cause ESRD. Thus, our results suggest that multiple loci underlie susceptibility to kidney disease in African Americans with type 2 diabetes and some may also contribute to all-cause ESRD.

The functional G143E variant of carboxylesterase 1 is associated with increased clopidogrel active metabolite levels and greater clopidogrel response

Introduction Carboxylesterase 1 (CES1) is the primary enzyme responsible for converting clopidogrel into biologically inactive carboxylic acid metabolites. Methods We genotyped a functional variant in CES1, G143E, in participants of the Pharmacogenomics of Anti-Platelet Intervention (PAPI) study (n=566) and in 350 patients with coronary heart disease treated with clopidogrel, and carried out an association analysis of bioactive metabolite levels, on-clopidogrel ADP-stimulated platelet aggregation, and cardiovascular outcomes. Results The levels of clopidogrel active metabolite were significantly greater in CES1 143E-allele carriers (P=0.001). Consistent with these findings, individuals who carried the CES1 143E-allele showed a better clopidogrel response as measured by ADP-stimulated platelet aggregation in both participants of the PAPI study (P=0.003) and clopidogrel-treated coronary heart disease patients (P=0.03). No association was found between this single nucleotide polymorphism and baseline measures of platelet aggregation in either cohort. Conclusion Taken together, these findings suggest, for the first time, that genetic variation in CES1 may be an important determinant of the efficacy of clopidogrel.

Paraoxonase 1 (PON1) Gene Variants Are Not Associated With Clopidogrel Response

A common functional variant in paraoxonase 1 (PON1), Q192R, was recently reported to be a major determinant of clopidogrel response. This variant was genotyped in 566 participants of the Amish Pharmacogenomics of Anti–Platelet Intervention (PAPI) study and in 227 percutaneous coronary intervention (PCI) patients. Serum paraoxonase activity was measured in a subset of 79 PAPI participants. PON1 Q192R was not associated with pre– or post–clopidogrel platelet aggregation in the PAPI study (P = 0.16 and P = 0.21, respectively) or the PCI cohort (P = 0.47 and P = 0.91, respectively). The Q192 allele was not associated with cardiovascular events (hazard ratio (HR) 0.46, 95% confidence interval (CI) 0.20–1.06; P = 0.07). No correlation was observed between paraoxonase activity and post–clopidogrel platelet aggregation (r2 < 0.01, P = 0.78). None of 49 additional PON1 variants evaluated was associated with post–clopidogrel platelet aggregation. These findings do not support a role for PON1 as a determinant of clopidogrel response.

Genetic Variation in PEAR1 Is Associated With Platelet Aggregation and Cardiovascular Outcomes

Background—Aspirin or dual antiplatelet therapy with aspirin and clopidogrel is a standard therapy for patients who are 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 dual antiplatelet therapy in individuals (n=565) from the Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study and conducted a genome-wide association study of drug response. Significant findings were extended by examining genotype and cardiovascular outcomes in 2 independent aspirin-treated cohorts: 227 percutaneous coronary intervention patients and 1000 patients of the International Verapamil SR/Trandolapril Study (INVEST) Genetic Substudy (INVEST-GENES). Results from the genome-wide association study revealed a strong association between single-nucleotide polymorphisms on chromosome 1q23 and post–dual antiplatelet therapyplatelet aggregation. Further genotyping revealed rs12041331 in the platelet endothelial aggregation receptor-1 (PEAR1) gene to be most strongly associated with dual antiplatelet therapy response (P=7.66×10−9). In white and black patients undergoing percutaneous coronary intervention, A-allele carriers of rs12041331 were more likely to experience a cardiovascular event or death compared with GG homozygotes (hazard ratio, 2.62; 95% confidence interval, 0.96–7.10; P=0.059; and hazard ratio, 3.97; 95% confidence interval, 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 with GG homozygotes (odds ratio, 2.03; 95% confidence interval, 1.01–4.09; P=0.048). Conclusion—Common genetic variation in PEAR1 may be a determinant of platelet response and cardiovascular events in patients on aspirin alone or in combination with clopidogrel. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00799396 and NCT00370045

Pharmacogenomics: application to the management of cardiovascular disease.

The past decade has seen substantial advances in cardiovascular pharmacogenomics. Genetic determinants of response to clopidogrel and warfarin have been defined, resulting in changes to the product labels for these drugs that suggest the use of genetic information as a guide for therapy. Genetic tests are available, as are guidelines for incorporation of genetic information into patient‐care decisions. These guidelines and the literature supporting them are reviewed herein. Significant advances have also been made in the pharmacogenomics of statin‐induced myopathy and the response to β‐blockers in heart failure, although the clinical applications of these findings are less clear. Other areas hold promise, including the pharmacogenomics of antihypertensive drugs, aspirin, and drug‐induced long‐QT syndrome (diLQTS). The potential value of pharmacogenomics in the discovery and development of new drugs is also described. In summary, pharmacogenomics has current applications in the management of cardiovascular disease, with clinically relevant data continuing to mount.

Purine Pathway Implicated in Mechanism of Resistance to Aspirin Therapy: Pharmacometabolomics-Informed Pharmacogenomics

Although aspirin is a well‐established antiplatelet agent, the mechanisms of aspirin resistance remain poorly understood. Metabolomics allows for measurement of hundreds of small molecules in biological samples, enabling detailed mapping of pathways involved in drug response. We defined the metabolic signature of aspirin exposure in subjects from the Heredity and Phenotype Intervention Heart Study. Many metabolites, including known aspirin catabolites, changed on exposure to aspirin, and pathway enrichment analysis identified purine metabolism as significantly affected by drug exposure. Furthermore, purines were associated with aspirin response, and poor responders had higher postaspirin adenosine and inosine levels than did good responders (n = 76; both P < 4 × 10−3). Using our established “pharmacometabolomics‐informed pharmacogenomics” approach, we identified genetic variants in adenosine kinase associated with aspirin response. Combining metabolomics and genomics allowed for more comprehensive interrogation of mechanisms of variation in aspirin response—an important step toward personalized treatment approaches for cardiovascular disease.

The CYP2C19*17 variant is not independently associated with clopidogrel response

Cytochrome P450 2C19 (CYP2C19) is the principal enzyme responsible for converting clopidogrel into its active metabolite, and common genetic variants have been identified, most notably CYP2C19*2 and CYP2C19*17, that are believed to alter its activity and expression, respectively.

Heterogeneity in gene loci associated with type 2 diabetes on human chromosome 20q13.1

Human chromosome 20q12-q13.1 has been linked to type 2 diabetes mellitus (T2DM) in multiple studies. We screened a 5.795-Mb region for diabetes-related susceptibility genes in a Caucasian cohort of 310 controls and 300 cases with T2DM and end-stage renal disease (ESRD), testing 390 SNPs for association with T2DM-ESRD. The most significant SNPs were found in the perigenic regions: HNF4A (hepatocyte nuclear factor 4alpha), SLC12A5 (potassium-chloride cotransporter member 5), CDH22 (cadherin-like 22), ELMO2 (engulfment and cell motility 2), SLC13A3 (sodium-dependent dicarboxylate transporter member 3), and PREX1 (phosphatidylinositol 3,4,5-triphosphate-dependent RAC exchanger 1). Haplotype analysis found six haplotype blocks globally associated with disease (p<0.05). We replicated the PREX1 SNP association in an independent case-control T2DM population and inferred replication of CDH22, ELMO2, SLC13A3, SLC12A5, and PREX1 using in silico perigenic analysis of two T2DM Genome-Wide Association Study data sets. We found substantial heterogeneity between study results.

Pharmacometabolomics reveals that serotonin is implicated in aspirin response variability.

While aspirin is generally effective for prevention of cardiovascular disease, considerable variation in drug response exists, resulting in some individuals displaying high on‐treatment platelet reactivity. We used pharmacometabolomics to define pathways implicated in variation of response to treatment. We profiled serum samples from healthy subjects pre‐ and postaspirin (14 days, 81 mg/day) using mass spectrometry. We established a strong signature of aspirin exposure independent of response (15/34 metabolites changed). In our discovery (N = 80) and replication (N = 125) cohorts, higher serotonin levels pre‐ and postaspirin correlated with high, postaspirin, collagen‐induced platelet aggregation. In a third cohort, platelets from subjects with the highest levels of serotonin preaspirin retained higher reactivity after incubation with aspirin than platelets from subjects with the lowest serotonin levels preaspirin (72 ± 8 vs. 61 ± 11%, P = 0.02, N = 20). Finally, ex vivo, serotonin strongly increased platelet reactivity after platelet incubation with aspirin (+20%, P = 4.9 × 10−4, N = 12). These results suggest that serotonin is implicated in aspirin response variability.