Genomewide Association Study Identifies Copy Number Variants Associated With Warfarin Dose Response and Risk of Venous Thromboembolism in African Americans.

The anticoagulant warfarin is commonly used to control and prevent thrombotic disorders, such as venous thromboembolism (VTE), which disproportionately afflicts African Americans. Despite the importance of copy number variants (CNVs), few studies have focused on characterizing and understanding their role in drug response and disease risk among African Americans. In this study, we conduct the first genome-wide analysis of CNVs to more comprehensively account for the contribution of genetic variation in warfarin dose requirement and VTE risk among African Americans. We used hidden Markov models to detect CNVs from high-throughput single-nucleotide polymorphism arrays for 340 African American participants in the International Warfarin Pharmacogenetics Consortium. We identified 11,570 CNVs resulting in 2,038 copy number variable regions (CNVRs) and found 3 CNVRs associated with warfarin dose requirement and 3 CNVRs associated with VTE risk in African Americans. CNVRs 1q31.2del and 6q14.1del were associated with increased warfarin dose requirement (ß = 11.18 and 4.94, respectively; Pemp  = < 0.002); CNVR 19p13.31del was associated with decreased warfarin dose requirement (ß = -1.41, Pemp  = 0.0004); CNVRs (2p22.1del and 5q35.1-q35.2del) were found to be associated with increased risk of VTE (odds ratios (ORs) = 1.88 and 14.9, respectively; Pemp ≤0.02); and CNVR 10q26.12del was associated with a decreased risk of VTE (OR = 0.6; Pemp  = 0.05). Modeling of the 10q26.12del in HepG2 cells revealed that this deletion results in decreased fibrinogen gene expression, decreased fibrinogen and WDR11 protein levels, and decreased secretion of fibrinogen into the extracellular matrix. We found robust evidence that CNVRs could contribute to warfarin dose requirement and risk of VTE in African Americans and for 10q26.3del describe a possible pathogenic mechanism.

Genetic loci associated with skin pigmentation in African Americans and their effects on vitamin D deficiency.

A recent genome-wide association study (GWAS) in African descent populations identified novel loci associated with skin pigmentation. However, how genomic variations affect skin pigmentation and how these skin pigmentation gene variants affect serum 25(OH) vitamin D variation has not been explored in African Americans (AAs). In order to further understand genetic factors that affect human skin pigmentation and serum 25(OH)D variation, we performed a GWAS for skin pigmentation with 395 AAs and a replication study with 681 AAs. Then, we tested if the identified variants are associated with serum 25(OH) D concentrations in a subset of AAs (n = 591). Skin pigmentation, Melanin Index (M-Index), was measured using a narrow-band reflectometer. Multiple regression analysis was performed to identify variants associated with M-Index and to assess their role in serum 25(OH)D variation adjusting for population stratification and relevant confounding variables. A variant near the SLC24A5 gene (rs2675345) showed the strongest signal of association with M-Index (P = 4.0 x 10-30 in the pooled dataset). Variants in SLC24A5, SLC45A2 and OCA2 together account for a large proportion of skin pigmentation variance (11%). The effects of these variants on M-Index was modified by sex (P for interaction = 0.009). However, West African Ancestry (WAA) also accounts for a large proportion of M-Index variance (23%). M-Index also varies among AAs with high WAA and high Genetic Score calculated from top variants associated with M-Index, suggesting that other unknown genomic factors related to WAA are likely contributing to skin pigmentation variation. M-Index was not associated with serum 25(OH)D concentrations, but the Genetic Score was significantly associated with vitamin D deficiency (serum 25(OH)D levels less than 12 ng/mL) (OR, 1.30; 95% CI, 1.04-1.64). The findings support the hypothesis suggesting that skin pigmentation evolved responding to increased demand for subcutaneous vitamin D synthesis in high latitude environments.

Genetic ancestry, skin color and social attainment: The four cities study.

INTRODUCTION: The Black population in the US is heterogeneous but is often treated as monolithic in research, with skin pigmentation being the primary indicator of racial classification. Objective: This paper examines the differences among Blacks by comparing genetic ancestry, skin color and social attainment of 259 residents across four US cities-Norman, Oklahoma; Cincinnati, Ohio; Harlem, New York; and Washington, District of Columbia. METHODS: Participants were recruited between 2004 and 2006 at community-based forums. Cross-sectional data were analyzed using chi-square tests, correlation analyses and logistic regression. RESULTS: There were variations in ancestry, melanin index and social attainment across some cities. Overall, men with darker skin color, and women with lighter skin color were significantly more likely to be married. Darker skin individuals with significantly more West African ancestry reported attainment of graduate degrees, and professional occupations than lighter skin individuals. CONCLUSIONS: Our findings suggest differences in skin pigmentation by geography and support regional variations in ancestry of US Blacks. Biomedical research should consider genetic ancestry and local historical/social context rather than relying solely on skin pigmentation as a proxy for race.

Pharmacogenomic genotypes define genetic ancestry in patients and enable population-specific genomic implementation.

The importance of genetic ancestry characterization is increasing in genomic implementation efforts, and clinical pharmacogenomic guidelines are being published that include population-specific recommendations. Our aim was to test the ability of focused clinical pharmacogenomic SNP panels to estimate individual genetic ancestry (IGA) and implement population-specific pharmacogenomic clinical decision-support (CDS) tools. Principle components and STRUCTURE were utilized to assess differences in genetic composition and estimate IGA among 1572 individuals from 1000 Genomes, two independent cohorts of Caucasians and African Americans (AAs), plus a real-world validation population of patients undergoing pharmacogenomic genotyping. We found that clinical pharmacogenomic SNP panels accurately estimate IGA compared to genome-wide genotyping and identify AAs with ≥70 African ancestry (sensitivity >82%, specificity >80%, PPV >95%, NPV >47%). We also validated a new AA-specific warfarin dosing algorithm for patients with ≥70% African ancestry and implemented it at our institution as a novel CDS tool. Consideration of IGA to develop an institutional CDS tool was accomplished to enable population-specific pharmacogenomic guidance at the point-of-care. These capabilities were immediately applied for guidance of warfarin dosing in AAs versus Caucasians, but also provide a real-world model that can be extended to other populations and drugs as actionable genomic evidence accumulates.

Association of Genetic Variants With Warfarin-Associated Bleeding Among Patients of African Descent.

Importance: Major warfarin-related bleeding occurs more frequently in African Americans than in other populations. Identification of potential genetic factors related to this adverse event may help identify at-risk patients. Objective: To identify genetic factors associated with warfarin-related bleeding in patients of African descent at an international normalized ratio (INR) of less than 4. Design, Setting, and Participants: A case-control genome-wide association study involving patients of African descent taking warfarin was conducted in a discovery cohort (University of Chicago [2009-2011] and the University of Illinois at Chicago [2002-2011]), and associations were confirmed in a replication cohort (University of Chicago [2015-2016]). Potential population stratification was examined in the discovery cohort by principal component analysis. Odds ratios (ORs) and 95% CIs were computed for bleeding risk by logistic regression analysis. Summary statistics from the discovery and the replication cohorts were analyzed with a fixed effects meta-analysis. The potential influence of single-nucleotide polymorphisms (SNPs) on gene expression was studied by luciferase expression assays. Exposures: Single-nucleotide polymorphisms associated with warfarin-related bleeding. Main Outcomes and Measures: Major bleeding-defined as bleeding requiring hospitalization, causing a decrease in hemoglobin level of more than 2 g/dL, requiring blood transfusion, or any combination of the 3-while taking warfarin at an INR of less than 4. Results: The discovery cohort consisted of 31 cases (mean age, 60.1 years [SD, 14.9 years], 26 women [83.9%]) and 184 warfarin-treated controls (mean age, 57.1 years [SD, 15.7 years]) with no documented bleeding. The replication cohort consisted of 40 cases (mean age, 55.6 years [SD, 17.3 years], 27 women [67.5%]), and 148 warfarin-treated controls (mean age, 55.4 years [SD, 17.1 years]; 98 women [66.2%]) with no documented bleeding. In the discovery cohort, 4 SNPs in linkage disequilibrium on chromosome 6 (rs115112393, rs16871327, rs78132896, and rs114504854) were associated with warfarin-related bleeding but did not reach genome-wide significance. The SNP rs78132896 occurred in 11 cases (35.5%) and 9 controls (4.9%) in the discovery cohort (OR, 8.31; 95% CI, 3.2-21.5; P < 6.21 × 10-8), and the association was confirmed in the replication cohort (the SNP was present in 14 cases [35.0%] and 7 controls [4.8%]; OR, 8.24; 95% CI, 3.1-25.3, P = 5.64 × 10-5). Genome-wide significance of this SNP was achieved when the cohorts were combined via meta-analysis (OR, 8.27; 95% CI, 4.18-16.38; P = 2.05 × 10-11). These SNPs are found only in people of African descent. In vitro luciferase expression assays demonstrated that rs16871327 (enhancer SNP) and rs78132896 (promoter SNP) risk alleles together increased EPHA7 gene (Entrez Gene 2045) transcription by a mean of 14.95 (SD, 1.7) compared with wild-type alleles (mean, 9.56 [SD, 0.84]; difference, 5.39; 95% CI, 4.1-6.6; P < .001). Conclusions and Relevance: In this preliminary study involving patients of African descent taking warfarin, 4 single-nucleotide polymorphisms in linkage disequilibrium on chromosome 6 were associated with an increased risk of major bleeding at INR of less than 4. Validation of these findings in an independent prospective cohort is required.

Novel genetic predictors of venous thromboembolism risk in African Americans.

Venous thromboembolism (VTE) is the third most common life-threatening cardiovascular condition in the United States, with African Americans (AAs) having a 30% to 60% higher incidence compared with other ethnicities. The mechanisms underlying population differences in the risk of VTE are poorly understood. We conducted the first genome-wide association study in AAs, comprising 578 subjects, followed by replication of highly significant findings in an independent cohort of 159 AA subjects. Logistic regression was used to estimate the association between genetic variants and VTE risk. Through bioinformatics analysis of the top signals, we identified expression quantitative trait loci (eQTLs) in whole blood and investigated the messenger RNA expression differences in VTE cases and controls. We identified and replicated single-nucleotide polymorphisms on chromosome 20 (rs2144940, rs2567617, and rs1998081) that increased risk of VTE by 2.3-fold (P< 6 × 10(-7)). These risk variants were found in higher frequency among populations of African descent (>20%) compared with other ethnic groups (<10%). We demonstrate that SNPs on chromosome 20 are cis-eQTLs for thrombomodulin (THBD), and the expression of THBD is lower among VTE cases compared with controls (P= 9.87 × 10(-6)). We have identified novel polymorphisms associated with increased risk of VTE in AAs. These polymorphisms are predominantly found among populations of African descent and are associated with THBD gene expression. Our findings provide new molecular insight into a mechanism regulating VTE susceptibility and identify common genetic variants that increase the risk of VTE in AAs, a population disproportionately affected by this disease.

Factors influencing pharmacokinetics of warfarin in African-Americans: implications for pharmacogenetic dosing algorithms.

AIM: This study attempted to identify predictors of S-warfarin clearance (CL[S]) and to make a pharmacokinetic evaluation of genotype-based dosing algorithms in African-Americans. METHODS: Using plasma S-warfarin concentration (Cp[S]) at a steady state and eight SNPs previously shown to influence warfarin dose in African-Americans, CL(S) and its predictors were estimated by population pharmacokinetic analysis in 60 African-Americans. The time courses of Cp(S) following either the loading dose or maintenance dose were simulated using the population pharmacokinetic estimates. RESULTS: CYP2C9*8 and body surface area or body weight were predictors of CL(S) (-30 and -5% per -0.1 m(2)/-10 kg reduction in CL[S], respectively) in African-Americans. Simulations of Cp(S) showed that Cp(S) at steady state was 1.4-times higher in patients with CYP2C9*8 than in those with CYP2C9*1/*1, irrespective of the algorithm for loading dose or maintenance dose. CONCLUSION: African-Americans possess independent predictors of CL(S), possibly leading to a prediction error of any dosing algorithm that excludes African-specific variant(s). Original submitted 3 September 2014; Revision submitted 3 November 2014.

Novel single nucleotide polymorphism in CYP2C9 is associated with changes in warfarin clearance and CYP2C9 expression levels in African Americans.

Warfarin is a widely used anticoagulant whose active S-enantiomer is primarily metabolized by the CYP2C9 enzyme. The CYP2C9*2 and CYP2C9*3 alleles are associated with lower warfarin dose requirement and decreased enzyme activity. In contrast, we previously identified a novel single-nucleotide polymorphism (SNP) (rs7089580A > T) in CYP2C9 that is associated with higher warfarin dose requirement in African Americans (AAs). In this study, we examine the effect of rs7089580 on warfarin pharmacokinetics and CYP2C9 expression in 63 AA patients and 32 AA liver tissues, respectively. We found oral clearance of S-warfarin to be higher among carriers of the minor rs7089580 allele (T) compared with wild-type homozygotes (3.73 ± 1.46 vs 2.95 ± 1.39 mL/min; P = 0.04). CYP2C9 messenger RNA expression in liver tissue was also higher among A/T and T/T genotypes compared with A/A (P < 0.02). Our findings indicate that rs7089580 is associated with higher S-warfarin clearance and CYP2C9 expression and may help explain the higher dose requirement of warfarin in AAs. Furthermore, rs7089580 is in complete linkage disequilibrium with the promoter SNP rs12251841 in AAs, which may provide a biologically plausible explanation for the observed effect on CYP2C9 expression levels. Given the many clinically relevant substrates of CYP2C9, identifying polymorphisms that affect expression levels and metabolism across ethnicities is essential for individualization of doses with a narrow therapeutic index.

Regional differences in awareness and attitudes regarding genetic testing for disease risk and ancestry.

Little is known about the lay public's awareness and attitudes concerning genetic testing and what factors influence their perspectives. The existing literature focuses mainly on ethnic and socioeconomic differences; however, here we focus on how awareness and attitudes regarding genetic testing differ by geographical regions in the US. We compared awareness and attitudes concerning genetic testing for disease risk and ancestry among 452 adults (41% Black and 67% female) in four major US cities, Norman, OK; Cincinnati, OH; Harlem, NY; and Washington, DC; prior to their participation in genetic ancestry testing. The OK participants reported more detail about their personal ancestries (p = 0.02) and valued ancestry testing over disease testing more than all other sites (p < 0.01). The NY participants were more likely than other sites to seek genetic testing for disease (p = 0.01) and to see benefit in finding out more about one's ancestry (p = 0.02), while the DC participants reported reading and hearing more about genetic testing for African ancestry than all other sites (p < 0.01). These site differences were not better accounted for by sex, age, education, self-reported ethnicity, religion, or previous experience with genetic testing/counseling. Regional differences in awareness and attitudes transcend traditional demographic predictors, such as ethnicity, age and education. Local sociocultural factors, more than ethnicity and socioeconomic status, may influence the public's awareness and belief systems, particularly with respect to genetics.

Replication of prostate cancer risk loci on 8q24, 11q13, 17q12, 19q33, and Xp11 in African Americans.

BACKGROUND: Prostate cancer (Pca) is a common malignancy that disproportionately affects African American men (AA). Recently there have been several genome-wide association studies (GWAS) implicating new prostate cancer risk loci along chromosomes 2, 3, 6, 7, 8, 10, 11, 12, 17, 19, and X in populations of European ancestry. Given the higher incidence and mortality for AAs, and differences in allele frequencies and haplotype structures between African and European descent populations, it is important to assess the impact of these candidate risk loci in AAs. METHODS: Here we evaluated 20 single nucleotide polymorphisms (SNPs) associated with prostate cancer risk in recent GWAS studies, in AA prostate cancer cases and controls. RESULTS: We replicated five of the SNPs in our AA population, rs10896449 on 11q13.2 (P = 0.009), rs2735839 on 19q33.33 region, (P = 0.04), rs443076 on chromosome 17q12 (P = 0.008), rs5945572 on Xp11.22 (P = 0.05), as well as the rare variant specific to west African ancestry, bd11934905 in region 2 of 8q24 (P = 1 x 10(-4)). CONCLUSIONS: While we were able to replicate a few of the previous GWAS SNPs, we were not able to confirm the vast majority of these associations in our AA population. This finding further supports the need to perform GWAS and additional fine mapping in AAs to locate additional susceptibility loci.

Association of HPC2/ELAC2 and RNASEL non-synonymous variants with prostate cancer risk in African American familial and sporadic cases.

INTRODUCTION: The RNASEL and HPC2/ELAC2 genes have been implicated in hereditary prostate cancer. Further assessment of the role of these genes in sporadic prostate cancer in African American men (AAM) is warranted. METHODS: Genotyping of HPC2/ELAC2 variants (S217L, A541T), along with RNASEL variants (R462Q and E541D) was completed in 155 African American sporadic and 88 familial prostate cancer cases, and 296 healthy male controls. Logistic regression analysis was performed and odds ratios (OR) were calculated, while correcting for both age and population stratification using admixture informative markers. RESULTS: The HPC2/ELAC2 217L allele was significantly associated with risk of prostate cancer when taking all cases into account (OR = 1.6; 1.0-2.6; P = 0.03). The RNASEL 541D allele was associated with a decrease in risk of prostate cancer in sporadic cases (OR = 0.4; 0.2-0.8; P = 0.01). We did not detect an association between prostate cancer risk and the RNASEL R462Q variant. Results from haplotype analyses of the two RNASEL variants revealed highly significant differences in haplotype allele frequencies between cases and controls suggesting a synergistic effect at the RNASEL locus. One haplotype in particular (462R-541D) is far more frequent in our control population and shows a strong protective effect against prostate cancer (OR = 0.47, P = 8.1 x 10(-9)). CONCLUSIONS: These results suggest that HPC2/ELAC2 and RNASEL may play a role, however minor, in prostate cancer risk among AAM.

Confirmation study of prostate cancer risk variants at 8q24 in African Americans identifies a novel risk locus.

Prostate cancer is a common complex disease that disproportionately affects men of African descent. Recently, several different common variants on chromosome 8q24 have been shown to be associated with prostate cancer in multiple studies and ethnic groups. The objective of this study was to confirm the association of 8q24 markers with prostate cancer in African Americans. We genotyped 24 markers along 8q24 and 80 unlinked ancestry informative markers in a hospital-based case-control sample of 1057 African American men (490 prostate cancer cases and 567 controls). Association analyses of 8q24 markers with prostate cancer risk were adjusted for both global and local 8q24 admixture stratification using estimates from ancestry informative markers. We report that rs7008482, which maps to the 8q24.13 region, is an additional independent prostate cancer risk variant (P = 5 x 10(-4)), and we also replicate the association of rs16901979 with prostate cancer (P = 0.002). Other published risk variants in the region such as rs1447295 and rs6983267 showed a similar direction and magnitude of effect, but were not significant in our population. Both rs7008482 and rs16901979 independently predicted risk and remained significant (P < 0.001) after controlling for each other. Our data combined with additional replications of 8q24 markers provide compelling support for multiple regions of risk for prostate cancer on 8q24.

IGF-1 and IGFBP-3 gene variants influence on serum levels and prostate cancer risk in African-Americans.

Insulin-like growth factor (IGF)-1 and Insulin-like growth factor binding protein-3 (IGFBP-3) are strong inhibitors of apoptosis and play a role in mediating the effects of growth hormone. Both IGF-1 and IGFBP-3 serum levels have been linked to cancer risk. Here, we explore the relationship between three common IGF polymorphisms [C/T single-nucleotide polymorphism (SNP) (rs7965399) and a dinucleotide repeat (CA)n within the 5' regulatory region of the IGF-1 gene and the -202 A/C SNP in the IGFBP-3 gene], serum levels and prostate cancer (Pca) risk in 767 African-Americans enrolled in a clinic-based case-control study. IGF-1 and IGFBP-3 levels were measured using immunochemiluminometric assay and the three polymorphisms were typed for 401 Pca cases and 366 age- and ethnicity-matched controls. Multiple linear regression and multivariable unconditional logistic regression were used to test for associations between genotypes and circulating IGF levels and Pca risk, respectively. The presence of at least one copy of the IGFBP-3 -202 C allele was strongly associated with lower IGFBP-3 serum levels (3532 versus 3106 ng/ml; P = 0.008). We also observed a 2-fold increase in Pca risk for individuals homozygous for the IGFBP-3 -202 C allele [odds ratio = 2.4; 95% confidence interval = 1.2-4.8). Furthermore, IGF-1 (CA)19 genotypes were significantly associated with lower IGFBP-3 serum levels (P = 0.003). Our results reveal that variation in the 5'-untranslated region of the IGF-1 and IGFBP-3 genes may be influencing IGF serum levels and Pca risk in African-Americans and suggest a need to explore this variation across diverse populations. Our study adds clarity and further support to the previous findings, implicating serum IGFBP-3 levels and the IGFBP-3 -202 A/C SNP in prostate carcinogenesis.

ICAM gene cluster SNPs and prostate cancer risk in African Americans.

Intercellular adhesion molecules (ICAMs) are known to be involved in various human cancers. An ICAM gene cluster lying within a 26 kb region on chromosome 19p13.2, and containing ICAM1, ICAM4, and ICAM5 has recently been identified as harboring a breast and prostate cancer susceptibility locus in two populations of European ancestry from Germany and Australia. The objective of this study was to confirm the ICAM association with prostate cancer in a sample of African American prostate cancer cases (N = 286) and controls (N = 391). Six single nucleotide polymorphisms (SNPs) within the three ICAM genes were genotyped. To control for potential population stratification an ancestry-adjusted association analysis was performed. We found that ICAM1 SNPs, -9A/C (rs5490) and K469E (rs5498) were associated with prostate cancer risk in men with a family history of prostate cancer (P = 0.008). Specifically, increased risk was observed for individuals who possessed the CC genotype of the -9 A/C variant (odds ratio = 2.5; 95% CI = 1.0-6.3) and at least one G allele of non-synonymous K469E variant (odds ratio = 1.8; 95% CI = 1.2-3.1). Strong linkage disequilibrium was observed across the ICAM region (P < 0.001). A common haplotype within the ICAM gene cluster, harboring the -9A/C variant was significantly associated with prostate cancer (P = 0.03), mainly due to men with family history (P = 0.01). Our results replicate previous findings of association of the ICAM gene cluster with prostate cancer and suggest that common genetic variation within ICAM1 and not ICAM5 may be an important risk factor for prostate cancer.