Colin A. McKenzie

Large-scale test of hypothesised associations between the angiotensin-converting-enzyme insertion/deletion polymorphism and myocardial infarction in about 5000 cases and 6000 controls

BACKGROUND The original report of a possible association between myocardial infarction and the insertion/deletion (I/D) polymorphism of the gene for the angiotensin-1-converting enzyme (ACE) indicated a risk ratio for myocardial infarction with the DD genotype of 1.34 (95% CI 1.05-1.70), and the association was claimed to be particularly strong in a retrospectively defined low-risk subgroup (3.2 [95% CI 1.7-5.9). Subsequent investigations reached varying conclusions, but all were small, and much larger studies were needed. METHODS 4629 myocardial infarction cases and 5934 controls were compared. Cases were UK men aged 30-54 years and women aged 30-64 years recruited on presentation to hospital with confirmed myocardial infarction. Controls were aged 30-64 years with no history of cardiovascular disease, but were siblings or children of myocardial infarction survivors, or spouses of such relatives. All risk-ratio calculations allow for this relatedness of some of the controls. An updated meta-analysis of previous studies was also conducted. FINDINGS The ACE DD genotype was found in 1359 (29.4%) of the myocardial infarction cases and in 1637 (27.6%) of the controls (risk ratio 1.10 [95% CI 1.00-1.21]). The association between myocardial infarction and the DD genotype did not seem to be stronger in the subgroup defined as low risk by previously used criteria (234 [28%] of 836 cases and 911 [28%] of 3253 controls: risk ratio 1.04 [95% CI 0.87-1.24]), or in any other subgroup. Nor was the ACE I/D genotype predictive of subsequent survival. INTERPRETATION This study involved many more cases than any previously reported study of this question, but did not confirm the existence of any substantial association. In an updated meta-analysis of these results with those of previously published studies, the risk ratio for myocardial infarction with the DD genotype seems to lie in the range 1.0 to about 1.1. Although an increase in risk of up to about 10-15% cannot be ruled out, substantially more extreme risks can be. Moreover, there are not especially strong associations in the subgroups previously selected for emphasis. These findings illustrate the need for some studies of candidate genes to involve much larger populations than is customary, without undue emphasis on retrospectively defined subgroups.

Linkage and Association Analysis of Angiotensin I–Converting Enzyme (ACE)–Gene Polymorphisms with ACE Concentration and Blood Pressure

Considerable effort has been expended to determine whether the gene for angiotensin I-converting enzyme (ACE) confers susceptibility to cardiovascular disease. In this study, we genotyped 13 polymorphisms in the ACE gene in 1,343 Nigerians from 332 families. To localize the genetic effect, we first performed linkage and association analysis of all the markers with ACE concentration. In multipoint variance-component analysis, this region was strongly linked to ACE concentration (maximum LOD score 7.5). Likewise, most of the polymorphisms in the ACE gene were significantly associated with ACE (P<.0013). The two most highly associated polymorphisms, ACE4 and ACE8, accounted for 6% and 19% of the variance in ACE, respectively. A two-locus additive model with an additive x additive interaction of these polymorphisms explained most of the ACE variation associated with this region. We next analyzed the relationship between these two polymorphisms (ACE4 and ACE8) and blood pressure (BP). Although no evidence of linkage was detected, significant association was found for both systolic and diastolic BP when a two-locus additive model developed for ACE concentration was used. Further analyses demonstrated that an epistasis model provided the best fit to the BP variation. In conclusion, we found that the two polymorphisms explaining the greatest variation in ACE concentration are significantly associated with BP, through interaction, in this African population sample. Our study also demonstrates that greater statistical power can be anticipated with association analysis versus linkage, when markers in strong linkage disequilibrium with a trait locus have been identified. Furthermore, allelic interaction may play an important role in the dissection of complex traits such as BP.

Genome-Wide Association Study of Coronary Heart Disease and Its Risk Factors in 8,090 African Americans: The NHLBI CARe Project

Coronary heart disease (CHD) is the leading cause of mortality in African Americans. To identify common genetic polymorphisms associated with CHD and its risk factors (LDL- and HDL-cholesterol (LDL-C and HDL-C), hypertension, smoking, and type-2 diabetes) in individuals of African ancestry, we performed a genome-wide association study (GWAS) in 8,090 African Americans from five population-based cohorts. We replicated 17 loci previously associated with CHD or its risk factors in Caucasians. For five of these regions (CHD: CDKN2A/CDKN2B; HDL-C: FADS1-3, PLTP, LPL, and ABCA1), we could leverage the distinct linkage disequilibrium (LD) patterns in African Americans to identify DNA polymorphisms more strongly associated with the phenotypes than the previously reported index SNPs found in Caucasian populations. We also developed a new approach for association testing in admixed populations that uses allelic and local ancestry variation. Using this method, we discovered several loci that would have been missed using the basic allelic and global ancestry information only. Our conclusions suggest that no major loci uniquely explain the high prevalence of CHD in African Americans. Our project has developed resources and methods that address both admixture- and SNP-association to maximize power for genetic discovery in even larger African-American consortia.

Genome-wide association of anthropometric traits in African- and African-derived populations

Genome-wide association (GWA) studies have identified common variants that are associated with a variety of traits and diseases, but most studies have been performed in European-derived populations. Here, we describe the first genome-wide analyses of imputed genotype and copy number variants (CNVs) for anthropometric measures in African-derived populations: 1188 Nigerians from Igbo-Ora and Ibadan, Nigeria, and 743 African-Americans from Maywood, IL. To improve the reach of our study, we used imputation to estimate genotypes at approximately 2.1 million single-nucleotide polymorphisms (SNPs) and also tested CNVs for association. No SNPs or common CNVs reached a genome-wide significance level for association with height or body mass index (BMI), and the best signals from a meta-analysis of the two cohorts did not replicate in approximately 3700 African-Americans and Jamaicans. However, several loci previously confirmed in European populations showed evidence of replication in our GWA panel of African-derived populations, including variants near IHH and DLEU7 for height and MC4R for BMI. Analysis of global burden of rare CNVs suggested that lean individuals possess greater total burden of CNVs, but this finding was not supported in an independent European population. Our results suggest that there are not multiple loci with strong effects on anthropometric traits in African-derived populations and that sample sizes comparable to those needed in European GWA studies will be required to identify replicable associations. Meta-analysis of this data set with additional studies in African-ancestry populations will be helpful to improve power to detect novel associations.

Fine mapping of the association with obesity at the FTO locus in African-derived populations

Genome-wide association studies have identified many common genetic variants that are associated with polygenic traits, and have typically been performed with individuals of recent European ancestry. In these populations, many common variants are tightly correlated, with the perfect or near-perfect proxies for the functional or true variant showing equivalent evidence of association, considerably limiting the resolution of fine mapping. Populations with recent African ancestry often have less extensive and/or different patterns of linkage disequilibrium (LD), and have been proposed to be useful in fine-mapping studies. Here, we strongly replicate and fine map in populations of predominantly African ancestry the association between variation at the FTO locus and body mass index (BMI) that is well established in populations of European ancestry. We genotyped single nucleotide polymorphisms that are correlated with the signal of association in individuals of European ancestry but that have varying degrees of correlation in African-derived individuals. Most of the variants, including one previously proposed as functionally important, have no significant association with BMI, but two variants, rs3751812 and rs9941349, show strong evidence of association (P = 2.58 x 10(-6) and 3.61 x 10(-6) in a meta-analysis of 9881 individuals). Thus, we have both strongly replicated this association in African-ancestry populations and narrowed the list of potentially causal variants to those that are correlated with rs3751812 and rs9941349 in African-derived populations. This study illustrates the potential of using populations with different LD patterns to fine map associations and helps pave the way for genetically guided functional studies at the FTO locus.

Genetic Variation on Chromosome 6 Influences F Cell Levels in Healthy Individuals of African Descent and HbF Levels in Sickle Cell Patients

Fetal haemoglobin (HbF) is a major ameliorating factor in sickle cell disease. We investigated if a quantitative trait locus on chromosome 6q23 was significantly associated with HbF and F cell levels in individuals of African descent. Single nucleotide polymorphisms (SNPs) in a 24-kb intergenic region, 33-kb upstream of the HBS1L gene and 80-kb upstream of the MYB gene, were typed in 177 healthy Afro-Caribbean subjects (AC) of approximately 7% European admixture, 631 healthy Afro-Germans (AG, a group of African and German descendents located in rural Jamaica with about 20% European admixture), 87 West African and Afro-Caribbean individuals with sickle cell anaemia (HbSS), as well as 75 Northern Europeans, which served as a contrasting population. Association with a tag SNP for the locus was detected in all four groups (AC, P = 0.005, AG, P = 0.002, HbSS patients, P = 0.019, Europeans, P = 1.5×10−7). The association signal varied across the interval in the African-descended groups, while it is more uniform in Europeans. The 6q QTL for HbF traits is present in populations of African origin and is also acting in sickle cell anaemia patients. We have started to distinguish effects originating from European and African ancestral populations in our admixed study populations.

Large-scale evidence that the cardiotoxicity of smoking is not significantly modified by the apolipoprotein E ε2/ε3/ε4 genotype

Summary Results from two small studies, involving a total of only 174 cases, have suggested that the increased risk of coronary heart disease conferred by cigarette smoking is substantially affected by genotype at the apolipoprotein E ( APOE ) e2/e3/e4 polymorphism. We have established APOE genotypes in 4484 patients with acute myocardial infarction diagnosed before the age of 55 years for male and 65 years for female patients, and in 5757 controls with no history of cardiovascular disease. On average, the hazard ratio for myocardial infarction was 1·17 (95% CI 1·09–1·25; p APOE genotypes (χ 2 2 =0·69; p=0·7). When differences in risk between different genotypes are not extreme (as with this APOE polymorphism), reliable assessment of hypothesised gene-environment interactions will often require the study of many thousands of disease cases.

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

Single-Trait and Multi-Trait Genome-Wide Association Analyses Identify Novel Loci for Blood Pressure in African-Ancestry Populations

Hypertension is a leading cause of global disease, mortality, and disability. While individuals of African descent suffer a disproportionate burden of hypertension and its complications, they have been underrepresented in genetic studies. To identify novel susceptibility loci for blood pressure and hypertension in people of African ancestry, we performed both single and multiple-trait genome-wide association analyses. We analyzed 21 genome-wide association studies comprised of 31,968 individuals of African ancestry, and validated our results with additional 54,395 individuals from multi-ethnic studies. These analyses identified nine loci with eleven independent variants which reached genome-wide significance (P < 1.25×10−8) for either systolic and diastolic blood pressure, hypertension, or for combined traits. Single-trait analyses identified two loci (TARID/TCF21 and LLPH/TMBIM4) and multiple-trait analyses identified one novel locus (FRMD3) for blood pressure. At these three loci, as well as at GRP20/CDH17, associated variants had alleles common only in African-ancestry populations. Functional annotation showed enrichment for genes expressed in immune and kidney cells, as well as in heart and vascular cells/tissues. Experiments driven by these findings and using angiotensin-II induced hypertension in mice showed altered kidney mRNA expression of six genes, suggesting their potential role in hypertension. Our study provides new evidence for genes related to hypertension susceptibility, and the need to study African-ancestry populations in order to identify biologic factors contributing to hypertension.

Angiotensin I-converting enzyme polymorphisms, ACE level and blood pressure among Nigerians, Jamaicans and African-Americans

The genes in the renin–angiotensin system are important physiologic candidates in studies of the genetic susceptibility to hypertension. Limited information has been available in most studies on the extent of variation in the candidate loci or the modifying effects of different environmental settings. We consequently genotyped 13 polymorphisms at the angiotensin I-converting enzyme (ACE) locus at an average distance of 2 kb in 2776 family members from Nigeria, Jamaica and an African-American community in the US. Allele and haplotype frequencies were similar in the three populations, with modest evidence of European admixture in the US. Two markers were consistently associated with ACE level in the three samples and the proportion of variance accounted for by ACE8 was similar in the three groups. No evidence of consistent association of single markers was noted with blood pressure across the three population samples, however. Likewise, in a haplotype-based analysis, despite significant associations within each population, the findings were not replicated consistently across all three samples. We did observe, however, that the overtransmitted haplotypes among hypertensives were drawn from a single clade, suggesting that susceptibility may cluster in patterns not captured directly by our markers.