Correction: Single-trait and multi-trait genome-wide association analyses identify novel loci for blood pressure in African-ancestry populations.

[This corrects the article DOI: 10.1371/journal.pgen.1006728.].

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.

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.

Quantitative variation in plasma angiotensin-I converting enzyme activity shows allelic heterogeneity in the ABO blood group locus.

Angiotensin-I converting enzyme (ACE) occupies a pivotal role in cardiovascular homeostasis. Major loci for plasma ACE have been identified at ACE on Chromosome 17 and at ABO on Chromosome 9. We sought to characterise the genetic architecture of plasma ACE at finer resolution in two populations. We carried out a GWAS in 1810 individuals of Japanese ethnicity; this identified signals at ACE and ABO that together accounted for nearly half of the population variability of the trait. We conducted measured haplotype analysis at the ABO locus in 1425 members of 248 British families using haplotypes of three SNPs, which together tagged the alleles responsible for the principal blood group antigens A1, A2, B and O. Type O alleles were associated with intermediate plasma ACE activity compared to Type A1 alleles (in whom plasma ACE activity was ∼36% lower) and Type B alleles (in whom plasma ACE activity was ∼36% higher). We demonstrated heterogeneity among A alleles: A2 alleles were associated with plasma ACE activity that was very similar to the O alleles. Variation at ACE accounted for 35% of the trait variance, and variation at ABO accounted for 15%. A further 10% could be ascribed to polygenic effects.

Rapid assessment of genetic ancestry in populations of unknown origin by genome-wide genotyping of pooled samples.

As we move forward from the current generation of genome-wide association (GWA) studies, additional cohorts of different ancestries will be studied to increase power, fine map association signals, and generalize association results to additional populations. Knowledge of genetic ancestry as well as population substructure will become increasingly important for GWA studies in populations of unknown ancestry. Here we propose genotyping pooled DNA samples using genome-wide SNP arrays as a viable option to efficiently and inexpensively estimate admixture proportion and identify ancestry informative markers (AIMs) in populations of unknown origin. We constructed DNA pools from African American, Native Hawaiian, Latina, and Jamaican samples and genotyped them using the Affymetrix 6.0 array. Aided by individual genotype data from the African American cohort, we established quality control filters to remove poorly performing SNPs and estimated allele frequencies for the remaining SNPs in each panel. We then applied a regression-based method to estimate the proportion of admixture in each cohort using the allele frequencies estimated from pooling and populations from the International HapMap Consortium as reference panels, and identified AIMs unique to each population. In this study, we demonstrated that genotyping pooled DNA samples yields estimates of admixture proportion that are both consistent with our knowledge of population history and similar to those obtained by genotyping known AIMs. Furthermore, through validation by individual genotyping, we demonstrated that pooling is quite effective for identifying SNPs with large allele frequency differences (i.e., AIMs) and that these AIMs are able to differentiate two closely related populations (HapMap JPT and CHB).

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.

The efficacy of detecting variants with small effects on the Affymetrix 6.0 platform using pooled DNA.

Genome-wide genotyping of a cohort using pools rather than individual samples has long been proposed as a cost-saving alternative for performing genome-wide association (GWA) studies. However, successful disease gene mapping using pooled genotyping has thus far been limited to detecting common variants with large effect sizes, which tend not to exist for many complex common diseases or traits. Therefore, for DNA pooling to be a viable strategy for conducting GWA studies, it is important to determine whether commonly used genome-wide SNP array platforms such as the Affymetrix 6.0 array can reliably detect common variants of small effect sizes using pooled DNA. Taking obesity and age at menarche as examples of human complex traits, we assessed the feasibility of genome-wide genotyping of pooled DNA as a single-stage design for phenotype association. By individually genotyping the top associations identified by pooling, we obtained a 14- to 16-fold enrichment of SNPs nominally associated with the phenotype, but we likely missed the top true associations. In addition, we assessed whether genotyping pooled DNA can serve as an inexpensive screen as the second stage of a multi-stage design with a large number of samples by comparing the most cost-effective 3-stage designs with 80% power to detect common variants with genotypic relative risk of 1.1, with and without pooling. Given the current state of the specific technology we employed and the associated genotyping costs, we showed through simulation that a design involving pooling would be 1.07 times more expensive than a design without pooling. Thus, while a significant amount of information exists within the data from pooled DNA, our analysis does not support genotyping pooled DNA as a means to efficiently identify common variants contributing small effects to phenotypes of interest. While our conclusions were based on the specific technology and study design we employed, the approach presented here will be useful for evaluating the utility of other or future genome-wide genotyping platforms in pooled DNA studies.

Ethnic differences in F cell levels in Jamaica: a potential tool for identifying new genetic loci controlling fetal haemoglobin.

High levels of fetal haemoglobin (HbF) are protective in beta-haemoglobinopathies. The proportion of erythrocytes containing HbF (F-cells, FC) was measured in healthy adults of African and Caucasian ancestry to assess the feasibility of localizing genes for the FC trait using admixture mapping. Participants were Afro-Caribbean (AC) blood donors and residents of a rural enclave with a history of recent German admixture (Afro-German, AG) recruited in Jamaica, and Caucasian Europeans recruited in Jamaica and the UK. FC levels were significantly different between groups (P < 0.001); the geometric mean FC level in the AC sample (n = 176) was 3.75% [95% confidence interval (CI) 3.36-4.18], AG sample (n = 631) was 2.77% (95% CI 2.63-2.92), and among Caucasians (n = 1099) was 3.26% (95% CI 3.13-3.39). After adjustment for age, sex, haemoglobin electrophoresis pattern, and HBG2 genotype, FC levels in the AC group remained significantly different (P < 0.001) from those in the Caucasian and the AG group but the difference between the Caucasian and AG groups became non-significant (P = 0.46) despite substantial differences in average ancestry. The data confirm ethnic differences in FC levels and indicate the potential usefulness of these populations for admixture mapping of genes for FC levels.

Edematous severe acute malnutrition is characterized by hypomethylation of DNA.

Edematous severe acute childhood malnutrition (edematous SAM or ESAM), which includes kwashiorkor, presents with more overt multi-organ dysfunction than non-edematous SAM (NESAM). Reduced concentrations and methyl-flux of methionine in 1-carbon metabolism have been reported in acute, but not recovered, ESAM, suggesting downstream DNA methylation changes could be relevant to differences in SAM pathogenesis. Here, we assess genome-wide DNA methylation in buccal cells of 309 SAM children using the 450 K microarray. Relative to NESAM, ESAM is characterized by multiple significantly hypomethylated loci, which is not observed among SAM-recovered adults. Gene expression and methylation show both positive and negative correlation, suggesting a complex transcriptional response to SAM. Hypomethylated loci link to disorders of nutrition and metabolism, including fatty liver and diabetes, and appear to be influenced by genetic variation. Our epigenetic findings provide a potential molecular link to reported aberrant 1-carbon metabolism in ESAM and support consideration of methyl-group supplementation in ESAM.

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.

Vitamin D levels are low in adult patients with sickle cell disease in Jamaica and West Africa.

BACKGROUND: Patients with sickle cell disease in the USA have been noted to have lower levels of vitamin D - measured as 25-hydroxyvitamin D (25(OH)D) - compared to controls. Average serum 25(OH)D levels are also substantially lower in African Americans than whites, while population distributions of 25(OH)D among Jamaicans of African descent and West Africans are the same as among USA whites. The purpose of this study was to examine whether adult patients with sickle cell disease living in tropical regions had reduced 25(OH)D relative to the general population. METHODS: We analyzed serum 25(OH)D in stored samples collected from studies in Jamaica and West Africa of adult patients with sickle cell disease and adult population controls. RESULTS: In samples of 20 Jamaicans and 50 West Africans with sickle cell disease mean values of 25(OH)D were 37% and 39% lower than controls, respectively. Metabolic abnormalities in the absorption and conversion pathways are possible causes for the consistent relative deficiency of 25(OH)D in sickle cell disease. CONCLUSIONS: Low 25(OH)D levels in tropical Africa where the burden of sickle cell disease is highest, deserve further investigation, and a randomized trial is warranted to address efficacy of supplementation.

Genetic variation in APOL1 and MYH9 genes is associated with chronic kidney disease among Nigerians.

PURPOSE: A region of chromosome 22 which includes APOL1 and MYH9 genes was recently identified as a risk locus for non-diabetic forms of kidney disease, including idiopathic and HIV-associated focal segmental glomerular sclerosis and kidney disease clinically attributed to hypertension among African Americans. The purposes of the current study were, therefore, to examine the frequency of these variants and to determine whether they are associated with chronic kidney disease (CKD) among native Africans. METHODS: To investigate the possible evidence of association between variants in these genes and non-diabetic CKD among West Africans, we performed a case/control analysis in a sample of 166 Nigerians without history of European admixture. Our study included a total of 9 variants on APOL1 (n = 4) and MYH9 (n = 5) genes. RESULTS: We observed significantly strong associations with previously reported APOL1 variants rs73885319 and rs60910145, and their two-allele "G1" haplotype (P < 0.005). We did not observe significant evidence of association between non-diabetic CKD and any of the MYH9 variants or haplotypes after accounting for multiple testing in our sample. CONCLUSIONS: In conclusion, APOL1 risk variants are associated with non-diabetic forms of CKD among Nigerians of Yoruba ethnicity. Further information on APOL1/MYH9 variants may lead to screening programs, which could lead to earlier detection and interventions for non-diabetic kidney disease.

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 x 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.

A genome-wide search replicates evidence of a quantitative trait locus for circulating angiotensin I-converting enzyme (ACE) unlinked to the ACE gene.

BACKGROUND: Angiotensin I-converting enzyme (ACE) plays an important role in cardiovascular homeostasis. There is evidence from different ethnic groups that circulating ACE levels are influenced by a quantitative trait locus (QTL) at the ACE gene on chromosome 17. The finding of significant residual familial correlations in different ethnic groups, after accounting for this QTL, and the finding of support for linkage to a locus on chromosome 4 in Mexican-American families strongly suggest that there may well be QTLs for ACE unlinked to the ACE gene. METHODS: A genome-wide panel of microsatellite markers, and a panel of biallelic polymorphisms in the ACE gene were typed in Nigerian families. Single locus models with fixed parameters were used to test for linkage to circulating ACE with and without adjustment for the effects of the ACE gene polymorphisms. RESULTS: Strong evidence was found for D17S2193 (Zmax = 3.5); other nearby markers on chromosome 17 also showed modest support. After adjustment for the effects of the ACE gene locus, evidence of "suggestive linkage" to circulating ACE was found for D4S1629 (Zmax = 2.2); this marker is very close to a locus previously shown to be linked to circulating ACE levels in Mexican-American families. CONCLUSION: In this report we have provided further support for the notion that there are QTLs for ACE unlinked to the ACE gene; our findings for chromosome 4, which appear to replicate the findings of a previous independent study, should be considered strong grounds for a more detailed examination of this region in the search for genes/variants which influence ACE levels.The poor yields, thus far, in defining the genetic determinants of hypertension risk suggest a need to look beyond simple relationships between genotypes and the ultimate phenotype. In addition to incorporating information on important environmental exposures, a better understanding of the factors which influence the building blocks of the blood pressure homeostatic network is also required. Detailed studies of the genetic determinants of ACE, an important component of the renin-angiotensin system, have the potential to contribute to this strategic objective.

Childhood malnutrition is associated with a reduction in the total melanin content of scalp hair.

Childhood malnutrition is known to be associated with visible lightening of hair colour (hypochromotrichia). Nevertheless, no systematic investigations have been carried out to determine the biochemical basis of this change. We used an HPLC method to measure melanins in the scalp hair of thirteen Jamaican children, diagnosed as having primary malnutrition, during various stages of their treatment and after recovery. During treatment for malnutrition, a progressive decrease in total melanin content along the hair shaft from tip to root (root:tip ratio: 0.62 (sd 0.31)) was observed. This ratio was significantly different (P = 0.003) from the ratio observed among children sampled several months after discharge from hospital (0.93 (sd 0.23)) and among normal control children (0.97 (sd 0.12)). Thus, it appears that a decrease in melanin content is associated with periods of malnutrition. The low root:tip ratio during malnutrition presumably arises because the tips reflect prior hair growth during 'normal' nutrition and the roots reflect hair growth during malnutrition; a return of the root:tip ratio to that seen among controls reflects 'recovery' from malnutrition. It is possible that reduced intake or availability of tyrosine, a key substrate in melanin synthesis, may play a role in the reduction of hair melanin content during periods of malnutrition. The precise mechanisms by which melanin content is reduced, and the role of aromatic amino acid availability in hair colour change and other features of childhood malnutrition remain to be explored.

Glutathione S-transferase polymorphisms may be associated with risk of oedematous severe childhood malnutrition.

It has been estimated that more than 50 % of deaths before the age of 5 years have undernutrition as an underlying cause. Severe childhood malnutrition, an extreme form of undernutrition, occurs as oedematous and non-oedematous syndromes. The reasons why only some children develop oedematous severe childhood malnutrition (OSCM) have remained elusive, but the heterogeneity of clinical appearances among children from relatively homogeneous backgrounds suggests that interindividual variation in susceptibility to OSCM may exist. We investigated variants of four glutathione S-transferase (GST) genes in a retrospective study among subjects (n 136) previously admitted to the Tropical Metabolism Research Unit, Jamaica, for the treatment of either OSCM (cases) or non-oedematous severe childhood malnutrition (controls). We found that GSTP1 Val(105) homozygotes were significantly more common among the cases (odds ratio (OR) 3.5; 95 % CI 1.1, 10.8). We also found an association of borderline significance between non-deletion GSTT1 genotypes (i.e. +/+ or +/0) and OSCM (OR 2.4; 95 % CI 1.0, 5.9). There was no significant association between OSCM and any of the other GST variants. These preliminary findings suggest that genetic variation within the GST superfamily may contribute to the risk of OSCM. Additional, larger data sets and studies of variants in other candidate genes are required in order to properly assess the true contribution, if any, of genetic variation to risk of OSCM. Such studies may improve our understanding of the causes of clinical heterogeneity in malnutrition.

Polymorphisms in genes involved in folate metabolism as risk factors for oedematous severe childhood malnutrition: a hypothesis-generating study.

BACKGROUND: Severe childhood malnutrition (SCM) occurs as both oedematous and non-oedematous syndromes. The reasons why some children develop oedematous SCM (OSCM) have remained elusive but differences in clinical presentation among malnourished children from similar backgrounds suggests that there might be inter-individual variation in susceptibility to OSCM. AIM: To estimate the strength of the association between variants of three genes involved in folate/methyl group metabolism [methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and cystathionine beta-synthase (CBS)] and risk of OSCM. METHODS: Patients previously admitted to the Tropical Metabolism Research Unit (TMRU) for treatment of either OSCM (cases, n = 74) or non-oedematous SCM (NOSCM, controls, n = 50) were recruited. Genotypes at four sites within the three genes (MTHFR C677T, MTHFR A1298C, MTR A2756G and CBS 844ins68) were determined using PCR-based assays. RESULTS: The MTHFR 677T [odds ratio (OR) 0.63, 95% CI 0.2-1.7] and MTR 2756G (OR 0.74, 95% CI 0.4-1.4) alleles were associated with moderate reduction in risk of OSCM whereas the CBS 844ins68 allele (OR 1.4, 0.7-2.4) was associated with an increased risk. None of these risks was significant at the 5% level. CONCLUSIONS: Genetic variation within folate/methyl group metabolic pathways might have a small but potentially important influence on risk of OSCM. Additional, larger data-sets will be required to test the specific hypotheses (about the putative effect size and direction of association) generated in this preliminary study. Such observations have the potential to improve our understanding of the pathogenesis of clinical heterogeneity in severe malnutrition.

UGT1A1 variation and gallstone formation in sickle cell disease.

Pigment gallstones are a common clinical complication of sickle cell (SS) disease. Genetic variation in the promoter of uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) underlies Gilbert syndrome, a chronic form of unconjugated hyperbilirubinemia, and appears to be a risk factor for gallstone formation. We investigated the association between UGT1A1 (TA)(n) genotype, hyperbilirubinemia, and gallstones in a sample of Jamaicans with SS disease. Subjects were from the Jamaican Sickle Cell Cohort Study (cohort sample, n = 209) and the Sickle Cell Clinic at the University of the West Indies, Kingston, Jamaica (clinic sample, n = 357). The UGT1A1 (TA)(n) promoter region was sequenced in 541 SS disease subjects and 111 healthy controls (control sample). Indirect bilirubin levels for (TA)(7)/(TA)(7) and (TA)(7)/(TA)(8) genotypes were elevated compared with (TA)(6)/(TA)(6) (clinic sample, P < 10(-5); cohort sample, P < 10(-3)). The (TA)(7)/(TA)(7) genotype was also associated with symptomatic presentation and gallstones in the clinic sample (odds ratio [OR] = 11.3; P = 7.0 x 10(-4)) but not in the younger cohort sample. These unexpected findings indicate that the temporal evolution of symptomatic gallstones may involve factors other than the bilirubin level. Although further studies of the pathogenesis of gallstones in SS disease are required, the (TA)(7)/(TA)(7) genotype may be a risk factor for symptomatic gallstones in older people with SS disease.