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1.  GWAS of blood cell traits identifies novel associated loci and epistatic interactions in Caucasian and African-American children 
Human Molecular Genetics  2012;22(7):1457-1464.
Hematological traits are important clinical indicators, the genetic determinants of which have not been fully investigated. Common measures of hematological traits include red blood cell (RBC) count, hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), mean corpuscular volume (MCV), platelet count (PLT) and white blood cell (WBC) count. We carried out a genome-wide association study of the eight common hematological traits among 7943 African-American children and 6234 Caucasian children. In African Americans, we report five novel associations of HBE1 variants with HCT and MCHC, the alpha-globin gene cluster variants with RBC and MCHC, and a variant at the ARHGEF3 locus with PLT, as well as replication of four previously reported loci at genome-wide significance. In Caucasians, we report a novel association of variants at the COPZ1 locus with PLT as well as replication of four previously reported loci at genome-wide significance. Extended analysis of an association observed between MCH and the alpha-globin gene cluster variants demonstrated independent effects and epistatic interaction at the locus, impacting the risk of iron deficiency anemia in African Americans with specific genotype states. In summary, we extend the understanding of genetic variants underlying hematological traits based on analyses in African-American children.
PMCID: PMC3657475  PMID: 23263863
2.  Genome-wide association analysis of red blood cell traits in African Americans: the COGENT Network 
Human Molecular Genetics  2013;22(12):2529-2538.
Laboratory red blood cell (RBC) measurements are clinically important, heritable and differ among ethnic groups. To identify genetic variants that contribute to RBC phenotypes in African Americans (AAs), we conducted a genome-wide association study in up to ∼16 500 AAs. The alpha-globin locus on chromosome 16pter [lead SNP rs13335629 in ITFG3 gene; P < 1E−13 for hemoglobin (Hgb), RBC count, mean corpuscular volume (MCV), MCH and MCHC] and the G6PD locus on Xq28 [lead SNP rs1050828; P < 1E − 13 for Hgb, hematocrit (Hct), MCV, RBC count and red cell distribution width (RDW)] were each associated with multiple RBC traits. At the alpha-globin region, both the common African 3.7 kb deletion and common single nucleotide polymorphisms (SNPs) appear to contribute independently to RBC phenotypes among AAs. In the 2p21 region, we identified a novel variant of PRKCE distinctly associated with Hct in AAs. In a genome-wide admixture mapping scan, local European ancestry at the 6p22 region containing HFE and LRRC16A was associated with higher Hgb. LRRC16A has been previously associated with the platelet count and mean platelet volume in AAs, but not with Hgb. Finally, we extended to AAs the findings of association of erythrocyte traits with several loci previously reported in Europeans and/or Asians, including CD164 and HBS1L-MYB. In summary, this large-scale genome-wide analysis in AAs has extended the importance of several RBC-associated genetic loci to AAs and identified allelic heterogeneity and pleiotropy at several previously known genetic loci associated with blood cell traits in AAs.
PMCID: PMC3658166  PMID: 23446634
3.  Genetic Variants That Confer Resistance to Malaria Are Associated with Red Blood Cell Traits in African-Americans: An Electronic Medical Record-based Genome-Wide Association Study 
G3: Genes|Genomes|Genetics  2013;3(7):1061-1068.
To identify novel genetic loci influencing interindividual variation in red blood cell (RBC) traits in African-Americans, we conducted a genome-wide association study (GWAS) in 2315 individuals, divided into discovery (n = 1904) and replication (n = 411) cohorts. The traits included hemoglobin concentration (HGB), hematocrit (HCT), RBC count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Patients were participants in the electronic MEdical Records and GEnomics (eMERGE) network and underwent genotyping of ~1.2 million single-nucleotide polymorphisms on the Illumina Human1M-Duo array. Association analyses were performed adjusting for age, sex, site, and population stratification. Three loci previously associated with resistance to malaria—HBB (11p15.4), HBA1/HBA2 (16p13.3), and G6PD (Xq28)—were associated (P ≤ 1 × 10−6) with RBC traits in the discovery cohort. The loci replicated in the replication cohort (P ≤ 0.02), and were significant at a genome-wide significance level (P < 5 × 10−8) in the combined cohort. The proportions of variance in RBC traits explained by significant variants at these loci were as follows: rs7120391 (near HBB) 1.3% of MCHC, rs9924561 (near HBA1/A2) 5.5% of MCV, 6.9% of MCH and 2.9% of MCHC, and rs1050828 (in G6PD) 2.4% of RBC count, 2.9% of MCV, and 1.4% of MCH, respectively. We were not able to replicate loci identified by a previous GWAS of RBC traits in a European ancestry cohort of similar sample size, suggesting that the genetic architecture of RBC traits differs by race. In conclusion, genetic variants that confer resistance to malaria are associated with RBC traits in African-Americans.
PMCID: PMC3704235  PMID: 23696099
red blood cell (RBC) traits; genome-wide association study; African-Americans; natural selection; informatics; electronic medical record
4.  Genome-wide association and linkage analyses of hemostatic factors and hematological phenotypes in the Framingham Heart Study 
BMC Medical Genetics  2007;8(Suppl 1):S12.
Increased circulating levels of hemostatic factors as well as anemia have been associated with increased risk of cardiovascular disease (CVD). Known associations between hemostatic factors and sequence variants at genes encoding these factors explain only a small proportion of total phenotypic variation. We sought to confirm known putative loci and identify novel loci that may influence either trait in genome-wide association and linkage analyses using the Affymetrix GeneChip 100K single nucleotide polymorphism (SNP) set.
Plasma levels of circulating hemostatic factors (fibrinogen, factor VII, plasminogen activator inhibitor-1, von Willebrand factor, tissue plasminogen activator, D-dimer) and hematological phenotypes (platelet aggregation, viscosity, hemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin concentration) were obtained in approximately 1000 Framingham Heart Study (FHS) participants from 310 families. Population-based association analyses using the generalized estimating equations (GEE), family-based association test (FBAT), and multipoint variance components linkage analyses were performed on the multivariable adjusted residuals of hemostatic and hematological phenotypes.
In association analysis, the lowest GEE p-value for hemostatic factors was p = 4.5*10-16 for factor VII at SNP rs561241, a variant located near the F7 gene and in complete linkage disequilibrium (LD) (r2 = 1) with the Arg353Gln F7 SNP previously shown to account for 9% of total phenotypic variance. The lowest GEE p-value for hematological phenotypes was 7*10-8 at SNP rs2412522 on chromosome 4 for mean corpuscular hemoglobin concentration. We presented top 25 most significant GEE results with p-values in the range of 10-6 to 10-5 for hemostatic or hematological phenotypes. In relating 100K SNPs to known candidate genes, we identified two SNPs (rs1582055, rs4897475) in erythrocyte membrane protein band 4.1-like 2 (EPB41L2) associated with hematological phenotypes (GEE p < 10-3). In linkage analyses, the highest linkage LOD score for hemostatic factors was 3.3 for factor VII on chromosome 10 around 15 Mb, and for hematological phenotypes, LOD 3.4 for hemoglobin on chromosome 4 around 55 Mb. All GEE and FBAT association and variance components linkage results can be found at
Using genome-wide association methodology, we have successfully identified a SNP in complete LD with a sequence variant previously shown to be strongly associated with factor VII, providing proof of principle for this approach. Further study of additional strongly associated SNPs and linked regions may identify novel variants that influence the inter-individual variability in hemostatic factors and hematological phenotypes.
PMCID: PMC1995619  PMID: 17903294
5.  Association between the ABO locus and hematological traits in Korean 
BMC Genetics  2012;13:78.
Recently, genome-wide association studies identified a pleiotropic gene locus, ABO, as being significantly associated with hematological traits. To confirm the effects of ABO on hematological traits, we examined the link between the ABO locus and hematological traits in Korean population-based cohorts.
Six tagging SNPs for ABO were analyzed with regard to their effects on hematological traits [white blood cell count (WBC), red blood cell count (RBC), platelet (Plat), mean corpuscular volume (MCV), and mean corpuscular haemoglobin concentration (MCHC)]. Linear regression analyses were performed, controlling for recruitment center, sex, and age as covariates. Of the 6 tagging SNPs, 3 (rs2073823, rs8176720, and rs495828) and 3 (rs2073823, rs8176717, and rs687289) were significantly associated with RBC and MCV, respectively (Bonferroni correction p-value criteria < 0.05/6 = 0.008). rs2073823 and a reported SNP (rs8176746), as well as rs495828 and a reported SNP (rs651007), showed perfect linkage disequilibrium status (r2s = 0.99). Of the remaining 3 SNPs (rs8176720, rs8176717 and rs687289), rs8176717 generated an independent signal with moderate p-value (= 0.045) when it was adjusted for by rs2073823 (the most significant SNP). We also identified a copy number variation (CNV) that was tagged by the SNP rs8176717, the minor allele of which correlated with the deletion allele of CNV. Our haplotype analysis indicated that the haplotype that contained the CNV deletion was significantly associated with MCV (β ± se = 0.363 ± 0.118, p =2.09 × 10-3).
Our findings confirm that ABO is one of the genetic factors that are associated with hematological traits in the Korean population. This result is notable, because GWASs fail to evaluate the link between a CNV and phenotype traits.
PMCID: PMC3472170  PMID: 22963146
ABO; GWAS; CNV; Hematological trait; Korean
6.  Genome-wide Association Study of Porcine Hematological Parameters in a Large White × Minzhu F2 Resource Population 
Hematological traits, which are important indicators of immune function in animals, have been commonly examined as biomarkers of disease and disease severity in humans and animals. Genome-wide significant quantitative trait loci (QTLs) provide important information for use in breeding programs of animals such as pigs. QTLs for hematological parameters (hematological traits) have been detected in pig chromosomes, although these are often mapped by linkage analysis to large intervals making identification of the underlying mutation problematic. Single nucleotide polymorphisms (SNPs) are the common form of genetic variation among individuals and are thought to account for the majority of inherited traits. In this study, a genome-wide association study (GWAS) was performed to detect regions of association with hematological traits in a three-generation resource population produced by intercrossing Large White boars and Minzhu sows during the period from 2007 to 2011. Illumina PorcineSNP60 BeadChip technology was used to genotype each animal and seven hematological parameters were measured (hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC) and red blood cell volume distribution width (RDW)). Data were analyzed in a three step Genome-wide Rapid Association using the Mixed Model and Regression-Genomic Control (GRAMMAR-GC) method. A total of 62 genome-wide significant and three chromosome-wide significant SNPs associated with hematological parameters were detected in this GWAS. Seven and five SNPs were associated with HCT and HGB, respectively. These SNPs were all located within the region of 34.6-36.5 Mb on SSC7. Four SNPs within the region of 43.7-47.0 Mb and fifty-five SNPs within the region of 42.2-73.8 Mb on SSC8 showed significant association with MCH and MCV, respectively. At chromosome-wide significant level, one SNP at 29.2 Mb on SSC1 and two SNPs within the region of 26.0-26.2 Mb were found to be significantly associated with RBC and RDW, respectively. Many of the SNPs were located within previously reported QTL regions and appeared to narrow down the regions compared with previously described QTL intervals. In current research, a total of seven significant SNPs were found within six candidate genes SCUBE3, KDR, TDO, IGFBP7, ADAMTS3 and AFP. In addition, the KIT gene, which has been previously reported to relate to hematological parameters, was located within the region significantly associated with MCH and MCV and could be a candidate gene. These results of this study may lead to a better understanding of the molecular mechanisms of hematological parameters in pigs.
PMCID: PMC3385009  PMID: 22745577
genome-wide association study; porcine; hematological parameters
7.  A Genome-Wide Association Study of Red Blood Cell Traits Using the Electronic Medical Record 
PLoS ONE  2010;5(9):e13011.
The Electronic Medical Record (EMR) is a potential source for high throughput phenotyping to conduct genome-wide association studies (GWAS), including those of medically relevant quantitative traits. We describe use of the Mayo Clinic EMR to conduct a GWAS of red blood cell (RBC) traits in a cohort of patients with peripheral arterial disease (PAD) and controls without PAD.
Methodology and Principal Findings
Results for hemoglobin level, hematocrit, RBC count, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were extracted from the EMR from January 1994 to September 2009. Out of 35,159 RBC trait values in 3,411 patients, we excluded 12,864 values in 1,165 patients that had been measured during hospitalization or in the setting of hematological disease, malignancy, or use of drugs that affect RBC traits, leaving a final genotyped sample of 3,012, 80% of whom had ≥2 measurements. The median of each RBC trait was used in the genetic analyses, which were conducted using an additive model that adjusted for age, sex, and PAD status. We identified four genomic loci that were associated (P<5×10−8) with one or more of the RBC traits (HBLS1/MYB on 6q23.3, TMPRSS6 on 22q12.3, HFE on 6p22.1, and SLC17A1 on 6p22.2). Three of these loci (HBLS1/MYB, TMPRSS6, and HFE) had been identified in recent GWAS and the allele frequencies, effect sizes, and the directions of effects of the replicated SNPs were similar to the prior studies.
Our results demonstrate feasibility of using the EMR to conduct high throughput genomic studies of medically relevant quantitative traits.
PMCID: PMC2946914  PMID: 20927387
8.  Breed-Specific Hematological Phenotypes in the Dog: A Natural Resource for the Genetic Dissection of Hematological Parameters in a Mammalian Species 
PLoS ONE  2013;8(11):e81288.
Remarkably little has been published on hematological phenotypes of the domestic dog, the most polymorphic species on the planet. Information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database. Normal hematological profiles were available for 6046 dogs, 5447 of which also had machine platelet concentrations within the reference interval. Seventy-five pure breeds plus a mixed breed control group were represented by 10 or more dogs. All measured parameters except mean corpuscular hemoglobin concentration (MCHC) varied with age. Concentrations of white blood cells (WBCs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex. Neutering status had an impact on hemoglobin concentration, mean corpuscular hemoglobin (MCH), MCHC, and concentrations of WBCs, neutrophils, monocytes, lymphocytes and platelets. Principal component analysis of hematological data revealed 37 pure breeds with distinctive phenotypes. Furthermore, all hematological parameters except MCHC showed significant differences between specific individual breeds and the mixed breed group. Twenty-nine breeds had distinctive phenotypes when assessed in this way, of which 19 had already been identified by principal component analysis. Tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. This study represents the first large-scale analysis of hematological phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of hematological traits, triangulating phenotype, breed and genetic predisposition.
PMCID: PMC3840015  PMID: 24282579
9.  A Meta-Analysis and Genome-Wide Association Study of Platelet Count and Mean Platelet Volume in African Americans 
PLoS Genetics  2012;8(3):e1002491.
Several genetic variants associated with platelet count and mean platelet volume (MPV) were recently reported in people of European ancestry. In this meta-analysis of 7 genome-wide association studies (GWAS) enrolling African Americans, our aim was to identify novel genetic variants associated with platelet count and MPV. For all cohorts, GWAS analysis was performed using additive models after adjusting for age, sex, and population stratification. For both platelet phenotypes, meta-analyses were conducted using inverse-variance weighted fixed-effect models. Platelet aggregation assays in whole blood were performed in the participants of the GeneSTAR cohort. Genetic variants in ten independent regions were associated with platelet count (N = 16,388) with p<5×10−8 of which 5 have not been associated with platelet count in previous GWAS. The novel genetic variants associated with platelet count were in the following regions (the most significant SNP, closest gene, and p-value): 6p22 (rs12526480, LRRC16A, p = 9.1×10−9), 7q11 (rs13236689, CD36, p = 2.8×10−9), 10q21 (rs7896518, JMJD1C, p = 2.3×10−12), 11q13 (rs477895, BAD, p = 4.9×10−8), and 20q13 (rs151361, SLMO2, p = 9.4×10−9). Three of these loci (10q21, 11q13, and 20q13) were replicated in European Americans (N = 14,909) and one (11q13) in Hispanic Americans (N = 3,462). For MPV (N = 4,531), genetic variants in 3 regions were significant at p<5×10−8, two of which were also associated with platelet count. Previously reported regions that were also significant in this study were 6p21, 6q23, 7q22, 12q24, and 19p13 for platelet count and 7q22, 17q11, and 19p13 for MPV. The most significant SNP in 1 region was also associated with ADP-induced maximal platelet aggregation in whole blood (12q24). Thus through a meta-analysis of GWAS enrolling African Americans, we have identified 5 novel regions associated with platelet count of which 3 were replicated in other ethnic groups. In addition, we also found one region associated with platelet aggregation that may play a potential role in atherothrombosis.
Author Summary
The majority of the variation in platelet count and mean platelet volume between individuals is heritable. We performed genome-wide association studies in more than 16,000 African American participants from seven population-based cohorts to identify genetic variants that correlate with variation in platelet count and mean platelet volume. We observed statistically significant evidence (p-value<5×10−8) that 10 genomic regions were associated with platelet count and 3 were associated with mean platelet volume. Of the regions that were significantly associated, we found 5 novel regions that were not reported previously in other populations. Three of these 5 regions were also associated with platelet count in European Americans and Hispanic Americans. All these regions contain genes that are either known to have or potentially may have a role in determining platelet count and/or mean platelet volume. We further found that one of these regions was also associated with agonist-induced platelet aggregation. Further studies will determine the exact role played by these genomic regions in platelet biology. The knowledge generated by this and other studies will not only help us better understand platelet biology but can also lead us to the discovery of new anti-platelet drugs.
PMCID: PMC3299192  PMID: 22423221
10.  Reduced Neutrophil Count in People of African Descent Is Due To a Regulatory Variant in the Duffy Antigen Receptor for Chemokines Gene 
PLoS Genetics  2009;5(1):e1000360.
Persistently low white blood cell count (WBC) and neutrophil count is a well-described phenomenon in persons of African ancestry, whose etiology remains unknown. We recently used admixture mapping to identify an approximately 1-megabase region on chromosome 1, where ancestry status (African or European) almost entirely accounted for the difference in WBC between African Americans and European Americans. To identify the specific genetic change responsible for this association, we analyzed genotype and phenotype data from 6,005 African Americans from the Jackson Heart Study (JHS), the Health, Aging and Body Composition (Health ABC) Study, and the Atherosclerosis Risk in Communities (ARIC) Study. We demonstrate that the causal variant must be at least 91% different in frequency between West Africans and European Americans. An excellent candidate is the Duffy Null polymorphism (SNP rs2814778 at chromosome 1q23.2), which is the only polymorphism in the region known to be so differentiated in frequency and is already known to protect against Plasmodium vivax malaria. We confirm that rs2814778 is predictive of WBC and neutrophil count in African Americans above beyond the previously described admixture association (P = 3.8×10−5), establishing a novel phenotype for this genetic variant.
Author Summary
Many African Americans have white blood cell counts (WBC) that are persistently below the normal range for people of European descent, a condition called “benign ethnic neutropenia.” Because most African Americans have both African and European ancestors, selected genetic variants can be analyzed to assign probable African or European origin to each region of each such person's chromosomes. Previously, we found a region on chromosome 1 where increased local African ancestry completely accounted for differences in WBC between African and European Americans, suggesting the presence of an African-derived variant causing low WBC. Here, we show that low neutrophil count is predominantly responsible for low WBC; that a dominant, European-derived allele contributes to high neutrophil count; and that the frequency of this allele differs in Africans and Europeans by >91%. Across the chromosome 1 locus, only the well-characterized “Duffy” polymorphism was this differentiated. Neutrophil count was more strongly associated to the Duffy variant than to ancestry, suggesting that the variant itself causes benign ethnic neutropenia. The African, or “null,” form of this variant abolishes expression of the “Duffy Antigen Receptor for Chemokines” on red blood cells, perhaps altering the concentrations and distribution of chemokines that regulate neutrophil production or migration.
PMCID: PMC2628742  PMID: 19180233
11.  Hematological profile of East African short-horn zebu calves from birth to 51 weeks of age 
Comparative Clinical Pathology  2012;22(5):1029-1036.
This paper is the first attempt to accurately describe the hematological parameters for any African breed of cattle, by capturing the changes in these parameters over the first 12 months of an animal’s life using a population-based sample of calves reared under field conditions and natural disease challenge. Using a longitudinal study design, a stratified clustered random sample of newborn calves was recruited into the IDEAL study and monitored at 5-weekly intervals until 51 weeks of age. The blood cell analysis performed at each visit included: packed cell volume; red cell count; red cell distribution width; mean corpuscular volume; mean corpuscular hemoglobin concentration; hemoglobin concentration; white cell count; absolute lymphocyte, eosinophil, monocyte, and neutrophil counts; platelet count; mean platelet volume; and total serum protein. The most significant age-related change in the red cell parameters was a rise in red cell count and hemoglobin concentration during the neonatal period. This is in contrast to what is reported for other ruminants, including European cattle breeds where the neonatal period is marked by a fall in the red cell parameters. There is a need to establish breed-specific reference ranges for blood parameters for indigenous cattle breeds. The possible role of the postnatal rise in the red cell parameters in the adaptability to environmental constraints and innate disease resistance warrants further research into the dynamics of blood cell parameters of these breeds.
PMCID: PMC3765850  PMID: 24032003
Hematology; East African; Zebu; Calves; Longitudinal study
12.  Molecular analysis and association with clinical and laboratory manifestations in children with sickle cell anemia 
To analyze the frequency of βS-globin haplotypes and alpha-thalassemia, and their influence on clinical manifestations and the hematological profile of children with sickle cell anemia.
The frequency of βS-globin haplotypes and alpha-thalassemia and any association with clinical and laboratorial manifestations were determined in 117 sickle cell anemia children aged 3–71 months. The confirmation of hemoglobin SS and determination of the haplotypes were achieved by polymerase chain reaction-restriction fragment length polymorphism, and alpha-thalassemia genotyping was by multiplex polymerase chain reaction (single-tube multiplex-polymerase chain reaction).
The genotype distribution of haplotypes was 43 (36.7%) Central African Republic/Benin, 41 (35.0%) Central African Republic/Central African Republic, 20 (17.0%) Rare/atypical, and 13 (11.1%) Benin/Benin. The frequency of the α3.7 deletion was 1.71% as homozygous (−α3.7/−α3.7) and 11.9% as heterozygous (−α3.7/αα). The only significant association in respect to haplotypes was related to the mean corpuscular volume. The presence of alpha-thalassemia was significantly associated to decreases in mean corpuscular volume, mean corpuscular hemoglobin and reticulocyte count and to an increase in the red blood cell count. There were no significant associations of βS-globin haplotypes and alpha-thalassemia with clinical manifestations.
In the study population, the frequency of alpha-thalassemia was similar to published data in Brazil with the Central African Republic haplotype being the most common, followed by the Benin haplotype. βS-globin haplotypes and interaction between alpha-thalassemia and sickle cell anemia did not influence fetal hemoglobin concentrations or the number of clinical manifestations.
PMCID: PMC4318370  PMID: 25305165
Anemia, Sickle Cell; Alpha-thalassemia; Beta-globins; Haplotypes; Child
13.  Genome-Wide Association Study of Coronary Heart Disease and Its Risk Factors in 8,090 African Americans: The NHLBI CARe Project 
PLoS Genetics  2011;7(2):e1001300.
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.
Author Summary
To date, most large-scale genome-wide association studies (GWAS) carried out to identify risk factors for complex human diseases and traits have focused on population of European ancestry. It is currently unknown whether the same loci associated with complex diseases and traits in Caucasians will replicate in population of African ancestry. Here, we conducted a large GWAS to identify common DNA polymorphisms associated with coronary heart disease (CHD) and its risk factors (type-2 diabetes, hypertension, smoking status, and LDL- and HDL-cholesterol) in 8,090 African Americans as part of the NHLBI Candidate gene Association Resource (CARe) Project. We replicated 17 associations previously reported in Caucasians, suggesting that the same loci carry common DNA sequence variants associated with CHD and its risk factors in Caucasians and African Americans. At five of these 17 loci, we used the different patterns of linkage disequilibrium between populations of European and African ancestry to identify DNA sequence variants more strongly associated with phenotypes than the index SNPs found in Caucasians, suggesting smaller genomic intervals to search for causal alleles. We also used the CARe data to develop new statistical methods to perform association studies in admixed populations. The CARe Project data represent an extraordinary resource to expand our understanding of the genetics of complex diseases and traits in non-European-derived populations.
PMCID: PMC3037413  PMID: 21347282
14.  Quantitative trait locus linkage analysis in a large Amish pedigree identifies novel candidate loci for erythrocyte traits 
We characterized a large Amish pedigree and, in 384 pedigree members, analyzed the genetic variance components with covariate screen as well as genome-wide quantitative trait locus (QTL) linkage analysis of red blood cell count (RBC), hemoglobin (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count (PLT), and white blood cell count (WBC) using SOLAR. Age and gender were found to be significant covariates in many CBC traits. We obtained significant heritability estimates for RBC, MCV, MCH, MCHC, RDW, PLT, and WBC. We report four candidate loci with Logarithm of the odds (LOD) scores above 2.0: 6q25 (MCH), 9q33 (WBC), 10p12 (RDW), and 20q13 (MCV). We also report eleven candidate loci with LOD scores between 1.5 and <2.0. Bivariate linkage analysis of MCV and MCH on chromosome 20 resulted in a higher maximum LOD score of 3.14. Linkage signals on chromosomes 4q28, 6p22, 6q25, and 20q13 are concomitant with previously reported QTL. All other linkage signals reported herein represent novel evidence of candidate QTL. Interestingly rs1800562, the most common causal variant of hereditary hemochromatosis in HFE (6p22) was associated with MCH and MCHC in this family. Linkage studies like the one presented here will allow investigators to focus the search for rare variants amidst the noise encountered in the large amounts of data generated by whole-genome sequencing.
PMCID: PMC3775389  PMID: 24058921
Amish; erythrocytes; linkage; QTL
15.  Quantitative trait locus linkage analysis in a large Amish pedigree identifies novel candidate loci for erythrocyte traits 
We characterized a large Amish pedigree and, in 384 pedigree members, analyzed the genetic variance components with covariate screen as well as genome-wide quantitative trait locus (QTL) linkage analysis of red blood cell count (RBC), hemoglobin (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count (PLT), and white blood cell count (WBC) using SOLAR. Age and gender were found to be significant covariates in many CBC traits. We obtained significant heritability estimates for RBC, MCV, MCH, MCHC, RDW, PLT, and WBC. We report four candidate loci with LOD scores above 2.0: 6q25 (MCH), 9q33 (WBC), 10p12 (RDW), and 20q13 (MCV). We also report eleven candidate loci with LOD scores between 1.5 and < 2.0. Bivariate linkage analysis of MCV and MCH on chromosome 20 resulted in a higher maximum LOD score of 3.14. Linkage signals on chromosomes 4q28, 6p22, 6q25, and 20q13 are concomitant with previously reported QTL. All other linkage signals reported herein represent novel evidence of candidate QTL. Interestingly rs1800562, the most common causal variant of hereditary hemochromatosis in HFE (6p22) was associated with MCH and MCHC in this family. Linkage studies like the one presented here will allow investigators to focus the search for rare variants amidst the noise encountered in the large amounts of data generated by whole genome sequencing.
PMCID: PMC3775389  PMID: 24058921
16.  Mean Platelet Volume (MPV) Predicts Middle Distance Running Performance 
PLoS ONE  2014;9(11):e112892.
Running economy and performance in middle distance running depend on several physiological factors, which include anthropometric variables, functional characteristics, training volume and intensity. Since little information is available about hematological predictors of middle distance running time, we investigated whether some hematological parameters may be associated with middle distance running performance in a large sample of recreational runners.
The study population consisted in 43 amateur runners (15 females, 28 males; median age 47 years), who successfully concluded a 21.1 km half-marathon at 75–85% of their maximal aerobic power (VO2max). Whole blood was collected 10 min before the run started and immediately thereafter, and hematological testing was completed within 2 hours after sample collection.
The values of lymphocytes and eosinophils exhibited a significant decrease compared to pre-run values, whereas those of mean corpuscular volume (MCV), platelets, mean platelet volume (MPV), white blood cells (WBCs), neutrophils and monocytes were significantly increased after the run. In univariate analysis, significant associations with running time were found for pre-run values of hematocrit, hemoglobin, mean corpuscular hemoglobin (MCH), red blood cell distribution width (RDW), MPV, reticulocyte hemoglobin concentration (RetCHR), and post-run values of MCH, RDW, MPV, monocytes and RetCHR. In multivariate analysis, in which running time was entered as dependent variable whereas age, sex, blood lactate, body mass index, VO2max, mean training regimen and the hematological parameters significantly associated with running performance in univariate analysis were entered as independent variables, only MPV values before and after the trial remained significantly associated with running time. After adjustment for platelet count, the MPV value before the run (p = 0.042), but not thereafter (p = 0.247), remained significantly associated with running performance.
The significant association between baseline MPV and running time suggest that hyperactive platelets may exert some pleiotropic effects on endurance performance.
PMCID: PMC4227876  PMID: 25386658
17.  Association Between Hematological Indices and Coronary Calcification in Symptomatic Patients without History of Coronary Artery Disease 
Atherosclerotic coronary artery disease (CAD) has long been shown to involve chronic low-grade subclinical inflammation. However, whether there is association between hematological indices assessed by complete blood count (CBC) and coronary atherosclerotic burden has not been well studied.
Materials and Methods:
Consecutive 868 patients without known CAD who presented with acute chest pain to emergency department and underwent coronary artery calcium (CAC) scoring evaluation by multi-detector cardiac computed tomography were included in our study. Clinical characteristics and CBC indices were compared among different CAC groups.
The cohort comprised 60% male with a mean age of 61 (SD = 14) years. Median Framingham risk of CAD was 4% (range 1-16%). Median CAC score was 0 (IQR 0-43). Higher CAC groups had significantly higher Framingham risk of CAD than lower CAC groups (P < 0.001). Among different CAC categories, there was no statistically significant difference in hemoglobin level (p 0.45), mean corpuscular volume (p 0.43), mean corpuscular hemoglobin (p 0.28), mean corpuscular hemoglobin volume (p 0.36), red cell distribution width (0.42), total white blood cell counts (p 0.291), neutrophil counts (p 0.352), lymphocyte counts (p 0.92), neutrophil to lymphocyte ratio (p 0.68), monocyte count (p 0.48), and platelet counts (p 0.25).
Our study did not detect significant association between hematological indices assessed with CBC and coronary calcification in symptomatic patients without known CAD.
PMCID: PMC4193148  PMID: 25317386
Coronary artery disease; Inflammation; Neutrophil-to-lymphocyte ratio; Red cell distribution width; White blood cell
18.  Population-Based Biochemistry, Immunologic and Hematological Reference Values for Adolescents and Young Adults in a Rural Population in Western Kenya 
PLoS ONE  2011;6(6):e21040.
There is need for locally-derived age-specific clinical laboratory reference ranges of healthy Africans in sub-Saharan Africa. Reference values from North American and European populations are being used for African subjects despite previous studies showing significant differences. Our aim was to establish clinical laboratory reference values for African adolescents and young adults that can be used in clinical trials and for patient management.
Methods and Findings
A panel of 298, HIV-seronegative individuals aged 13–34 years was randomly selected from participants in two population-based cross-sectional surveys assessing HIV prevalence and other sexually transmitted infections in western Kenya. The adolescent (<18 years)-to-adults (≥18 years) ratio and the male-to-female ratio was 1∶1. Median and 95% reference ranges were calculated for immunohematological and biochemistry values. Compared with U.S-derived reference ranges, we detected lower hemoglobin (HB), hematocrit (HCT), red blood cells (RBC), mean corpuscular volume (MCV), neutrophil, glucose, and blood urea nitrogen values but elevated eosinophil and total bilirubin values. Significant gender variation was observed in hematological parameters in addition to T-bilirubin and creatinine indices in all age groups, AST in the younger and neutrophil, platelet and CD4 indices among the older age group. Age variation was also observed, mainly in hematological parameters among males. Applying U.S. NIH Division of AIDS (DAIDS) toxicity grading to our results, 40% of otherwise healthy study participants were classified as having an abnormal laboratory parameter (grade 1–4) which would exclude them from participating in clinical trials.
Hematological and biochemistry reference values from African population differ from those derived from a North American population, showing the need to develop region-specific reference values. Our data also show variations in hematological indices between adolescent and adult males which should be considered when developing reference ranges. This study provides the first locally-derived clinical laboratory reference ranges for adolescents and young adults in western Kenya.
PMCID: PMC3119664  PMID: 21713038
19.  Effect of aqueous leaves extract of Ocimum gratissimum on hematological parameters in rats 
This study aims to elucidate the effect of Ocimum gratissimum on hematological parameters in rats.
Materials and Methods:
Thirty male albino Wistar rats were randomly assigned into three groups of ten rats each. Group 1 was control, while groups 2 (LD) and 3 (HD) received 500 mg/kg and 1000 mg/kg body weight, respectively, of the extract orally once daily. Rats in all three groups received normal rat chow and drinking water ad libitum for 28 days. Complete blood count was done using an automatic counter.
The HD group had significantly (P<.05) higher red blood cell (RBC) counts, packed cell volume (PCV), hemoglobin (Hb), and platelet counts as compared with the control and LD groups. No significant changes were observed in the total white blood cell (WBC) count of the three groups, but significantly (P<.05) lower lymphocyte and higher neutrophil counts were observed in the HD group compared with the LD group. The mean platelet volume (MPV), platelet–large cell ratio (P–LCR), and platelet distribution width (PDW) were significantly (P<.05) reduced in the HD compared with the LD group. The mean corpuscular volume (MCV) and RBC distribution width–standard deviation were significantly (P<.05) lower in the HD group than in control. No significant changes were observed in levels of mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and RBC distribution width–coefficient of variation among the groups.
We conclude that oral administration of O gratissimum increases RBC, PCV, Hb, platelet count, and neutrophils and also leads to a decrease in platelet indices (i.e., MPV, P–LCR, and PDW).
PMCID: PMC3657984  PMID: 23776807
Blood indices; differential white blood cell; Hb; O gratissimum; packed cell volume; platelet; red blood cell; total WBC
20.  Maps of Open Chromatin Guide the Functional Follow-Up of Genome-Wide Association Signals: Application to Hematological Traits 
PLoS Genetics  2011;7(6):e1002139.
Turning genetic discoveries identified in genome-wide association (GWA) studies into biological mechanisms is an important challenge in human genetics. Many GWA signals map outside exons, suggesting that the associated variants may lie within regulatory regions. We applied the formaldehyde-assisted isolation of regulatory elements (FAIRE) method in a megakaryocytic and an erythroblastoid cell line to map active regulatory elements at known loci associated with hematological quantitative traits, coronary artery disease, and myocardial infarction. We showed that the two cell types exhibit distinct patterns of open chromatin and that cell-specific open chromatin can guide the finding of functional variants. We identified an open chromatin region at chromosome 7q22.3 in megakaryocytes but not erythroblasts, which harbors the common non-coding sequence variant rs342293 known to be associated with platelet volume and function. Resequencing of this open chromatin region in 643 individuals provided strong evidence that rs342293 is the only putative causative variant in this region. We demonstrated that the C- and G-alleles differentially bind the transcription factor EVI1 affecting PIK3CG gene expression in platelets and macrophages. A protein–protein interaction network including up- and down-regulated genes in Pik3cg knockout mice indicated that PIK3CG is associated with gene pathways with an established role in platelet membrane biogenesis and thrombus formation. Thus, rs342293 is the functional common variant at this locus; to the best of our knowledge this is the first such variant to be elucidated among the known platelet quantitative trait loci (QTLs). Our data suggested a molecular mechanism by which a non-coding GWA index SNP modulates platelet phenotype.
Author Summary
Genome-wide scans have revealed multiple genetic regions underlying complex traits. However, the transition from an initial association signal to identifying the functional DNA change(s) has proved challenging. Many of the DNA changes discovered are located outside protein-coding regions and may exert their effects through gene regulation. We screened genetic regions associated with hematological traits in erythroblasts (red blood cells) and megakaryocytes (platelet-producing cells) and mapped sites of open chromatin, which harbor active gene regulatory elements. We investigated a DNA sequence change located within a site of open chromatin at chromosome 7 in megakaryocytes, but not erythroblasts, known to be associated with platelet volume. We showed that this DNA change is functional due to alteration of the binding site of a transcription factor, which regulates the expression of a gene that affects platelet characteristics. Mice lacking this gene revealed significant differences in expression of several important platelet genes compared to wild-type mice. The approach described here can be applied in different cell types to functionally follow-up association signals with many other biological traits by identification of the causative base change and how it affects gene function, thus paving the way to clinical benefit.
PMCID: PMC3128100  PMID: 21738486
21.  Variation of Red Blood Cell Distribution Width and Mean Platelet Volume after Moderate Endurance Exercise 
Advances in Hematology  2014;2014:192173.
Although physical exercise strongly influences several laboratory parameters, data about the hematological changes after medium distance running are scarce. We studied 31 middle-trained athletes (mean training regimen 217 ± 32 min/week) who performed a 21.1 km, half-marathon run. Blood samples were collected before the run, at the end, and 3 and 20 hours thereafter. The complete blood count was performed on Advia 2120 and included red blood cell (RBC), reticulocyte, and platelet counts; hemoglobin; mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); reticulocyte haemoglobin content (Ret CHR); RBC distribution width (RDW), mean platelet volume (MPV). No significant variations were observed for MCH and Ret CHR. The RBC, reticulocyte, and hemoglobin values modestly decreased after the run. The MCV significantly increased at the end of running but returned to baseline 3 hours thereafter. The RDW constantly increased, reaching a peak 20 hours after the run. The platelet count and MPV both increased after the run and returned to baseline 3 hours thereafter. These results may have implications for definition of reference ranges and antidoping testing, and may also contribute to explaining the relationship between endurance exercise and mortality, since previous studies reported that RDW and MPV may be significantly associated with cardiovascular disease.
PMCID: PMC4147199  PMID: 25197280
22.  Trpc2 Depletion Protects RBC from Oxidative Stress-Induced Hemolysis 
Experimental hematology  2011;40(1):71-83.
Transient receptor potential channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca2+]i) through TRPC2 and TRPC3. Because modulation of [Ca2+]i is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild type littermate controls. Although the erythropoietin induced increase in [Ca2+]i was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenyhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. While erythropoietin induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.
PMCID: PMC3237850  PMID: 21924222
TRP Channels; Trpc2; Trpc3; erythropoietin; oxidative stress
23.  Hematological profile of normal pregnant women in Lagos, Nigeria 
Hematological profile is considered one of the factors affecting pregnancy and its outcome. Anemia is the most common hematological problem in pregnancy, followed by thrombocytopenia. Leukocytosis is almost always associated with pregnancy. The study reported here was designed to evaluate the overall mean values of seven major hematological parameters and their mean values at different trimesters of pregnancy.
Subjects and methods
This examination was a cross-sectional study of 274 pregnant women who registered to attend the Lagos University Teaching Hospital or Lagos State University Teaching Hospital antenatal clinics between their first and third trimester. Blood (4.5 mL) was collected from each participant into a tube containing the anticoagulant ethylenediaminetetraacetic acid (EDTA). A full blood count was performed on each sample and the results were analyzed.
Overall, the values obtained were (mean ± standard deviation [SD]): hematocrit level, 30.16% ± 5.55%; hemoglobin concentration, 10.94 ± 1.86 g/dL; white blood cells, 7.81 ± 2.34 × 109; platelets, 228.29 ± 65.6 × 109; cell volume 78.30 ± 5.70 fL, corpuscular hemoglobin, 28.57 ± 2.48 pg; and corpuscular hemoglobin concentration, 36.45 ± 1.10 g/dL. When grouped by trimester, the mean ± SD value of packed cell volume at first trimester was 32.07% ± 6.80%; of second trimester, 29.76% ± 5.21%; and of third, 33.04% ± 3.88%. The mean ± SD hemoglobin concentration values were 11.59 ± 2.35 g/dL, 10.81 ± 1.72 g/dL, and 10.38 ± 1.27 g/dL for women in their first, second, and third trimester, respectively. Mean ± SD white blood cell concentration for first, second, and third trimesters were 7.31 ± 2.38 × 109, 7.88 ± 2.33 × 109, and 8.37 ± 2.15 × 109, respectively, while the mean ± SD platelet values for first, second, and third trimesters were 231.50 ± 79.10 × 109, 227.57 ± 63 × 109, and 200.82 ± 94.42 × 109, respectively. A statistically significant relationship was found to exist between packed cell volume and white blood cell count with increase in gestational age (P = 0.010 and 0.001, respectively). However, there was no statistically significant association between platelet count and increase in gestational age (P = 0.296).
These findings reinforce the need for supplementation and provide additional information on hematological reference values in pregnancy in Nigeria.
PMCID: PMC3647602  PMID: 23662089
anemia; thrombocytopenia; hematology; normal pregnancy; trimester
24.  Comparative Hematoxicity of Fusirium Mycotoxin in Experimental Sprague-Dawley Rats 
Toxicology International  2013;20(1):25-29.
Mycotoxins are fungal toxin and contaminated to human through food-stuffs. Hematological abnormality, mainly thrombocytopenia and leukopenia are induced after consumption of mycotoxin. Experiments were conducted to evaluate the hematotoxicity of trichothecenes mycotoxins in Sprague-Dawley rats. Hematological parameters viz. Hemoglobin, hematocrit, erythrocyte count (RBC), white blood cell count (WBC), lymphocytes, monocytes, neutrophils, eosinophils, basophils, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width, mean platelet volume, plateletcrit and platelet distribution width were determined at 0, 6, 12 and 24 h after injection of 0.5 ml of T-2, Deoxynivalenol (DON), nivalenol, zearalenone, neosolaniol, ochratoxin-B mycotoxin equivalent to 1 × 10-3 μg/μl to Sprague-Dawley rats. Experiments showed that trichothecenes toxin produces severe hematological alternation. The reductions of RBC and WBC were observed in all Fusarium mycotoxins treated group. T-2 toxin group shows severe toxicity as compared to other mycotoxin treated group. DON is the least hematotoxicity and T-2 the most.
PMCID: PMC3702123  PMID: 23833434
Hematotoxicity; mycotoxins; Sprague-Dawley rats
25.  Racial or ethnic differences in allele frequencies of single‐nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene and their influence on response to methotrexate in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2006;65(9):1213-1218.
The anti‐folate drug methotrexate (MTX) is commonly used to treat rheumatoid arthritis.
To determine the allele frequencies of five common coding single‐nucleotide polymorphisms (SNPs) in the methylenetetrahydrofolate reductase (MTHFR) gene in African‐Americans and Caucasians with rheumatoid arthritis and controls to assess whether there are differences in allele frequencies among these ethnic or racial groups and whether these SNPs differentially affect the efficacy or toxicity of MTX.
Allele frequencies in the 677, 1298 and 3 additional SNPs in the MTHFR coding region in 223 (193 Caucasians and 30 African‐Americans) patients with rheumatoid arthritis who previously participated in one of two prospective clinical trials were characterised, and genotypes were correlated with the efficacy and toxicity of MTX. Another 308 subjects with rheumatoid arthritis who participated in observational studies, one group predominantly Caucasian and the other African‐American, as well as 103 normal controls (53 African‐Americans and 50 Caucasians) were used to characterise allele frequencies of these SNPs and their associated haplotypes.
Significantly different allele frequencies were seen in three of the five SNPs and haplotype frequencies between Caucasians and African‐Americans. Allele frequencies were similar between patients with rheumatoid arthritis and controls of the same racial or ethnic group. Frequencies of the rs4846051C, 677T and 1298C alleles were 0.33, 0.11 and 0.13, respectively, among African‐Americans with rheumatoid arthritis. Among Caucasians with rheumatoid arthritis, these allele frequencies were 0.08 (p<0.001 compared with African‐Americans with rheumatoid arthritis), 0.30 (p = 0.002) and 0.34 (p<0.001), respectively. There was no association between SNP alleles or haplotypes and response to MTX as measured by the mean change in the 28‐joint Disease Activity Score from baseline values. In Caucasians, the 1298 A (major) allele was associated with a significant increase in MTX‐related adverse events characteristic of a recessive genetic effect (odds ratio 15.86, 95% confidence interval 1.51 to 167.01; p = 0.021), confirming previous reports. There was an association between scores of MTX toxicity and the rs4846051 C allele, and haplotypes containing this allele, in African‐Americans, but not in Caucasians.
: These results, although preliminary, highlight racial or ethnic differences in frequencies of common MTHFR SNPs. The MTHFR 1298 A and the rs4846051 C alleles were associated with MTX‐related adverse events in Caucasians and African‐Americans, respectively, but these findings should be replicated in larger studies. The rs4846051 SNP, which is far more common in African‐Americans than in Caucasians, can also be proved to be a useful ancestry informative marker in future studies on genetic admixture.
PMCID: PMC1798268  PMID: 16439441

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