Genome-wide association studies (GWAS) are a useful approach in the study of the genetic components of complex phenotypes. Aside from large cohorts, GWAS have generally been limited to the study of one or a few diseases or traits. The emergence of biobanks linked to electronic medical records (EMRs) allows the efficient re-use of genetic data to yield meaningful genotype-phenotype associations for multiple phenotypes or traits. Phase I of the electronic MEdical Records and GEnomics (eMERGE-I) Network is a National Human Genome Research Institute (NHGRI)-supported consortium composed of five sites to perform various genetic association studies using DNA repositories and EMR systems. Each eMERGE site has developed EMR-based algorithms to comprise a core set of fourteen phenotypes for extraction of study samples from each site’s DNA repository. Each eMERGE site selected samples for a specific phenotype, and these samples were genotyped at either the Broad Institute or at the Center for Inherited Disease Research (CIDR) using the Illumina Infinium BeadChip technology. In all, approximately 17,000 samples from across the five sites were genotyped. A unified quality control (QC) pipeline was developed by the eMERGE Genomics Working Group and used to ensure thorough cleaning of the data. This process includes examination of sample quality, marker quality, and various batch effects. Upon completion of the genotyping and QC analyses for each site’s primary study, the eMERGE Coordinating Center merged the datasets from all five sites. This larger merged dataset re-entered the established eMERGE QC pipeline. Based on lessons learned during the process, additional analyses and QC checkpoints were added to the pipeline to ensure proper merging. Here we explore the challenges associated with combining datasets from different genotyping centers and describe the expansion to the eMERGE QC pipeline for merged datasets. These additional steps will be useful as the eMERGE project expands to include additional sites in eMERGE-II and also serve as a starting point for investigators merging multiple genotype data sets accessible through the National Center for Biotechnology Information (NCBI) in the database of Genotypes and Phenotypes (dbGaP). Our experience demonstrates that merging multiple datasets after additional QC can be an efficient use of genotype data despite new challenges that appear in the process.

Genetic variants in intron 1 of the fat mass– and obesity-associated (FTO) gene have been consistently associated with body mass index (BMI) in Europeans. However, follow-up studies in African Americans (AA) have shown no support for some of the most consistently BMI–associated FTO index single nucleotide polymorphisms (SNPs). This is most likely explained by different race-specific linkage disequilibrium (LD) patterns and lower correlation overall in AA, which provides the opportunity to fine-map this region and narrow in on the functional variant. To comprehensively explore the 16q12.2/FTO locus and to search for second independent signals in the broader region, we fine-mapped a 646–kb region, encompassing the large FTO gene and the flanking gene RPGRIP1L by investigating a total of 3,756 variants (1,529 genotyped and 2,227 imputed variants) in 20,488 AAs across five studies. We observed associations between BMI and variants in the known FTO intron 1 locus: the SNP with the most significant p-value, rs56137030 (8.3×10−6) had not been highlighted in previous studies. While rs56137030was correlated at r2>0.5 with 103 SNPs in Europeans (including the GWAS index SNPs), this number was reduced to 28 SNPs in AA. Among rs56137030 and the 28 correlated SNPs, six were located within candidate intronic regulatory elements, including rs1421085, for which we predicted allele-specific binding affinity for the transcription factor CUX1, which has recently been implicated in the regulation of FTO. We did not find strong evidence for a second independent signal in the broader region. In summary, this large fine-mapping study in AA has substantially reduced the number of common alleles that are likely to be functional candidates of the known FTO locus. Importantly our study demonstrated that comprehensive fine-mapping in AA provides a powerful approach to narrow in on the functional candidate(s) underlying the initial GWAS findings in European populations.

Author Summary

Genetic variants within the fat mass– and obesity-associated (FTO) gene are associated with increased risk of obesity. To better understand which specific genetic variant(s) in this genetic region is associated with obesity risk, we attempt to genotype or impute all known genetic variants in the region and test for association with body mass index as a measurement of obesity in over 20,000 African Americans. We identified 29 potential candidate variants, of which one variant (rs1421085) is a particularly interesting candidate for future functional follow-up studies. Our example shows the powerful approach of studying a large African American population, substantially reducing the number of possible functional variants compared with European descent populations.

Background

Systematic study of clinical phenotypes is important for a better understanding of the genetic basis of human diseases and more effective gene-based disease management. A key aspect in facilitating such studies requires standardized representation of the phenotype data using common data elements (CDEs) and controlled biomedical vocabularies. In this study, the authors analyzed how a limited subset of phenotypic data is amenable to common definition and standardized collection, as well as how their adoption in large-scale epidemiological and genome-wide studies can significantly facilitate cross-study analysis.

Methods

The authors mapped phenotype data dictionaries from five different eMERGE (Electronic Medical Records and Genomics) Network sites studying multiple diseases such as peripheral arterial disease and type 2 diabetes. For mapping, standardized terminological and metadata repository resources, such as the caDSR (Cancer Data Standards Registry and Repository) and SNOMED CT (Systematized Nomenclature of Medicine), were used. The mapping process comprised both lexical (via searching for relevant pre-coordinated concepts and data elements) and semantic (via post-coordination) techniques. Where feasible, new data elements were curated to enhance the coverage during mapping. A web-based application was also developed to uniformly represent and query the mapped data elements from different eMERGE studies.

Results

Approximately 60% of the target data elements (95 out of 157) could be mapped using simple lexical analysis techniques on pre-coordinated terms and concepts before any additional curation of terminology and metadata resources was initiated by eMERGE investigators. After curation of 54 new caDSR CDEs and nine new NCI thesaurus concepts and using post-coordination, the authors were able to map the remaining 40% of data elements to caDSR and SNOMED CT. A web-based tool was also implemented to assist in semi-automatic mapping of data elements.

Conclusion

This study emphasizes the requirement for standardized representation of clinical research data using existing metadata and terminology resources and provides simple techniques and software for data element mapping using experiences from the eMERGE Network.

Genome-wide association studies (GWAS) are being conducted at an unprecedented rate in population-based cohorts and have increased our understanding of the pathophysiology of complex disease. The recent application of GWAS to clinic-based cohorts has also yielded genetic predictors of clinical outcomes. Regardless of context, the practical utility of this information will ultimately depend upon the quality of the original data. Quality control (QC) procedures for GWAS are computationally intensive, operationally challenging, and constantly evolving. With each new dataset, new realities are discovered about GWAS data and best practices continue to be developed. The Genomics Workgroup of the National Human Genome Research Institute (NHGRI) funded electronic Medical Records and Genomics (eMERGE) network has invested considerable effort in developing strategies for QC of these data. The lessons learned by this group will be valuable for other investigators dealing with large scale genomic datasets. Here we enumerate some of the challenges in QC of GWAS data and describe the approaches that the eMERGE network is using for quality assurance in GWAS data, thereby minimizing potential bias and error in GWAS results. In this protocol we discuss common issues associated with QC of GWAS data, including data file formats, software packages for data manipulation and analysis, sex chromosome anomalies, sample identity, sample relatedness, population substructure, batch effects, and marker quality. We propose best practices and discuss areas of ongoing and future research.

Telomere shortening is a marker of cellular aging and has been associated with risk of Alzheimer’s disease. Few studies have determined if telomere length is associated with cognitive decline in non-demented elders. We prospectively studied 2,734 non-demented elders (mean age: 74 years). We measured cognition with the Modified Mini-Mental State Exam (3MS) and Digit Symbol Substitution Test (DSST) repeatedly over 7 years. Baseline telomere length was measured in blood leukocytes and classified by tertile as “short”, “medium”, or “long”. At baseline, longer telomere length was associated with better DSST score (36.4, 34.9 and 34.4 points for long, medium and short, p <0.01) but not for change in score. However, seven-year 3MS change scores were less among those with longer telomere length (−1.7 points vs. −2.5 and −2.9, p = 0.01). Findings were similar after multivariable adjustment for age, gender, race, education, assay batch, and baseline score. There was a borderline statistically significant interaction for telomere length and APOE e4 on 3MS change score (p=0.06). Thus, telomere length may serve as a biomarker for cognitive aging.

Chromosome 3p21–22 harbors two clusters of chemokine receptor genes, several of which serve as major or minor coreceptors of HIV-1. Although the genetic association of CCR5 and CCR2 variants with HIV-1 pathogenesis is well known, the role of variation in other nearby chemokine receptor genes remain unresolved. We genotyped exonic single nucleotide polymorphisms (SNPs) in chemokine receptor genes: CCR3, CCRL2, and CXCR6 (at 3p21) and CCR8 and CX3CR1 (at 3p22), the majority of which were non-synonymous. The individual SNPs were tested for their effects on disease progression and outcomes in five treatment-naïve HIV-1/AIDS natural history cohorts. In addition to the known CCR5 and CCR2 associations, significant associations were identified for CCR3, CCR8, and CCRL2 on progression to AIDS. A multivariate survival analysis pointed to a previously undetected association of a non-conservative amino acid change F167Y in CCRL2 with AIDS progression: 167F is associated with accelerated progression to AIDS (RH = 1.90, P = 0.002, corrected). Further analysis indicated that CCRL2-167F was specifically associated with more rapid development of pneumocystis pneumonia (PCP) (RH = 2.84, 95% CI 1.28–6.31) among four major AIDS–defining conditions. Considering the newly defined role of CCRL2 in lung dendritic cell trafficking, this atypical chemokine receptor may affect PCP through immune regulation and inducing inflammation.

Author Summary

Human chemokine receptors are cell surface proteins that may be utilized by HIV-1 for entry into host cells. DNA variation in the HIV-1 major coreceptor CCR5 affects HIV-1 infection and progression. This study comprehensively assesses the role of genetic variation of multiple chemokine receptor genes clustered in the chromosome 3p21 and 3p22 on HIV-1 disease outcomes in HIV-1 natural history cohorts. The multivariate survival analyses identified functional variants that altered disease progression rate in CCRL2, CCR3, and CCR8. CCRL2-F167Y affects the rate to AIDS development through a specific protection against pneumocystis pneumonia (PCP), a common AIDS–defining condition. Our study identified this atypical chemokine receptor CCRL2 as a key factor involved in PCP, possibly through inducing inflammation in the lung.

BACKGROUND

Self-identified race or ethnic group is used to determine normal reference standards in the prediction of pulmonary function. We conducted a study to determine whether the genetically determined percentage of African ancestry is associated with lung function and whether its use could improve predictions of lung function among persons who identified themselves as African American.

METHODS

We assessed the ancestry of 777 participants self-identified as African American in the Coronary Artery Risk Development in Young Adults (CARDIA) study and evaluated the relation between pulmonary function and ancestry by means of linear regression. We performed similar analyses of data for two independent cohorts of subjects identifying themselves as African American: 813 participants in the Health, Aging, and Body Composition (HABC) study and 579 participants in the Cardiovascular Health Study (CHS). We compared the fit of two types of models to lung-function measurements: models based on the covariates used in standard prediction equations and models incorporating ancestry. We also evaluated the effect of the ancestry-based models on the classification of disease severity in two asthma-study populations.

RESULTS

African ancestry was inversely related to forced expiratory volume in 1 second (FEV1) and forced vital capacity in the CARDIA cohort. These relations were also seen in the HABC and CHS cohorts. In predicting lung function, the ancestry-based model fit the data better than standard models. Ancestry-based models resulted in the reclassification of asthma severity (based on the percentage of the predicted FEV1) in 4 to 5% of participants.

CONCLUSIONS

Current predictive equations, which rely on self-identified race alone, may misestimate lung function among subjects who identify themselves as African American. Incorporating ancestry into normative equations may improve lung-function estimates and more accurately categorize disease severity. (Funded by the National Institutes of Health and others.)

Objectives

We investigated the impact of polymorphisms in key renin angiotensin system genes on the association between angiotensin converting enzyme inhibitors (ACEINH) exposure and global and executive cognitive function in the Health, Aging and Body Composition study.

Design

Cohort study.

Setting

Community-based

Participants

3,075 participants: mean age: 73.6 years, 58% Caucasian, 52% women, 15% on ACEINH, 8 years of follow-up.

Measurements

The phenotypes were longitudinal change in Executive Clock Draw test-1 (CLOX1), the Digit Symbol Substitution test, and the Modified Mini Mental Status Examination. The genetic polymorphisms included the angiotensin converting enzyme insertion deletion (ACEID) in the angiotensin converting enzyme gene and the M235T and 6AG polymorphisms in the angiotensinogen gene (AGT).

Results

The 6AG and M235T polymorphisms in AGT had significant interaction with ACEINH exposure on the longitudinal change in CLOX1 scores in Caucasian participants (p=0.01 for both polymorphisms) independent of blood pressure levels. Specifically, ACEINH exposure was protective against CLOX1 score decline in carriers of the AA genotype of the 6AG and the CC genotype of the M235T (p-value for the ACEINH vs non-ACEINH groups =0.01 for 6AG and 0.005 for M235T) but not the other genotypes. These associations were not significant with other cognitive tests, with ACEID, or in African Americans.

Conclusion

ACEINH may provide a protective effect on executive function in Caucasians with AGT polymorphisms known to be associated with increased renin angiotensin system activity. If confirmed in a pharmacogenetic trial, ACEINH may have additional cognitive protection in a select group of elderly individuals.

Introduction

The eMERGE (electronic MEdical Records and GEnomics) Network is an NHGRI-supported consortium of five institutions to explore the utility of DNA repositories coupled to Electronic Medical Record (EMR) systems for advancing discovery in genome science. eMERGE also includes a special emphasis on the ethical, legal and social issues related to these endeavors.

Organization

The five sites are supported by an Administrative Coordinating Center. Setting of network goals is initiated by working groups: (1) Genomics, (2) Informatics, and (3) Consent & Community Consultation, which also includes active participation by investigators outside the eMERGE funded sites, and (4) Return of Results Oversight Committee. The Steering Committee, comprised of site PIs and representatives and NHGRI staff, meet three times per year, once per year with the External Scientific Panel.

Current progress

The primary site-specific phenotypes for which samples have undergone genome-wide association study (GWAS) genotyping are cataract and HDL, dementia, electrocardiographic QRS duration, peripheral arterial disease, and type 2 diabetes. A GWAS is also being undertaken for resistant hypertension in ≈2,000 additional samples identified across the network sites, to be added to data available for samples already genotyped. Funded by ARRA supplements, secondary phenotypes have been added at all sites to leverage the genotyping data, and hypothyroidism is being analyzed as a cross-network phenotype. Results are being posted in dbGaP. Other key eMERGE activities include evaluation of the issues associated with cross-site deployment of common algorithms to identify cases and controls in EMRs, data privacy of genomic and clinically-derived data, developing approaches for large-scale meta-analysis of GWAS data across five sites, and a community consultation and consent initiative at each site.

Future activities

Plans are underway to expand the network in diversity of populations and incorporation of GWAS findings into clinical care.

Summary

By combining advanced clinical informatics, genome science, and community consultation, eMERGE represents a first step in the development of data-driven approaches to incorporate genomic information into routine healthcare delivery.

The posttranscriptional gene regulation mediated by microRNA plays an important role in the development and function of male and female reproductive organs and germ cells in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in the testis and ovary in Holstein cattle by combining the Solexa sequencing with bioinformatics. In total 100 and 104 novel pre-miRNAs were identified in testicular and ovarian tissues, encoding 122 and 136 mature miRNAs, respectively. Of these, 6 miRNAs appear to be bovine-specific. A total of 246 known miRNAs were co-expressed in the testicular and ovarian tissues. Of the known miRNAs, twenty-one testis-specific and nine ovary-specific (1-23 reads) were found. Approximately 30.5% of the known bovine miRNAs in this study were found to have >2-fold differential expression within the two respective reproductive organ systems. The putative miRNA target genes of miRNAs were involved in pathways associated with reproductive physiology. Both known and novel tissue-specific miRNAs are expressed by Real-time quantitative PCR analysis in dairy cattle. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the bovine testicular and ovarian tissues, which provide important information on sex differences in miRNA expression. Diverse miRNAs may play an important regulatory role in the development of the reproductive organs in Holstein cattle.

Converging lines of evidence suggest that AKT1 is a major mediator of the responses to insulin, insulin-like growth factor 1 (IGF1), and glucose. AKT1 also plays a key role in the regulation of both muscle cell hypertrophy and atrophy. We hypothesized that AKT1 variants may play a role in the endophenotypes that make up metabolic syndrome. We studied a 12-kb region including the first exon of the AKT1 gene for association with metabolic syndrome-related phenotypes in four study populations [FAMUSS cohort (n = 574; age 23.7 ± 5.7 years), Strong Heart Study (SHS) (n = 2,134; age 55.5 ± 7.9 years), Dynamics of Health, Aging and Body Composition (Health ABC) (n = 3,075; age 73.6 ± 2.9 years), and Studies of a Targeted Risk Reduction Intervention through Defined Exercise (STRRIDE) (n = 175; age 40–65 years)]. We identified a three SNP haplotype that we call H1, which represents the ancestral alleles at the three loci and H2, which represents the derived alleles at the three loci. In young adult European Americans (FAMUSS), H1 was associated with higher fasting glucose levels in females. In middle age Native Americans (SHS), H1 carriers showed higher fasting insulin and HOMA in males, and higher BMI in females. In older African-American and European American subjects (Health ABC) H1 carriers showed a higher incidence of metabolic syndrome. Homozygotes for the H1 haplotype showed about twice the risk of metabolic syndrome in both males and females (p < 0.001). In middle-aged European Americans with insulin resistance (STRRIDE) studied by intravenous glucose tolerance test (IVGTT), H1 carriers showed increased insulin resistance due to the Sg component (p = 0.021). The 12-kb haplotype is a risk factor for metabolic syndrome and insulin resistance that needs to be explored in further populations.

Electronic supplementary material

The online version of this article (doi:10.1007/s00439-010-0910-8) contains supplementary material, which is available to authorized users.

Objectives

To assess blood aldosterone-renin ratio (ARR) and its relationship to ambulatory blood pressure (ABP) and left ventricular mass (LVM) in children.

Study design

A cross-sectional clinical study was conducted in 102 children (71.6% African American and 62.7% male) aged 7-18 years (mean=13.6, median=14). ABP (24-hour monitoring) was expressed as blood pressure index (BPI = mean BP/95th percentile by sex and height). LVM was measured by echocardiography and expressed as an index (LVMI=grams/ht2.7). Regression analyses were used to estimate associations.

Results

African American children had significantly lower serum aldosterone concentration and plasma renin activity compared with European American children (aldosterone: 5.9 ng/dl vs. 11.4 ng/dl, P = <0.0001 and renin: 1.6 ng/ml/h vs. 2.8 ng/ml/h, P = 0.01 respectively). However, ARR was not significantly different by race. ARR was not associated with 24-hour ABP, but was significantly associated with LVMI (β = 0.4 g/m2.7, P = 0.02) after adjustment for the ratio of 24-hour urine Na to creatinine excretion, BMI-z score, and ABPI.

Conclusion

The study observed a significant association between ARR and LVMI but not ABP in children, which suggested early cardiac remodeling associated with a high ARR.

Background

A common variant at chromosome 9p21 (tagged by the rs1333049 or rs10757278 SNP) is strongly associated with Myocardial Infarction (MI) and major arterial aneurysms. An association with Peripheral Arterial Disease (PAD) was also reported in a sample aged <75 years, but this disappeared on removal of respondents with a MI history, resulting in an odds ratio for PAD of 1.09 (p=0.075). We aimed to estimate the association of this variant with Ankle Brachial Index (ABI) and PAD in three older populations.

Methods and Results

We used data from the InCHIANTI, Baltimore Longitudinal Study of Aging and Health, Aging and Body Composition studies. In 2,630 Caucasian individuals (mean age 76.4 years) the C allele at rs1333049 was associated with lower mean ABI measures and with increased prevalence of PAD. These associations remained after removal of baseline and incident MI cases over a 6 year follow-up for both ABI (−0.017 ABI units, 95% CI: −0.03- −0.01, p=1.3×10−4) and PAD (per allele OR: 1.29, 95% CI: 1.06–1.56, p=0.012). These associations also remained after adjustment for known atherosclerosis risk factors including Diabetes Mellitus, smoking, hypercholesterolemia and hypertension.

Conclusions

The C allele at rs1333049 is associated with an increased prevalence of Peripheral Arterial Disease and lower mean Ankle Brachial Index. This association was independent of the presence of diagnosed MI and atherosclerotic risk factors in 3 older Caucasian populations.

Although telomere length (TL) is known to play a critical role in cellular senescence, the relationship of TL to aging and longevity in humans is not well understood. In a large biracial population-based cohort, we tested the hypotheses that elderly persons with shorter TL in peripheral white blood cells have poorer survival, shorter life span, and fewer years of healthy life (YHL). Associations were evaluated using Cox proportional hazard models and linear regression analyses where appropriate. TL (in kilo base pairs) was not associated with overall survival (hazard ratio 1.0; 95% confidence interval 0.9–1.1) or death from any specific underlying cause including infectious diseases, cancer, or cardiac and cerebrovascular diseases. TL, however, was positively associated with more YHL (β = 0.08 ± 0.04, p = .03). Findings suggest that TL may not be a strong biomarker of survival in older individuals, but it may be an informative biomarker of healthy aging.

Previous studies have reported associations of polymorphisms in the IGF1 gene with phenotypes of body composition (BC). The purpose of this study was to identify phenotypes of BC and physical function that were associated with the IGF1 promoter polymorphism (rs35767, −C1245T). Subjects from the Health, Aging, and Body Composition Study, white males and females (n = 925/836) and black males and females (533/705) aged 70–79 years were genotyped for the polymorphism. Phenotypes of muscle size and function, bone mineral density, and BC were analyzed for associations with this polymorphism. To validate and compare these findings, a cohort of young (mean age = 24.6, SD = 5.9) white men and women (n = 173/296) with similar phenotypic measurements were genotyped. An association with BC was identified in elderly females when significant covariates (physical activity, age, smoking status, body mass index) were included. White women with C/C genotype had 3% more trunk fat and 2% more total fat than those with C/T (P < 0.05). Black women with C/C genotype had 3% less total lean mass and 3% less muscle mass than their T/T counterparts (P < 0.05). Associations were identified with muscle strength in white women (P < 0.01) that were in agreement with the C/C genotype having lower muscle function. Thus, in an elderly population but not a young population, a polymorphism in the IGF1 gene may be predictive of differences in body composition, primarily in black females.

Electronic supplementary material

The online version of this article (doi:10.1007/s00421-010-1500-0) contains supplementary material, which is available to authorized users.

Objective

To determine whether variants in the estrogen receptor 1 (alpha) and 2 (beta) (ESR1 and ESR2) genes are associated with cognitive impairment in non-demented elderly men and women.

Background

Several single nucleotide polymorphisms (SNPs) on ESR1 and ESR 2 genes have been associated with a range of hormone sensitive diseases such as breast cancer and osteoporosis. Genetic variations in ESR may also influence cognitive aging but are less studied, especially among men.

Methods

We studied 2527 participants enrolled in an ongoing prospective study of community-dwelling elders. Four SNPs from ESR1 and four from ESR2 were analyzed. We measured cognitive function with the Modified Mini-Mental Status Examination (3MS) at baseline and biannually; cognitive impairment was defined as a decline of 5 or more points over 4 years. We calculated odds of developing cognitive impairment across SNPs using gender-stratified logistic regression and adjusted analyses for age, education, baseline 3MS score and in addition for race.

Results

1343 women (mean age 73.4) and 1184 men (mean age 73.7) comprised our cohort. Among women, after multivariate adjustment, 2 of the ESR1 SNPs (rs8179176, rs9340799) and 2 of the ESR2 SNPs (rs1256065, rs1256030) were associated with likelihood of developing cognitive impairment, although the association for rs8179176 was of trend level significance. In men, 1 of the ESR1 SNPs (rs728524) and 2 of the ESR2 (rs1255998, rs1256030) were associated with cognitive impairment. Further adjustment for race attenuated the results somewhat. There was no association between any ESR SNP and level of bioavailable estradiol but testosterone level did vary among 2 of the SNPs (p<0.05).

Conclusion

We found that among non-demented community elders, several SNPS in the ESR1 and ESR2 genes were associated with risk of developing cognitive impairment. These findings suggest that estrogen receptor genetic variants may play a role in cognitive aging.

Peripheral arterial disease (PAD) is associated with significant morbidity and mortality, and has a higher prevalence in African Americans than Caucasians. Ankle arm index (AAI) is the ratio of systolic blood pressure in the leg to that in the arm, and, when low, is a marker of PAD. We used an admixture mapping approach to search for genetic loci associated with low AAI. Using data from 1040 African-American participants in the observational, population-based Health, Aging, and Body Composition Study who were genotyped at 1322 single nucleotide polymorphisms(SNPs) that are informative for African versus European ancestry and span the entire genome, we estimated genetic ancestry in each chromosomal region and then tested the association between AAI and genetic ancestry at each locus. We found a region of chromosome 11 that reaches its peak between 80 and 82 Mb associated with low AAI (p<0.001 for rs12289502 and rs9665943, both within this region). 753 African-American participants in the observational, population-based Cardiovascular Health Study were genotyped at rs9665943 to test the reproducibility of this association, and this association was also statistically significant (odds ratio(OR) for homozygous African genotype 1.59 (95% confidence interval (CI) 1.12–2.27)). Another candidate SNP (rs1042602) in the same genomic region was tested in both populations, and was also found to be significantly associated with low AAI in both populations (OR for homozygous African genotype 1.89 (95% CI 1.29–2.76)). This study identifies a novel region of chromosome 11 representing an area with a potential candidate gene associated with PAD in African Americans.

Objective

The purpose of this study was to examine the association of the alpha-actinin-3 (ACTN3) R577X polymorphism on muscle function and physical performance in older adults.

Methods

We measured knee extensor torque, midthigh muscle cross-sectional area, muscle quality, short physical performance battery score, and 400-meter walk time at baseline and after 5 years in white older adults aged 70–79 years in the Health, Aging and Body Composition Study cohort (n = 1367). Incident persistent lower extremity limitation (PLL) over 5 years was additionally assessed. We also examined white men in the Osteoporotic Fractures in Men Study, a longitudinal, observational cohort (n = 1152) of men 65 years old or older as a validation cohort for certain phenotypes.

Results

There were no significant differences between genotype groups in men or women for adjusted baseline phenotypes. Male X-homozygotes had a significantly greater adjusted 5-year increase in their 400-meter walk time compared to R-homozygotes and heterozygotes (p = .03). In women, X-homozygotes had a ~35% greater risk of incident PLL compared to R-homozygotes (hazard ratio = 0.65, 95% confidence interval = 0.44–0.94). There were no other significant associations between any of the phenotypes and ACTN3 genotype with aging in either cohort.

Conclusions

The ACTN3 polymorphism may influence declines in certain measures of physical performance with aging in older white adults, based on longitudinal assessments. However, the influence of the ACTN3 R577X polymorphism does not appear to have a strong effect on skeletal muscle–related phenotypes based on the strength and consistency of the associations and lack of replication with regard to specific phenotypes.

Hyperuricemia is associated with primary hypertension (HTN) in adults and children. Furthermore, uric acid levels during childhood are associated with blood pressure (BP) levels in adulthood. We measured 24-h ambulatory BP and serum uric acid (SUA) in 104 children referred for possible hypertension. Mean age was 13.7 ± 2.6 y (range 7-18y) with 67 males and 37 females; 74 were African-American, 29 Caucasian and one Asian. SUA was associated with age (r=0.38, P=0.0001) and BMI Z-score (r=0.23, P=0.021). SUA was significantly associated with mean ambulatory systolic (S) and diastolic (D) BP. Mean ambulatory BP was normalized to gender- and height-specific reference standards using BP index. SUA was significantly associated with 24-h DBP index and nocturnal DBP index after adjusting for age, gender, race, BMI Z-score and urinary sodium excretion. SUA was also significantly associated with 24-h DBP load and nocturnal DBP load. Uric acid was significantly associated with increased likelihood for diastolic HTN (OR 2.1, CI 1.2, 3.7; P=0.0063) after adjusting for other co-variables. Among children at risk for HTN, the likelihood for diastolic HTN (as defined by ambulatory blood pressure monitoring) increases significantly as SUA increases. SUA may be associated with increased severity of HTN during youth.

The prevalence of obesity (body mass index (BMI) ≥30 kg/m2) is higher in African Americans than in European Americans, even after adjustment for socioeconomic factors, suggesting that genetic factors may explain some of the difference. To identify genetic loci influencing BMI, we carried out a pooled analysis of genome-wide admixture mapping scans in 15,280 African Americans from 14 epidemiologic studies. Samples were genotyped at a median of 1,411 ancestry-informative markers. After adjusting for age, sex, and study, BMI was analyzed both as a dichotomized (top 20% versus bottom 20%) and a continuous trait. We found that a higher percentage of European ancestry was significantly correlated with lower BMI (ρ = −0.042, P = 1.6×10−7). In the dichotomized analysis, we detected two loci on chromosome X as associated with increased African ancestry: the first at Xq25 (locus-specific LOD = 5.94; genome-wide score = 3.22; case-control Z = −3.94); and the second at Xq13.1 (locus-specific LOD = 2.22; case-control Z = −4.62). Quantitative analysis identified a third locus at 5q13.3 where higher BMI was highly significantly associated with greater European ancestry (locus-specific LOD = 6.27; genome-wide score = 3.46). Further mapping studies with dense sets of markers will be necessary to identify the alleles in these regions of chromosomes X and 5 that may be associated with variation in BMI.

Author Summary

Obesity is about 1.5-fold more prevalent in African Americans than European Americans. To determine whether genetic background may contribute to this observed disparity, we scanned the genomes of African Americans, searching for genomic regions where obese individuals have a difference from the average proportion of African ancestry. By examining genetic data from more than 15,000 African Americans, we show that the proportion of European ancestry is inversely correlated with BMI. In obese individuals, we detect two loci with increased African ancestry on chromosome X (Xq13.1 and Xq25) and one locus with increased European ancestry on chromosome 5 (5q13.3). The 5q13.3 and Xq25 regions both contain genes that are known to be involved in appetite regulation. Our results suggest that genetic factors may contribute to the difference in obesity prevalence between African Americans and European Americans. Further studies of the regions may identify the causative variants affecting susceptibility to obesity.

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.

Rationale: Plasminogen activator inhibitor (PAI)-1 inhibits urokinase and tissue plasminogen activator, required for host response to infection. Whether variation within the PAI-1 gene is associated with increased susceptibility to infection is unknown.

Objectives: To ascertain the role of the 4G/5G polymorphism and other genetic variants within the PAI-1 gene. We hypothesized that variants associated with increased PAI-1 expression would be associated with an increased occurrence of community-acquired pneumonia (CAP).

Methods: Longitudinal analysis (>12 yr) of the Health, Aging, and Body Composition cohort, aged 65–74 years at start of analysis.

Measurements and Main Results: We genotyped the 4G/5G PAI-1 polymorphism and six additional single nucleotide polymorphisms. Of the 3,075 subjects, 272 (8.8%) had at least one hospitalization for CAP. Among whites, variants at the PAI4G,5G, PAI2846, and PAI7343 sites had higher risk of CAP (P = 0.018, 0.021, and 0.021, respectively). At these sites, variants associated with higher PAI-1 expression were associated with increased CAP susceptibility. Compared with the 5G/5G genotypes at PAI4G,5G site, the 4G/4G and 4G/5G genotypes were associated with a 1.98-fold increased risk of CAP (95% confidence interval, 1.2–3.2; P = 0.006). In whole blood stimulation assay, subjects with a 4G allele had 3.3- and 1.9-fold increased PAI-1 expression (P = 0.043 and 0.034, respectively). In haplotype analysis, the 4G/G/C/A haplotype at the PAI4G,5G, PAI2846, PAI4588, and PAI7343 single nucleotide polymorphisms was associated with higher CAP susceptibility, whereas the 5G/G/C/A haplotype was associated with lower CAP susceptibility. No associations were seen among blacks.

Conclusions: Genotypes associated with increased expression of PAI-1 were associated with increased susceptibility to CAP in elderly whites.

There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts – cis effects, and elsewhere in the genome – trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10−57), CCL4L1 (p = 3.9×10−21), IL18 (p = 6.8×10−13), LPA (p = 4.4×10−10), GGT1 (p = 1.5×10−7), SHBG (p = 3.1×10−7), CRP (p = 6.4×10−6) and IL1RN (p = 7.3×10−6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8×10−40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These include the presence of strong genetic effects in cis locations. The identification of protein quantitative trait loci (pQTLs) may be a powerful complementary method of improving our understanding of disease pathways.

Author Summary

One of the central dogmas of molecular genetics is that DNA is transcribed to RNA which is translated to protein and alterations to proteins can influence human diseases. Genome-wide association studies have recently revealed many new DNA variants that influence human diseases. To complement these efforts, several genome-wide studies have established that DNA variation influences mRNA expression levels. Loci influencing mRNA levels have been termed “eQTLs”. In this study we have performed the first genome-wide association study of the third piece in this jigsaw – the role of DNA variation in relation to protein levels, or “pQTLs”. We analysed 42 proteins measured in blood fractions from the InCHIANTI study. We identified eight cis effects including common variants in or near the IL6R, CCL4, IL18, LPA, GGT1, SHBG, CRP and IL1RN genes, all associated with blood levels of their respective protein products. Mechanisms implicated included altered transcription (GGT1) but also rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA) and variation in gene copy number (CCL4). Blood levels of many of these proteins are correlated with human diseases and the identification of “pQTLs” may in turn help our understanding of disease.

BACKGROUND AND OBJECTIVES: The 894T allele in exon 7 of the endothelial nitric oxide synthase (eNOS) gene has been inconsistently associated with hypertension in different racial groups. Because high-normal blood pressure (BP) confers an increased risk for the development of hypertension and other cardiovascular disorders, including left ventricular hypertrophy (LVH), we tested the hypothesis that the allelic variation (894T) in the eNOS gene would directly correlate with alterations in LV mass (LVM) in individuals with high-normal BP. METHODS: Genotype distribution of G894T was compared between 20 African Americans (10 females/10 males) with high-normal BP (systolic BP of 130-139 and/or diastolic BP of 85-89 mmHg) and 64 counterparts (37 females/27 males) with normal BP (<130/85 mmHg). Echocardiographic LVM was calculated (Devereux formula) and indexed to body surface area to define the presence of LVH (LVMI >134/110 g/m2 for men/women). RESULTS: For the entire group, the 894T allelic frequencies (15, 48%) and G894T genotype distributions were consistent with the Hardy-Weinberg equilibrium expectations (estimated disequilibrium coefficient = 0.0118, P=0.40). LVMI was significantly higher in homozygous carriers (TT) of the rare 894T allele (n = 3 females/0 males) than in heterozygous GT (n = 13 females/7 males) and individuals bearing the GG (n=34 females/27 males) variant (124 +/- 70 vs. 82 +/- 24 and 82 +/- 19 g/m2, respectively, P < 0.05). The observed relationship between eNOS 894T allele and LVMI was restricted to individuals with high-normal BP (r = 0.94, P = 0.03) but not in those with normal BP (r = 0.39, P =0.64), by analysis of variance (ANOVA) after adjusting for age, gender, body mass index, smoking and systolic BP. CONCLUSION: These findings, not previously described, provide important preliminary evidence to suggest an increased susceptibility to LVH in African Americans who carry the 894T variant of the eNOS gene and have high-normal blood pressure.