A second generation human haplotype map of over 3.1 million SNPs.

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

Genome-wide detection and characterization of positive selection in human populations.

With the advent of dense maps of human genetic variation, it is now possible to detect positive natural selection across the human genome. Here we report an analysis of over 3 million polymorphisms from the International HapMap Project Phase 2 (HapMap2). We used 'long-range haplotype' methods, which were developed to identify alleles segregating in a population that have undergone recent selection, and we also developed new methods that are based on cross-population comparisons to discover alleles that have swept to near-fixation within a population. The analysis reveals more than 300 strong candidate regions. Focusing on the strongest 22 regions, we develop a heuristic for scrutinizing these regions to identify candidate targets of selection. In a complementary analysis, we identify 26 non-synonymous, coding, single nucleotide polymorphisms showing regional evidence of positive selection. Examination of these candidates highlights three cases in which two genes in a common biological process have apparently undergone positive selection in the same population:LARGE and DMD, both related to infection by the Lassa virus, in West Africa;SLC24A5 and SLC45A2, both involved in skin pigmentation, in Europe; and EDAR and EDA2R, both involved in development of hair follicles, in Asia.

Gene expression in fixed tissues and outcome in hepatocellular carcinoma.

BACKGROUND: It is a challenge to identify patients who, after undergoing potentially curative treatment for hepatocellular carcinoma, are at greatest risk for recurrence. Such high-risk patients could receive novel interventional measures. An obstacle to the development of genome-based predictors of outcome in patients with hepatocellular carcinoma has been the lack of a means to carry out genomewide expression profiling of fixed, as opposed to frozen, tissue. METHODS: We aimed to demonstrate the feasibility of gene-expression profiling of more than 6000 human genes in formalin-fixed, paraffin-embedded tissues. We applied the method to tissues from 307 patients with hepatocellular carcinoma, from four series of patients, to discover and validate a gene-expression signature associated with survival. RESULTS: The expression-profiling method for formalin-fixed, paraffin-embedded tissue was highly effective: samples from 90% of the patients yielded data of high quality, including samples that had been archived for more than 24 years. Gene-expression profiles of tumor tissue failed to yield a significant association with survival. In contrast, profiles of the surrounding nontumoral liver tissue were highly correlated with survival in a training set of tissue samples from 82 Japanese patients, and the signature was validated in tissues from an independent group of 225 patients from the United States and Europe (P=0.04). CONCLUSIONS: We have demonstrated the feasibility of genomewide expression profiling of formalin-fixed, paraffin-embedded tissues and have shown that a reproducible gene-expression signature correlated with survival is present in liver tissue adjacent to the tumor in patients with hepatocellular carcinoma.

Common genetic variation in KCNH2 is associated with QT interval duration: the Framingham Heart Study.

BACKGROUND: QT prolongation is associated with increased risk of sudden cardiac death in the general population and in people exposed to QT-prolonging drugs. Mutations in the KCNH2 gene encoding the HERG potassium channel cause 30% of long-QT syndrome, and binding to this channel leads to drug-induced QT prolongation. We tested common KCNH2 variants for association with continuous QT interval duration. METHODS AND RESULTS: We selected 17 single nucleotide polymorphisms and rs1805123, a previously associated missense single nucleotide polymorphism, for genotyping in 1730 unrelated men and women from the Framingham Heart Study. rs3807375 genotypes were associated with continuous QT interval duration in men and women (2-df P=0.002), with a dominant model suggested (P=0.0004). An independent sample of 871 Framingham Heart Study men and women replicated the association (1-sided dominant P=0.02). On combined analysis of 2123 subjects, individuals with AA or AG genotypes had a 0.14-SD (SE, 0.04) or 3.9-ms higher age-, sex- and RR-adjusted QT interval compared with GG individuals (P=0.00006). The previously reported association of rs1805123 (K897T) replicated under a dominant (AA/AC, 0.12 [corrected] SD [SE, 0.07] or 3.1 ms higher versus CC; 1-sided P=0.04) or additive model (0.06 SD [SE, 0.03] or 1.6 ms higher per A allele; 1-sided P=0.01). CONCLUSIONS: Two common genetic variants at the KCNH2 locus are associated with continuous QT interval duration in an unselected community-based sample. Studies to determine the influence of these variants on risk of sudden cardiac death and drug-induced arrhythmias should be considered.

Contribution of clinical correlates and 13 C-reactive protein gene polymorphisms to interindividual variability in serum C-reactive protein level.

BACKGROUND: Serum C-reactive protein (CRP) level is a heritable complex trait that predicts incident cardiovascular disease. We investigated the clinical and genetic sources of interindividual variability in serum CRP. METHODS AND RESULTS: We studied serum CRP in 3301 Framingham Heart Study (FHS) participants (mean age 61 years, 53% women). Twelve clinical covariates explained 26% of the variability in CRP level, with body mass index alone explaining 15% (P<0.0001) of the variance. To investigate the influence of genetic variation at the CRP gene on CRP levels, we first constructed a dense linkage disequilibrium map for common single-nucleotide polymorphisms (SNPs) spanning the CRP locus (1 SNP every 850 bases, 26 kilobase [kb] genomic region). Thirteen CRP SNPs were genotyped in 1640 unrelated FHS participants with measured CRP levels. After adjustment for clinical covariates, 9 of 13 SNPs were associated with CRP level (P<0.05). To account for correlation among SNPs, we conducted forward stepwise selection among all 13 SNPs; a triallelic SNP (rs3091244) remained associated with CRP level (stepwise P<0.0001). The triallelic SNP (C-->T-->A; allele frequencies 62%, 31%, and 7%), located in the promoter sequence, explained 1.4% of total serum CRP variation; haplotypes harboring the minor T and A alleles of this SNP were associated with higher CRP level (haplotype P=0.0002 and 0.004). CONCLUSIONS: In our community-based sample, clinical variables explained 26% of the interindividual variation in CRP, whereas a common triallelic CRP SNP contributed modestly. Studies of larger samples are warranted to assess the association of genetic variation in CRP and risk of cardiovascular disease.

Common genetic variation in five thrombosis genes and relations to plasma hemostatic protein level and cardiovascular disease risk.

OBJECTIVE: We undertook a linkage disequilibrium (LD)-based genetic approach to investigate the hypothesis that common sequence variants in 5 thrombosis genes influence plasma hemostatic protein levels or risk of cardiovascular disease (CVD). METHODS AND RESULTS: In a reference panel, we characterized LD structure at the fibrinogen gene cluster (fibrinogen-beta[FGB], FGA, and FGG), factor VII (F7), and tissue plasminogen activator (PLAT) loci. Forty-one tag single nucleotide polymorphisms (SNPs) were genotyped in 1811 unrelated Framingham Heart Study participants. There were significant associations of 9 FGB SNPs with fibrinogen level (minimum P=0.002) and of 7 F7 SNPs and factor VII level (minimum P<0.0001). SNPs at the PLAT locus were not associated with PLAT level. In stepwise analysis, a single FGB variant explained 1% of the residual variance in fibrinogen level, and 2 F7 SNPs together explained 10% of the residual variance in factor VII level. Two PLAT haplotypes were associated with CVD (multivariable-adjusted global P=0.0004). CONCLUSIONS: A comprehensive survey of common sequence variation demonstrates that cis-regulatory SNPs explain a modest proportion of the residual variance in circulating fibrinogen and factor VII level and PLAT haplotypes increase the risk of CVD. Additional studies are warranted to confirm the association of PLAT sequence variation and risk of CVD.

The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

Clinical and genetic correlates of aldosterone-to-renin ratio and relations to blood pressure in a community sample.

Aldosterone:renin ratio (ARR) is used to screen for hyperaldosteronism. Data regarding correlates of ambulatory ARR in the community and its relation to hypertension incidence are limited. We defined clinical correlates of ARR, determined its heritability, tested for association and linkage, and related ARR to blood pressure (BP) progression in nonhypertensive individuals among 3326 individuals from the Framingham Heart Study (53% women; mean age: 59 years). Ambulatory morning ARR (serum aldosterone and plasma renin concentrations) were related to clinical covariates, genetic variation across the REN locus, a 10-cM linkage map, and among nonhypertensive participants (n=1773) to progression of >or=1 Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure BP category (optimal: <120/80 mm Hg, normal: 120 to 129/80 to 84 mm Hg, high normal: 130 to 139/85 to 89 mm Hg, hypertension: >or=140/90 mm Hg), or incident hypertension (systolic BP: >or=140 mm Hg, diastolic BP: >or=90 mm Hg, or use of antihypertensive treatment). ARR was positively associated with age, female sex, untreated hypertension, total/high-density lipoprotein cholesterol ratio, hormone replacement therapy, and beta-blocker use, but negatively associated with angiotensin-converting enzyme inhibitor and diuretic use. ARR was heritable (h(2)=0.40), had modest linkage to chromosome 11p (logarithm of the odds: 1.89), but was not associated with 17 common variants in REN (n=1729). On follow-up (mean: 3 years), 607 nonhypertensive individuals (34.2%) developed BP progression, and 283 (16.0%) developed hypertension. Higher baseline logARR was associated with increased risk of BP progression (odds ratio per SD increment: 1.23; 95% CI: 1.11 to 1.37) and hypertension incidence (odds ratio per SD increment: 1.16; 95% CI: 1.00 to 1.33). ARR is a heritable trait influenced by clinical and genetic factors. There is a continuous gradient of increasing risk of BP progression across ARR levels in nonhypertensive individuals.

Vascular stiffness and genetic variation at the endothelial nitric oxide synthase locus: the Framingham Heart study.

Arterial stiffness is a moderately heritable trait that is affected by alterations in the bioavailability of NO. Previous studies have found associations between variants in the gene for endothelial NO synthase (NOS3) and arterial properties. We previously identified a linkage peak for forward pressure wave amplitude in the immediate vicinity of NOS3. Therefore, we evaluated relations between arterial stiffness measures and common genetic variants at this locus. Eighteen single nucleotide polymorphisms capturing approximately 90% of underlying common variation in NOS3 were genotyped in unrelated Framingham Heart Study participants (N=1157; 52.2% women; mean age: 62 years) with routinely ascertained tonometry data that provided 5 arterial phenotypes (forward and reflected pressure wave amplitude, central pulse pressure, carotid-femoral pulse wave velocity, and mean arterial pressure). In women but not men, the genotype for the common NOS3 missense mutation (Glu298Asp, rs1799983) was related to central pulse pressure (women: GG=53+/-0.9, GT=54+/-0.9, and TT=47+/-2.0 mm Hg, P=0.0047; men: GG=50+/-1.0, GT=49+/-0.9, and TT=47+/-1.8 mm Hg, P=0.30) and forward wave amplitude (women: GG=41+/-0.7, GT=42+/-0.7, and TT=38+/-1.6 mm Hg, P=0.029; men: GG=42+/-0.9, GT=41+/-0.8, and TT=39+/-1.5 mm Hg, P=0.47). The only other nominally significant sex-specific association in men but not women was between an intronic polymorphism (rs1800781) and reflected wave amplitude (women: AA=10.4+/-0.4, AG=11.1+/-0.6, and GG=8.9+/-2.2 mm Hg, P=0.50; men: AA=6.1+/-0.3, AG=7.3+/-0.5, and GG=11.3+/-2.3 mm Hg, P=0.014). After adjusting for multiple testing (18 polymorphisms and 5 phenotypes), these nominal associations were no longer significant. The present study was suggestive of modest relations between common genetic variants at the NOS3 locus and arterial stiffness.