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Background— Fibrinogen is both central to blood coagulation and an acute-phase reactant. We aimed to identify common variants influencing circulation fibrinogen levels. Methods and Results— We conducted a genome-wide association analysis on 6 population-based studies, the Rotterdam Study, the Framingham Heart Study, the Cardiovascular Health Study, the Atherosclerosis Risk in Communities Study, the Monitoring of Trends and Determinants in Cardiovascular Disease/KORA Augsburg Study, and the British 1958 Birth Cohort Study, including 22 096 participants of European ancestry. Four loci were marked by 1 or more single-nucleotide polymorphisms that demonstrated genome-wide significance ( P −8 ). These included a single-nucleotide polymorphism located in the fibrinogen β chain ( FGB ) gene and 3 single-nucleotide polymorphisms representing newly identified loci. The high-signal single-nucleotide polymorphisms were rs1800789 in exon 7 of FGB ( P =1.8�10 −30 ), rs2522056 downstream from the interferon regulatory factor 1 ( IRF1 ) gene ( P =1.3�10 −15 ), rs511154 within intron 1 of the propionyl coenzyme A carboxylase ( PCCB ) gene ( P =5.9�10 −10 ), and rs1539019 on the NLR family pyrin domain containing 3 isoforms ( NLRP3 ) gene ( P =1.04�10 −8 ). Conclusions— Our findings highlight biological pathways that may be important in regulation of inflammation underlying cardiovascular disease.

BACKGROUND Smoking is associated with a systemic inflammatory response. However, the role of genetic predisposition is not well known. We assessed whether circulatory acute phase reactants were associated with smoking and whether or not the association was modified by the major cytokine gene of the acute phase reaction, interleukin-6 (IL-6). METHODS In total, 1,003 postmyocardial infarction patients were recruited in six European cities and six repeated clinical examinations performed. C-reactive protein (CRP), interleukin 6 (IL-6), and fibrinogen levels were assayed at 5,659 subject visits. Genotyping of single nucleotide polymorphisms was performed in the IL-6 gene. RESULTS Cumulative smoking (pack-years) and time since smoking cessation were strongly associated with blood levels of all three inflammatory markers. Among subjects without any respiratory disorder, these associations remained statistically significant for CRP and IL-6. A polymorphism in the IL-6 gene (rs2069840) showed an interaction with smoking on CRP (p < .001) and IL-6 (p = .049) peripheral levels. CONCLUSIONS These results indicate a potential role of the IL-6 gene in the inflammatory response associated with smoking and suggest rs2069840 polymorphism deserves attention.

SummaryPrevious studies reported a gender-specific association between plasma fibrinogen concentrations and incident hypertension. We systematically analysed polymorphisms and haplotypes across the fibrinogen gene cluster with fibrinogen levels and assessed their contribution to prevalent hypertension in 2,200 men and 2,159 women from the population-based MONICA/KORA Augsburg study. Eleven tagging single nucleotide polymorphisms (SNPs) were systematically selected in the three fibrinogen genes and haplotypes were reconstructed. The minor alleles of two SNPs, rs2227401 (FGB) and rs2070016 (FGA) and the haplotypes tagged by those variants, were significantly associated with higher fibrinogen concentrations in both, men and women, explaining 1% of the total variance of fibrinogen concentrations. In addition, a FGG haplotype, tagged by rs1049636, was associated with lower concentrations of fibrinogen in women, but not in men. Regarding hypertension, we detected a significant association with a FGA promoter variant (rs2070008) in women only, whereas fibrinogen haplotypes were not associated with hyper-tension after correction for multiple comparisons in either men or women. In conclusion, our results suggest that variants in all three fibrinogen genes are significantly associated with differences in fibrinogen concentrations with modest contribution to phenotypic variance. It is likely that other genetic variants outside the fibrinogen gene loci are involved in the regulation of fibrinogen concentrations. In addition, one FGA promoter variant was significantly associated with hypertension in women. Confirmation of these findings by future studies is warranted.

Aims C-reactive protein represents the classical acute-phase protein produced in the liver in response to inflammatory stimuli. This study evaluated the association of gene polymorphisms with differences in C-reactive protein concentrations and assessed its intra-individual variability as a marker of individual response. Methods and results One thousand and three myocardial infarction (MI) survivors were recruited in six European cities, and C-reactive protein concentrations were measured repeatedly during a 6-month period. We investigated 114 polymorphisms in 13 genes, all involved in the innate inflammatory pathway. We found two polymorphisms within the C-reactive protein ( CRP ) gene rs1800947 and rs1205, of which the minor alleles were strongly associated with lower levels of C-reactive protein ( P < 10−6). A haplotype, identified by those two polymorphisms, was associated with the lowest C-reactive protein concentrations ( P < 10−6). Additionally, the minor alleles of several variants were significantly associated with greater individual variability of C-reactive protein concentrations ( P < 10−3). Conclusion The present study investigated the association of polymorphisms with inter- and intra-individual variability of C-reactive protein levels. Two minor alleles of C-reactive protein variants were associated with lower C-reactive protein concentrations. Regarding intra-individual variability, we observed associations with the minor alleles of several variants in selected candidate genes, including the CRP gene itself.

Interleukin-6 ( IL-6 ) is a pleiotropic cytokine which has been proposed as “cytokine for gerontologists” and linked to age-related metabolic disturbances such as the metabolic syndrome or type 2 diabetes. Polymorphisms located in the promoter region of IL-6 have been reported to be involved in the regulation of IL-6 transcription. This study investigates whether IL-6 promoter variants −174 G/C and −573 G/C are associated with quantitative traits related to the metabolic syndrome (International Diabetes Federation criteria) in a population of normoglycemic subjects ( n = 878) from the latest KORA survey (KORA S4). Genotyping was performed using MALDI-TOF MS. Besides lower height ( p = 0.01) the −174 CC genotype was independently associated with lower waist ( p = 0.002) and hip ( p = 0.01) circumferences in men. Furthermore, the −174 CC genotype was associated with BMI ( p = 0.004) when adjusted for waist and hip circumference. The present study does not suggest associations with further components of the metabolic syndrome. The association with height seems to be the central factor indicating an influence of IL-6 on growth through impaired bone metabolism. However, the complex relationships need further investigation.

As part of a project on environmental disasters in minority populations, this study aimed to evaluate differences in the sequence of N-acetyltransferase 2 (NAT2) as a metabolic susceptibility gene in yet unexplored ethnicities. Eight single nucleotide polymorphisms (SNP) in the NAT2 coding region and a variant in the 3′ flanking region were analyzed in 290 unrelated Kyrgyz and 140 unrelated Romanians by SNP-specific PCR analysis. The variants 341C, 481T, and 803G were less and 857A more prevalent in Kyrgyz (P < 0.0001). The variant at site 857 indicates Asian descent. 282C>T and 590G>A showed no significant variation by ethnicity. 364G>A and 411A>T turned out to be monomorphic. Database comparisons of the NAT2 minor allele frequencies support that Romanians belong to Caucasians and Kyrgyz are in between Caucasians and East Asians. The distributions of predicted haplotypes differed significantly between the two ethnicities where the Kyrgyz showed a higher genetic diversity. The haplotype without mutations was more common in Kyrgyz (40.1% in Kyrgyz, 29.3% in Romanians). Accordingly, the imputed slow acetylator phenotype was less prevalent in Kyrgyz (35.2% versus 51.4% in Romanians). We found pronounced ethnic differences in NAT2 genotypes with yet unknown effect on the health risks for environmental or occupational exposures in minority populations. (Cancer Epidemiol Biomarkers Prev 2006;(15)1:138–41)

Previous studies reported a gender-specific association between plasma fibrinogen concentrations and incident hypertension. We systematically analysed polymorphisms and haplotypes across the fibrinogen gene cluster with fibrinogen levels and assessed their contribution to prevalent hypertension in 2,200 men and 2,159 women from the population-based MONICA/KORA Augsburg study. Eleven tagging single nucleotide polymorphisms (SNPs) were systematically selected in the three fibrinogen genes and haplotypes were reconstructed. The minor alleles of two SNPs, rs2227401 (FGB) and rs2070016 (FGA) and the haplotypes tagged by those variants, were significantly associated with higher fibrinogen concentrations in both, men and women, explaining 1% of the total variance of fibrinogen concentrations. In addition, a FGG haplotype, tagged by rs1049636, was associated with lower concentrations of fibrinogen in women, but not in men. Regarding hypertension, we detected a significant association with a FGA promoter variant (rs2070008) in women only, whereas fibrinogen haplotypes were not associated with hypertension after correction for multiple comparisons in either men or women. In conclusion, our results suggest that variants in all three fibrinogen genes are significantly associated with differences in fibrinogen concentrations with modest contribution to phenotypic variance. It is likely that other genetic variants outside the fibrinogen gene loci are involved in the regulation of fibrinogen concentrations. In addition, one FGA promoter variant was significantly associated with hypertension in women. Confirmation of these findings by future studies is warranted.

Previously, estimation of genotype misclassification of single nucleotide polymorphisms (SNPs) as encountered in epidemiologic practice and involving thousands of subjects was lacking. The authors collected representative data on approximately 14,000 subjects from 8 studies and 646,558 genotypes assessed in 2005 by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Overall discordance among 57,805 double genotypes from routine quality control was 0.36%. Fitting different misclassification models by maximum likelihood assuming identical misclassification for all SNPs, the estimated misclassification probabilities ranged from 0.0000 to 0.0035. When applying the misclassification simulation and extrapolation (MC-SIMEX) method for the first time to genetic data to account for the misclassification in a reanalysis of adiponectin-encoding (APM1) gene SNP associations with plasma adiponectin in 1,770 subjects, the authors found no impact of this small error on association estimates but increased estimates for a more substantial error. This study is the first to provide large-scale epidemiologic data on SNP genotype misclassification. The estimated misclassification in this example was small and negligible for association estimates, which is reassuring and essential for detecting SNP associations. In situations with more substantial error, the presented approach using duplicate genotyping and the MC-SIMEX method is practical and helpful for quantifying the genotyping error and its impact.

As part of a project on environmental pollution, this study aimed to evaluate associations between blood lead (BPb) levels, hemoglobin (Hb) content, and single-nucleotide polymorphisms (SNPs) of delta-aminolevulinic acid dehydratase (ALAD) gene in 129 unrelated women from Romania. Five SNPs (rs1805313, rs2228083, rs1805312, rs1800435, rs1139488) were analyzed with respect to haplotype structure and impact on BPb levels and Hb content with proportional odds and analysis of covariance models. Combinations of SNPs were rare (16%). Low haplotype diversity was found with seven haplotypes. One rare haplotype implied the C allele of rs1800435, often referred to as the ALAD2 allele (frequency 8.6%). The putative risk genotype (CC) occurred in only one woman with BPb below 0.5 microg/dl. Median BPb was 4.8 microg/dl and differed markedly by community with a level of 12.5 microg/dl near a mining-spill region. Hb was regular (interquartile range 12.3-13.7 g/dl) and not correlated with BPb, although quantitatively lower in women living near the spill region. No significant associations were found for BPb or Hb with SNPs, haplotypes, or diplotypes. BPb levels were higher in this region than in populations from industrialized countries but without hematotoxic effects. An impact of ALAD2 on BPb or Hb was not seen in these women.

Introduction Increased levels of interleukin 6 (IL-6), a marker for systemic inflammation, have been associated with cardiovascular morbidity and mortality. Materials and Methods We investigated the influence of IL6 gene polymorphisms on mean level and variability of plasma IL-6 in a population of myocardial infarction survivors recruited in six European cities as part of the AIRGENE study. DNA from each individual was collected and genotyped for eight functional and tagging single nucleotide polymorphisms (SNPs) in the IL6 gene. Results We analyzed 946 subjects with 5520 repeated plasma samples for IL-6 levels. For four IL6 SNPs in high linkage disequilibrium, heterozygous and homozygous minor allele genotypes were associated with an increase in mean plasma IL-6 levels. SNP rs1800795 was associated with a 6.3% increase in IL-6 (95% confidence interval [CI] 1.7-11,2%) For these SNPs, we found that genotypes associated with higher IL-6 levels also tended to be associated to higher between-individual variability of IL-6 levels on the log-scale than other genotypes. Variability over time within individuals varied little by genotype. Conclusions We found four genetic polymorphisms in the IL6 gene associated with mean level and variability of plasma IL-6 between individuals in myocardial infarction survivors.

Local inflammation in airway diseases is well recognised, but less is known about the association between low-grade systemic inflammatory processes and lung function. The aim of the present study was to assess the association between inflammatory markers and lung function, taking into account polymorphisms in genes coding for inflammatory markers. In 134 post-myocardial infarction patients, six repeated measurements of C-reactive protein (CRP), interleukin (IL)-6 and fibrinogen in peripheral blood were assayed using high-sensitivity tests. Spirometry was conducted at baseline. Genotyping of single nucleotide polymorphisms was performed in genes coding for the inflammatory markers. CRP and IL-6 levels were negatively associated with forced expiratory volume in one second (FEV 1 ), forced vital capacity (FVC) and mean forced expiratory flow between 25 and 75% of FVC (FEF 25–75% ). In the CRP gene, both the polymorphism rs1205 and the haplotype 2 showed a protective association with FEV 1 and FEF 25–75% , and, to a lesser extent, with FVC. rs1205 and haplotype 2 were both negatively associated with CRP levels in peripheral blood. Analysis with instrumental variables also showed a protective effect between these CRP gene polymorphisms and lung function. Results are very suggestive that heritability of lung function is at least partly controlled by the CRP gene. Applying a Mendelian randomisation approach, the study supports a causal association between low-grade general inflammation and airway diseases.

Recently, significant associations between common variants of the transcription factor 7-like 2 gene ( TCF7L2) and type 2 diabetes have been reported. This study was designed to replicate the reported associations of the two highly correlated (r (2)=0.86) TCF7L2 single nucleotide polymorphisms rs12255372 and rs7903146 with type 2 diabetes in a case-control study of 2369 MONICA/KORA participants (678 cases/1691 controls from Augsburg, Germany). To further investigate the pathogenic mechanism underlying these associations, we extended our analyses to the metabolic syndrome (IDF, NCEP definitions) and its components in a population-based study comprising 1404 male and female KORA participants aged 55-74 years. Results of our analyses strongly confirmed the minor T alleles as risk variants for type 2 diabetes (rs7903146: OR (TvsC) [95% CI]=1.36 [1.18;1.58], p=0.00003, and rs12255372: OR (TvsG) [95% CI]=1.31 [1.13;1.51], p=0.0003). Moreover, the T allele at rs7903146 was inversely associated with log-transformed, HOMA-%B (beta=-0.07, p=0.005) as a measure of basal insulin secretion, and log-transformed fasting insulin (beta=-0.06, p=0.02). No association was found with insulin resistance (HOMA-IR) and the metabolic syndrome. These findings support replication evidence that TCF7L2 variants increase type 2 diabetes risk. TCF7L2 may primarily affect pancreatic beta cell function.

Apolipoprotein A5 (APOA5) gene variants were reported to be associated with two components of metabolic syndrome (MetS): higher TG levels and lower HDL levels. Moreover, a recent Japanese case-control study found variant −1131T>C associated with MetS itself. Thus, our study systematically analyzed the APOA5 gene for association with lipid parameters, any other features of MetS, including waist circumference, glucose-related parameters, blood pressure, uric acid, and MetS itself in Caucasians. Ten polymorphisms were analyzed in a large fasting sample of the population-based Cooperative Health Research in the Region of Augsburg (KORA) survey S4 (n = 1,354; southern Germany) and in a second fasting sample, the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study (n = 1,770; Austria). Minor alleles of variants −1131T>C, −3A>G, c.56C>G, 476G>A, and 1259T>C were significantly associated with higher TG levels in single polymorphism (P < 0.001) and haplotype (P ⩽ 6.6 × 10−6) analysis. Besides associations with lower HDL levels in SAPHIR (P ⩽ 0.001), there were no significant findings with any other features of MetS. Variant c.56C>G was associated with higher risk for MetS [odds ratio (95% confidence interval) = 1.43 (1.04, 1.99), P = 0.03 for KORA and 1.48 (1.10, 1.99), P = 0.009 for SAPHIR). Our study confirms the association of the APOA5 locus with TG and HDL levels in humans. Furthermore, the data suggest a different mechanism of APOA5 impact on MetS in Caucasians, as variant c.56C>G (not analyzed in the Japanese study) and not −1131T>C, as in the Japanese subjects, was associated with MetS.

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p = 5.2×10−201), ABCG2 (p = 3.1×10−26), SLC17A1 (p = 3.0×10−14), SLC22A11 (p = 6.7×10−14), SLC22A12 (p = 2.0×10−9), SLC16A9 (p = 1.1×10−8), GCKR (p = 1.4×10−9), LRRC16A (p = 8.5×10−9), and near PDZK1 (p = 2.7×10−9). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p = 4.0×10−26) and propionyl-L-carnitine (p = 5.0×10−8) concentrations, which in turn were associated with serum UA levels (p = 1.4×10−57 and p = 8.1×10−54, respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels., Author Summary Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. The regulation of serum uric acid levels is under a strong genetic control. This study describes the first meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent. We show that common DNA variants at nine different loci are associated with uric acid concentrations, five of which are novel. These variants are located within the genes coding for organic anion transporter 4 (SLC22A11), monocarboxylic acid transporter 9 (SLC16A9), glucokinase regulatory protein (GCKR), Carmil (LRRC16A), and near PDZ domain-containing 1 (PDZK1). Gender-specific effects are shown for variants within the recently identified genes coding for glucose transporter 9 (SLC2A9) and the ATP-binding cassette transporter (ABCG2). Based on screening of 163 metabolites, we show an association of one of the identified variants within SLC16A9 with DL-carnitine and propionyl-L-carnitine. Moreover, DL-carnitine and propionyl-L-carnitine were strongly correlated with serum UA levels, forming a triangle between SNP, metabolites and UA levels. Taken together, these associations highlight pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia.

Background Toll-like receptor 4 (TLR4), the signaling receptor for lipopolysaccharides, is an important member of the innate immunity system. Since several studies have suggested that type 2 diabetes might be associated with changes in the innate immune response, we sought to investigate the association between genetic variants in the TLR4 gene and incident type 2 diabetes. Methods A case-cohort study was conducted in initially healthy, middle-aged subjects from the MONICA/KORA Augsburg studies including 498 individuals with incident type 2 diabetes and 1,569 non-cases. Seven SNPs were systematically selected in the TLR4 gene and haplotypes were reconstructed. Results The effect of TLR4 SNPs on incident type 2 diabetes was modified by the ratio of total cholesterol to high-density lipoprotein cholesterol (TC/HDL-C). In men, four out of seven TLR4 variants showed significant interaction with TC/HDL-C after correction for multiple testing (p < 0.01). The influence of the minor alleles of those variants on the incidence of type 2 diabetes was observed particularly for male patients with high values of TC/HDL-C. Consistent with these findings, haplotype-based analyses also revealed that the effect of two haplotypes on incident type 2 diabetes was modified by TC/HDL-C in men (p < 10-3). However, none of the investigated variants or haplotypes was associated with type 2 diabetes in main effect models without assessment of effect modifications. Conclusion We conclude that minor alleles of several TLR4 variants, although not directly associated with type 2 diabetes might increase the risk for type 2 diabetes in subjects with high TC/HDL-C. Additionally, our results confirm previous studies reporting sex-related dissimilarities in the development of type 2 diabetes.