Combined effects of thrombosis pathway gene variants predict cardiovascular events.

The genetic background of complex diseases is proposed to consist of several low-penetrance risk loci. Addressing this complexity likely requires both large sample size and simultaneous analysis of different predisposing variants. We investigated the role of four thrombosis genes: coagulation factor V (F5), intercellular adhesion molecule 1 (ICAM1), protein C (PROC), and thrombomodulin (THBD) in cardiovascular diseases. Single allelic gene variants and their pair-wise combinations were analyzed in two independently sampled population cohorts from Finland. From among 14,140 FINRISK participants (FINRISK-92, n = 5,999 and FINRISK-97, n = 8,141), we selected for genotyping a sample of 2,222, including 528 incident cardiovascular disease (CVD) cases and random subcohorts totaling 786. To cover all known common haplotypes (>10%), 54 single nucleotide polymorphisms (SNPs) were genotyped. Classification-tree analysis identified 11 SNPs that were further analyzed in Cox's proportional hazard model as single variants and pair-wise combinations. Multiple testing was controlled by use of two independent cohorts and with false-discovery rate. Several CVD risk variants were identified: In women, the combination of F5 rs7542281 x THBD rs1042580, together with three single F5 SNPs, was associated with CVD events. Among men, PROC rs1041296, when combined with either ICAM1 rs5030341 or F5 rs2269648, was associated with total mortality. As a single variant, PROC rs1401296, together with the F5 Leiden mutation, was associated with ischemic stroke events. Our strategy to combine the classification-tree analysis with more traditional genetic models was successful in identifying SNPs-acting either in combination or as single variants--predisposing to CVD, and produced consistent results in two independent cohorts. These results suggest that variants in these four thrombosis genes contribute to arterial cardiovascular events at population level.

Lack of association between polymorphisms of the IL18R1 and IL18RAP genes and cardiovascular risk: the MORGAM Project.

BACKGROUND: Interleukin-18 is a pro-inflammatory cytokine suspected to be associated with atherosclerosis and its complications. We had previously shown that one single nucleotide polymorphism (SNP) of the IL18 gene was associated with cardiovascular disease (CVD) through an interaction with smoking. As a further step for elucidating the contribution of the IL-18 pathway to the etiology of CVD, we here investigated the association between the genetic variability of two IL-18 receptor genes, IL18R1 and IL18RAP, with the risk of developing CVD. METHODS: Eleven tagging SNPs, 5 in IL18R1 and 6 in IL18RAP, characterizing the haplotypic variability of the corresponding genes; were genotyped in 5 European prospective CVD cohorts including 1416 cases and 1772 non-cases, as part of the MORGAM project. Both single-locus and haplotypes analyses were carried out to investigate the association of these SNPs with CVD. RESULTS: We did not find any significant differences in allele, genotype and haplotype frequencies between cases and non-cases for either of the two genes. Moreover, the search for interactions between SNPs located in different genes, including 5 IL18 SNPs previously studied in the MORGAM project, and between SNPs and environmental factors remained unfruitful. CONCLUSION: Our analysis suggests that the variability of IL18R1 and IL18RAP genes are unlikely to contribute to modulate the risk of CVD.

Risk alleles of USF1 gene predict cardiovascular disease of women in two prospective studies.

Upstream transcription factor 1 (USF1) is a ubiquitously expressed transcription factor controlling several critical genes in lipid and glucose metabolism. Of some 40 genes regulated by USF1, several are involved in the molecular pathogenesis of cardiovascular disease (CVD). Although the USF1 gene has been shown to have a critical role in the etiology of familial combined hyperlipidemia, which predisposes to early CVD, the gene's potential role as a risk factor for CVD events at the population level has not been established. Here we report the results from a prospective genetic-epidemiological study of the association between the USF1 variants, CVD, and mortality in two large Finnish cohorts. Haplotype-tagging single nucleotide polymorphisms exposing all common allelic variants of USF1 were genotyped in a prospective case-cohort design with two distinct cohorts followed up during 1992-2001 and 1997-2003. The total number of follow-up years was 112,435 in 14,140 individuals, of which 2,225 were selected for genotyping based on the case-cohort study strategy. After adjustment for conventional risk factors, we observed an association of USF1 with CVD and mortality among females. In combined analysis of the two cohorts, female carriers of a USF1 risk haplotype had a 2-fold risk of a CVD event (hazard ratio [HR] 2.02; 95% confidence interval [CI] 1.16-3.53; p = 0.01) and an increased risk of all-cause mortality (HR 2.52; 95% CI 1.46-4.35; p = 0.0009). A putative protective haplotype of USF1 was also identified. Our study shows how a gene identified in exceptional families proves to be important also at the population level, implying that allelic variants of USF1 significantly influence the prospective risk of CVD and even all-cause mortality in females.

Haplotypic analysis of tag SNPs of the interleukin-18 gene in relation to cardiovascular disease events: the MORGAM Project.

Interleukin-18 (IL-18) is a key inflammatory molecule suspected of being involved in the etiology of cardiovascular diseases (CVD). Five single nucleotide polymorphisms (SNPs) capturing the common genetic variation of the IL-18 gene (tag SNPs) were genotyped in five European prospective CVD cohorts including 1933 cases and 1938 non-cases as part of the MORGAM Project. Not a single SNP was found associated with CVD. However, a significant (P=0.002) gene-smoking interaction was observed. In smokers, the -105T allele was more frequent in cases than in non-cases (0.29 vs 0.25) and associated with an increased risk of disease (odds ratio (OR)=1.25 (1.07-1.45), P=0.005), whereas the inverse relationship tended to be observed in non-smokers (OR=0.90 (0.78-1.02), P=0.131). The gene-smoking interaction was broadly homogenous across the cohorts and was also observed through haplotype analyses. In conclusion, using the concerted effort of several European prospective CVD cohorts, we are able to show that one IL-18 tag SNP interacts with smoking to modulate the risk of developing CVD.

Evaluating whole genome amplification via multiply-primed rolling circle amplification for SNP genotyping of samples with low DNA yield.

The amount of available DNA is often a limiting factor in pursuing genetic analyses of large-scale population cohorts. An association between higher DNA yield from blood and several phenotypes associated with inflammatory states has recently been demonstrated, suggesting that exclusion of samples with very low DNA yield may lead to biased results in statistical analyses. Whole genome amplification (WGA) could present a solution to the DNA concentration-dependent sample selection. The aim was to thoroughly assess WGA for samples with low DNA yield, using the multiply-primed rolling circle amplification method. Fifty-nine samples were selected with the lowest DNA yield (less than 7.5 microg) among 799 samples obtained for one population cohort. The genotypes obtained from two replicate WGA samples and the original genomic DNA were compared by typing 24 single nucleotide polymorphisms (SNPs). Multiple genotype discrepancies were identified for 13 of the 59 samples. The largest portion of discrepancies was due to allele dropout in heterozygous genotypes in WGA samples. Pooling the WGA DNA replicates prior to genotyping markedly improved genotyping reproducibility for the samples, with only 7 discrepancies identified in 4 samples. The nature of discrepancies was mostly homozygote genotypes in the genomic DNA and heterozygote genotypes in the WGA sample, suggesting possible allele dropout in the genomic DNA sample due to very low amounts of DNA template. Thus, WGA is applicable for low DNA yield samples, especially if using pooled WGA samples. A higher rate of genotyping errors requires that increased attention be paid to genotyping quality control, and caution when interpreting results.

Genetic variation of the interleukin-1 family and nongenetic factors determining the interleukin-1 receptor antagonist phenotypes.

The natural anti-inflammatory protein interleukin-1 receptor antagonist (IL-1Ra) inhibits the activity of IL-1 and is associated with vascular injury and metabolic disorders. We analyzed genetic and nongenetic determinants of the IL-1Ra phenotype. Fifteen haplotype-tagging single nucleotide polymorphisms (SNPs) in the IL-1α (IL1A), IL-1ß (IL1B), and IL-1 receptor antagonist (IL-1RN) genes were determined in the Health 2000 survey (n = 6771) and European myocardial infarction (MI) survivors (n = 972). Three SNPs were genotyped in the FINRISK97 (FR97) study (n = 7222). We found 3 IL1RN variants that were associated with the IL-1Ra phenotype in the study populations and remained significant after Bonferroni correction with increasing significance in meta-analysis (P values for rs3213448,rs315952, rs315949, respectively: 5.5 x 10(-11), 1.5 x 10(-11), and 4.0 x 10(-14)). Minor allele of the rare IL1B variant rs1143642 was associated with decreased IL-1Ra levels in the Health 2000 and FR97 populations, and the association strengthened in the meta-analysis (P = 9.4 x 10(-7)). The proportion of variance explained by the IL1RN variant was larger in MI survivors (5.0%) than in the unselected population (0.5%). Body mass index was the strongest nongenetic predictor of the IL-1Ra phenotype, explaining 11.8% of the variance in Health 2000, 18.1% in FR97, and 25% in MI survivors. In conclusion, 3 IL1RN SNPs and 1 IL1B variant were determining IL-1Ra phenotype independently of body mass index and other metabolic phenotypes. The proportion of phenotypic variation in IL-1Ra explained by the genetic variants was, however, modest compared with the proportion explained by the body mass index.

Association of variation in the interleukin-1 gene family with diabetes and glucose homeostasis.

OBJECTIVE: Proinflammatory cytokine IL-1beta is capable of decreasing insulin-induced glucose transport. Therefore, we hypothesized that genetic variation in the IL-1 gene family is associated with measures of glucose homeostasis and diabetes. DESIGN AND OUTCOME MEASURES: Fifteen haplotype-tagging single-nucleotide polymorphisms in the IL-1alpha, IL-1beta, and IL-1 receptor antagonist genes were determined in a Finnish population survey (n = 6771). Glucose and insulin concentrations were measured, and indices of insulin resistance and beta-cell function were calculated using the homeostasis model assessment. Two-hour oral glucose tolerance tests were carried out on a subsample of 1390 participants. Associations with prevalent diabetes were tested for replication in a sample of European myocardial infarction survivors (n = 972). RESULTS: The minor allele of the IL-1beta rs1143634(G-->A) was associated with higher blood glucose than the major allele: 5.37, 5.41, and 5.48 mmol/liter for the GG, AG, and AA genotypes, respectively (multivariate adjusted P for trend <0.0001; Bonferroni corrected P = 0.00096). The 2-h glucose was also higher (6.45 and 7.20 mmol/liter for the GG vs. AA; P = 0.003, Bonferroni corrected P = 0.045). The haplotype ACG of rs1143634, rs3917356, and rs16944 associated with higher glucose, higher homeostasis model assessment for insulin resistance index, higher 2-h insulin, and prevalent diabetes (adjusted rate ratio = 1.54; 95% confidence interval = 1.03-2.30; P = 0.037). The association with prevalent diabetes was replicated among European myocardial infarction survivors (rate ratio = 2.09; 95% confidence interval = 1.17-3.76; P = 0.013). CONCLUSIONS: These results suggest that genetic variation in the IL-1 gene family is associated with measures of glucose homeostasis and prevalent diabetes.

Gender differences in genetic risk profiles for cardiovascular disease.

BACKGROUND: Cardiovascular disease (CVD) incidence, complications and burden differ markedly between women and men. Although there is variation in the distribution of lifestyle factors between the genders, they do not fully explain the differences in CVD incidence and suggest the existence of gender-specific genetic risk factors. We aimed to estimate whether the genetic risk profiles of coronary heart disease (CHD), ischemic stroke and the composite end-point of CVD differ between the genders. METHODOLOGY/PRINCIPAL FINDINGS: We studied in two Finnish population cohorts, using the case-cohort design the association between common variation in 46 candidate genes and CHD, ischemic stroke, CVD, and CVD-related quantitative risk factors. We analyzed men and women jointly and also conducted genotype-gender interaction analysis. Several allelic variants conferred disease risk for men and women jointly, including rs1801020 in coagulation factor XII (HR = 1.31 (1.08-1.60) for CVD, uncorrected p = 0.006 multiplicative model). Variant rs11673407 in the fucosyltransferase 3 gene was strongly associated with waist/hip ratio (uncorrected p = 0.00005) in joint analysis. In interaction analysis we found statistical evidence of variant-gender interaction conferring risk of CHD and CVD: rs3742264 in the carboxypeptidase B2 gene, p(interaction) = 0.009 for CHD, and rs2774279 in the upstream stimulatory factor 1 gene, p(interaction) = 0.007 for CHD and CVD, showed strong association in women but not in men, while rs2069840 in interleukin 6 gene, p(interaction) = 0.004 for CVD, showed strong association in men but not in women (uncorrected p-values). Also, two variants in the selenoprotein S gene conferred risk for ischemic stroke in women, p(interaction) = 0.003 and 0.007. Importantly, we identified a larger number of gender-specific effects for women than for men. CONCLUSIONS/SIGNIFICANCE: A false discovery rate analysis suggests that we may expect half of the reported findings for combined gender analysis to be true positives, while at least third of the reported genotype-gender interaction results are true positives. The asymmetry in positive findings between the genders could imply that genetic risk loci for CVD are more readily detectable in women, while for men they are more confounded by environmental/lifestyle risk factors. The possible differences in genetic risk profiles between the genders should be addressed in more detail in genetic studies of CVD, and more focus on female CVD risk is also warranted in genome-wide association studies.

Variation in the selenoprotein S gene locus is associated with coronary heart disease and ischemic stroke in two independent Finnish cohorts.

Selenoprotein S (SEPS1) is a novel candidate gene involved in the regulation of inflammatory response and protection from oxidative damage. This study explored the genetic variation in the SEPS1 locus for an association with CVD as well as with quantitative phenotypes related to obesity and inflammation. We used the case-cohort design and time-to-event analysis in two separate prospectively followed population-based cohorts FINRISK 92 and 97 (n = 999 and 1,223 individuals, respectively) to study the associations of five single nucleotide polymorphisms with the risk for coronary heart disease (CHD) and ischemic stroke events. We found a significant association with increased CHD risk in females carrying the minor allele of rs8025174 in the combined analysis of both cohorts [hazard ratio (HR) 2.95 (95% confidence interval: 1.37-6.39)]. Another variant, rs7178239, increased the risk for ischemic stroke significantly in females [HR: 3.35 (1.66-6.76)] and in joint analysis of both sexes and both cohorts [HR: 1.75 (1.17-2.64)]. These results indicate that variation in the SEPS1 locus may have an effect on CVD morbidity, especially in females. This observation should stimulate further investigations of the role of this gene and protein in the pathogenesis of CVD.