Powerful identification of cis-regulatory SNPs in human primary monocytes using allele-specific gene expression.

A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.

Integrating genome-wide genetic variations and monocyte expression data reveals trans-regulated gene modules in humans.

One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns-independent component analysis-to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.

G/T substitution in intron 1 of the UNC13B gene is associated with increased risk of nephropathy in patients with type 1 diabetes.

OBJECTIVE: Genetic and environmental factors modulate the susceptibility to diabetic nephropathy, as initiating and/or progression factors. The objective of the European Rational Approach for the Genetics of Diabetic Complications (EURAGEDIC) study is to identify nephropathy susceptibility genes. We report molecular genetic studies for 127 candidate genes for nephropathy. RESEARCH DESIGN AND METHODS: Polymorphisms were identified through sequencing of promoter, exon, and flanking intron gene regions and a database search. A total of 344 nonredundant SNPs and nonsynonymous variants were tested for association with diabetic nephropathy (persistent albuminuria >/=300 mg/24 h) in a large type 1 diabetes case/control (1,176/1,323) study from three European populations. RESULTS: Only one SNP, rs2281999, located in the UNC13B gene, was significantly associated with nephropathy after correction for multiple testing. Analyses of 21 additional markers fully characterizing the haplotypic variability of the UNC13B gene showed consistent association of SNP rs13293564 (G/T) located in intron 1 of the gene with nephropathy in the three populations. The odds ratio (OR) for nephropathy associated with the TT genotype was 1.68 (95% CI 1.29-2.19) (P = 1.0 x 10(-4)). This association was replicated in an independent population of 412 case subjects and 614 control subjects (combined OR of 1.63 [95% CI 1.30-2.05], P = 2.3 x 10(-5)). CONCLUSIONS: We identified a polymorphism in the UNC13B gene associated with nephropathy. UNC13B mediates apopotosis in glomerular cells in the presence of hyperglycemia, an event occurring early in the development of nephropathy. We propose that this polymorphism could be a marker for the initiation of nephropathy. However, further studies are needed to clarify the role of UNC13B in nephropathy.

Performance comparison of two microarray platforms to assess differential gene expression in human monocyte and macrophage cells.

BACKGROUND: In this study we assessed the respective ability of Affymetrix and Illumina microarray methodologies to answer a relevant biological question, namely the change in gene expression between resting monocytes and macrophages derived from these monocytes. Five RNA samples for each type of cell were hybridized to the two platforms in parallel. In addition, a reference list of differentially expressed genes (DEG) was generated from a larger number of hybridizations (mRNA from 86 individuals) using the RNG/MRC two-color platform. RESULTS: Our results show an important overlap of the Illumina and Affymetrix DEG lists. In addition, more than 70% of the genes in these lists were also present in the reference list. Overall the two platforms had very similar performance in terms of biological significance, evaluated by the presence in the DEG lists of an excess of genes belonging to Gene Ontology (GO) categories relevant for the biology of monocytes and macrophages. Our results support the conclusion of the MicroArray Quality Control (MAQC) project that the criteria used to constitute the DEG lists strongly influence the degree of concordance among platforms. However the importance of prioritizing genes by magnitude of effect (fold change) rather than statistical significance (p-value) to enhance cross-platform reproducibility recommended by the MAQC authors was not supported by our data. CONCLUSION: Functional analysis based on GO enrichment demonstrates that the 2 compared technologies delivered very similar results and identified most of the relevant GO categories enriched in the reference list.

Polymorphisms in 33 inflammatory genes and risk of myocardial infarction--a system genetics approach.

The hypothesis of a causal link between inflammation and atherosclerosis would be strengthened if variants of inflammatory genes were associated with disease. Polymorphisms of 33 genes encoding inflammatory molecules were tested for association with myocardial infarction (MI). Patients with MI and a parental history of MI (n = 312) and controls from the UK (n = 317) were genotyped for 162 polymorphisms. Thirteen polymorphisms were associated with MI (P values ranging from 0.003 to 0.041). For three genes, ITGB1, SELP, and TNFRSF1B haplotype frequencies differed between patients and controls (P values < 0.01). We further assessed the simultaneous contribution of all polymorphisms and relevant covariates to MI using a two-step strategy of data mining relying on Random Forest and DICE algorithms. In a replication study involving two independent samples from the UK (n = 649) and France (n = 706), one interaction between the ITGA4/R898Q polymorphism and current smoking status was replicated. This study illustrates a strategy for assessing the joint effect of a large number of polymorphisms on a phenotype that may provide information that single locus or single gene analysis may fail to uncover. Overall, there was weak evidence for an implication of inflammatory polymorphisms on susceptibility to MI.

Differential haplotypic expression of the interleukin-18 gene.

Interleukin 18 (IL-18) is suspected to play an important role in atherosclerosis and plaque vulnerability. We had previously shown that haplotypes combining two IL18 gene polymorphisms in complete linkage disequilibrium, C-105T (rs360717) in 5'-untranslated region (UTR) and A+183G (rs5744292) in 3'-UTR, were related to IL-18 circulating levels and cardiovascular outcome, the C(-105) G(+183) haplotype being associated with lower IL-18 levels and lower cardiovascular risk. This study was aimed at investigating the functional role of the two polymorphisms and their haplotypes on IL18 expression levels. Allelic imbalance experiments conducted in 24 and 20 subjects heterozygous for the C-105T and the A+183G polymorphisms did not detect any difference when subjects were considered as a whole (-0.009+/-0.044, P=0.85 and +0.114+/-0.082, P=0.18, respectively). However, when splitting individuals according to their haplo-genotype, the haplotype C(-105) G(+183) was associated with a lower expression level than C(-105) A(+183) (-0.287+/-0.076, P=0.005), but did not differ from T(-105) A(+183) (-0.138+/-0.083, P=0.13). The lower expression associated with C(-105) G(+183) was confirmed by real-time reverse transcription-PCR. Transfection of different haplotypic versions of the 3'-UTR did not show any difference in the expression of an upstream reporter gene. A 10-h study of the mRNA degradation kinetics by allelic imbalance with the A+183G polymorphism did not show any differential allelic degradation. In conclusion, the haplotype associated with lower IL-18 circulating concentrations and a lower cardiovascular risk was consistently associated with decreased IL18 expression levels, although the exact functional mechanisms remain to be elucidated.

Haplotypes of the caspase-1 gene, plasma caspase-1 levels, and cardiovascular risk.

Caspase-1 processes the interleukin (IL)-1beta and IL-18 inactive precursors to the biologically active cytokines that are known to have proatherogenic effects. The present study investigated the genetic variability of the CASP1 gene and plasma levels of caspase-1 in relation to cardiovascular risk. In Europeans, 3 tag SNPs captured 4 common haplotypes of the CASP1 gene. Among these, the A(in6) allele of the G+7/in6A polymorphism was less frequent in 246 cases with myocardial infarction and a parental history of disease than in 253 controls free of familial history of disease (0.13+/-0.02 versus 0.20+/-0.02; P=0.005). However, in a larger case/control study (n=1774), these effects are borderline restricted to the UK population. In a prospective cohort of 1168 patients with coronary artery disease followed up during a median period of 6.0 years, the A(in6) allele exhibited a borderline association with future cardiovascular death (hazard ratio [HR]: 0.64, 0.41 to 1.01; P=0.053) and was associated with lower serum IL-18 levels (P=0.014). Baseline caspase-1 levels in the top quartile of the distribution were predictive of cardiovascular deaths (HR=3.62, 1.81 to 7.27; P=0.0003 compared with the bottom quartile). Finally, in vitro assays of allelic imbalance showed that the CASP1 haplotype carrying the A(in6) allele was associated with a lower mRNA expression. These results indicate that caspase-1 levels are predictive of future cardiovascular death in patients with coronary artery disease. The role of CASP1 genetic variations in the susceptibility to myocardial infarction requires further investigation.

Genetic analysis of the interleukin-18 system highlights the role of the interleukin-18 gene in cardiovascular disease.

BACKGROUND: Interleukin (IL)-18 plays a key role in atherosclerosis and its complications. The present study investigated the genetic variability of 4 genes of the IL-18 system-IL18, IL18R1, IL18RAP, and IL18BP-in relation to circulating IL-18 levels and cardiovascular mortality. METHODS AND RESULTS: Twenty-two polymorphisms were genotyped in 1288 patients with coronary artery disease prospectively followed up during a median period of 5.9 years. The end point was death from cardiovascular causes (n=142). Baseline IL-18 levels were predictive of cardiovascular deaths occurring during < or =4 years of follow-up (HR=2.96, 95% CI 1.54 to 5.70, P=0.001 for the top compared with the bottom quartile) but not of later deaths. Haplotypes of the IL18 gene were associated with IL-18 levels (P=0.002) and cardiovascular mortality (P=0.006) after adjustment for cardiovascular risk factors. The same haplotype was associated with both a 9% lowering effect on IL-18 levels and a protective effect on risk (HR=0.57, 95% CI 0.36 to 0.92). IL18 haplotypes explained only 2% of IL-18 variability. Adjustment for baseline IL-18 levels abolished the association of haplotypes with cardiovascular risk. The haplotype associated with phenotypes was the only one carrying the minor allele of the IL18/A+183G polymorphism located in the 3'untranslated region and potentially affecting mRNA stability. The other genes of the system were not related to IL-18 levels or cardiovascular outcome. CONCLUSIONS: Variations of the IL18 gene consistently influence circulating levels of IL-18 and clinical outcome in patients with coronary artery disease, which supports the hypothesis of a causal role of IL-18 in atherosclerosis and its complications.