Blood Eosinophil Count and Metabolic, Cardiac and Pulmonary Outcomes: A Mendelian Randomization Study.

Blood eosinophil count is associated with a variety of common complex outcomes in epidemiological observation. The aim of this study was to explore the causal association between determined blood eosinophil count and 20 common complex outcomes (10 metabolic, 6 cardiac, and 4 pulmonary). Through Mendelian randomization, we investigated genetic evidence for the genetically determined eosinophil in association with each outcomes using individual-level LifeLines cohort data (n = 13,301), where a weighted eosinophil genetic risk score comprising five eosinophil associated variants was created. We further examined the associations of the genetically determined eosinophil with those outcomes using summary statistics obtained from genome-wide association study consortia (6 consortia and 14 outcomes). Blood eosinophil count, by a 1-SD genetically increased, was not statistically associated with common complex outcomes in the LifeLines. Using the summary statistics, we showed that a higher genetically determined eosinophil count had a significant association with lower odds of obesity (odds ratio (OR) 0.81, 95% confidence interval (CI) [0.74, 0.89]) but not with the other traits and diseases. To conclude, an elevated eosinophil count is unlikely to be causally associated to higher risk of metabolic, cardiac, and pulmonary outcomes. Further studies with a stronger genetic risk score for eosinophil count may support these results.

Missing heritability: is the gap closing? An analysis of 32 complex traits in the Lifelines Cohort Study.

Despite the recent explosive rise in number of genetic markers for complex disease traits identified in genome-wide association studies, there is still a large gap between the known heritability of these traits and the part explained by these markers. To gauge whether this 'heritability gap' is closing, we first identified genome-wide significant SNPs from the literature and performed replication analyses for 32 highly relevant traits from five broad disease areas in 13 436 subjects of the Lifelines Cohort. Next, we calculated the variance explained by multi-SNP genetic risk scores (GRSs) for each trait, and compared it to their broad- and narrow-sense heritabilities captured by all common SNPs. The majority of all previously-associated SNPs (median=75%) were significantly associated with their respective traits. All GRSs were significant, with unweighted GRSs generally explaining less phenotypic variance than weighted GRSs, for which the explained variance was highest for height (15.5%) and varied between 0.02 and 6.7% for the other traits. Broad-sense common-SNP heritability estimates were significant for all traits, with the additive effect of common SNPs explaining 48.9% of the variance for height and between 5.6 and 39.2% for the other traits. Dominance effects were uniformly small (0-1.5%) and not significant. On average, the variance explained by the weighted GRSs accounted for only 10.7% of the common-SNP heritability of the 32 traits. These results indicate that GRSs may not yet be ready for accurate personalized prediction of complex disease traits limiting widespread adoption in clinical practice.

Eosinophil Count Is a Common Factor for Complex Metabolic and Pulmonary Traits and Diseases: The LifeLines Cohort Study.

There is ongoing debate on the association between eosinophil count and diseases, as previous studies were inconsistent. We studied the relationship of eosinophil count with 22 complex metabolic, cardiac, and pulmonary traits and diseases. From the population-based LifeLines Cohort Study (N = 167,729), 13,301 individuals were included. We focused on relationship of eosinophil count with three classes of metabolic (7 traits, 2 diseases), cardiac (6 traits, 2 diseases), and pulmonary (2 traits, 2 diseases) outcomes. Regression analyses were applied in overall, women and men, while adjusted for age, sex, BMI and smoking. A p-value of <0.00076 was considered statistically significant. 58.2% of population were women (mean±SD 51.3±11.1 years old). In overall, one-SD higher of ln-eosinophil count was associated with a 0.04 (±SE ±0.002;p = 6.0×10-6) SD higher levels in ln-BMI, 0.06 (±0.007;p = 3.1×10-12) SD in ln-TG, 0.04 (±0.003;p = 7.0×10-6) SD in TC, 0.04 (±0.004;p = 6.3×10-7) SD in LDL, 0.04 (±0.006;p = 6.0×10-6) SD in HbA1c; and with a 0.05 (±0.004;p = 1.7×10-8) SD lower levels in HDL, 0.05 (±0.007;p = 3.4×10-23) SD in FEV1, and 0.09 (±0.001;p = 6.6×10-28) SD in FEV1/FVC. A higher ln-eosinophil count was associated with 1.18 (95%CI 1.09-1.28;p = 2.0×10-5) odds ratio of obesity, 1.29 (1.19-1.39;p = 1.1×10-10) of metabolic syndrome, 1.40 (1.25-1.56;p = 2.7×10-9) of COPD and 1.81 (1.61-2.03;p = 1.0×10-23) of asthma. Similar results were found in women. We found no association between ln-eosinophil count either with blood pressure indices in overall, women and men; or with BMI, LDL, HbA1c and obesity in men. In a large population based cohort, we confirmed eosinophil count as a potential factor implicated in metabolic and pulmonary outcomes.

Common genes underlying asthma and COPD? Genome-wide analysis on the Dutch hypothesis.

Asthma and chronic obstructive pulmonary disease (COPD) are thought to share a genetic background ("Dutch hypothesis"). We investigated whether asthma and COPD have common underlying genetic factors, performing genome-wide association studies for both asthma and COPD and combining the results in meta-analyses. Three loci showed potential involvement in both diseases: chr2p24.3, chr5q23.1 and chr13q14.2, containing DDX1, COMMD10 (both participating in the nuclear factor (NF) κß pathway) and GNG5P5, respectively. Single nucleotide polymorphisms (SNPs) rs9534578 in GNG5P5 reached genome-wide significance after first replication phase (p=9.96×10(-9)). The second replication phase, in seven independent cohorts, provided no significant replication. Expression quantitative trait loci (eQTL) analysis in blood cells and lung tissue on the top 20 associated SNPs identified two SNPs in COMMD10 that influenced gene expression. Inflammatory processes differ in asthma and COPD and are mediated by NF-κß, which could be driven by the same underlying genes, COMMD10 and DDX1. None of the SNPs reached genome-wide significance. Our eQTL studies support a functional role for two COMMD10 SNPs, since they influence gene expression in both blood cells and lung tissue. Our findings suggest that there is either no common genetic component in asthma and COPD or, alternatively, different environmental factors, e.g. lifestyle and occupation in different countries and continents, which may have obscured the genetic common contribution.

Comparing the hub-and-spoke model practices of the LifeLines Study in the Netherlands and the H3Africa Initiative.

Biobank sample storage is critical in population health and epidemiology studies. Biobanks bridge two very different worlds: they connect to the participants and patients at an individual level, but they also aggregate information and represent the cutting edge of scientific discovery. In this brief report, we describe how the LifeLines study in the Netherlands manages its resources for communication and services, and how it can serve as a model for the Human Heredity and Health in Africa Initiative (H3Africa Initiative).

Ciliary genes are down-regulated in bronchial tissue of primary ciliary dyskinesia patients.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disease characterized by recurrent respiratory tract infections, sinusitis, bronchiectasis and male infertility. The pulmonary phenotype in PCD is caused by the impaired motility of cilia in the respiratory epithelium, due to ultrastructural defects of these organelles. We hypothesized that defects of multi-protein ciliary complexes should be reflected by gene expression changes in the respiratory epithelium. We have previously found that large group of genes functionally related to cilia share highly correlated expression pattern in PCD bronchial tissue. Here we performed an explorative analysis of differential gene expression in the bronchial tissue from six PCD patients and nine non-PCD controls, using Illumina HumanRef-12 Whole Genome BeadChips. We observed 1323 genes with at least 2-fold difference in the mean expression level between the two groups (t-test p-value <0.05). Annotation analysis showed that the genes down-regulated in PCD biopsies (602) were significantly enriched for terms related to cilia, whereas the up-regulated genes (721) were significantly enriched for terms related to cell cycle and mitosis. We assembled a list of human genes predicted to encode ciliary proteins, components of outer dynein arms, inner dynein arms, radial spokes, and intraflagellar transport proteins. A significant down-regulation of the expression of genes from all the four groups was observed in PCD, compared to non-PCD biopsies. Our data suggest that a coordinated down-regulation of the ciliome genes plays an important role in the molecular pathomechanism of PCD.

Large-scale gene-centric meta-analysis across 39 studies identifies type 2 diabetes loci.

To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 × 10(-9)) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 × 10(-6)). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 × 10(-7)) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 × 10(-15)). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 × 10(-8)). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups.

Comparative analysis of the human hepatic and adipose tissue transcriptomes during LPS-induced inflammation leads to the identification of differential biological pathways and candidate biomarkers.

BACKGROUND: Insulin resistance (IR) is accompanied by chronic low grade systemic inflammation, obesity, and deregulation of total body energy homeostasis. We induced inflammation in adipose and liver tissues in vitro in order to mimic inflammation in vivo with the aim to identify tissue-specific processes implicated in IR and to find biomarkers indicative for tissue-specific IR. METHODS: Human adipose and liver tissues were cultured in the absence or presence of LPS and DNA Microarray Technology was applied for their transcriptome analysis. Gene Ontology (GO), gene functional analysis, and prediction of genes encoding for secretome were performed using publicly available bioinformatics tools (DAVID, STRING, SecretomeP). The transcriptome data were validated by proteomics analysis of the inflamed adipose tissue secretome. RESULTS: LPS treatment significantly affected 667 and 483 genes in adipose and liver tissues respectively. The GO analysis revealed that during inflammation adipose tissue, compared to liver tissue, had more significantly upregulated genes, GO terms, and functional clusters related to inflammation and angiogenesis. The secretome prediction led to identification of 399 and 236 genes in adipose and liver tissue respectively. The secretomes of both tissues shared 66 genes and the remaining genes were the differential candidate biomarkers indicative for inflamed adipose or liver tissue. The transcriptome data of the inflamed adipose tissue secretome showed excellent correlation with the proteomics data. CONCLUSIONS: The higher number of altered proinflammatory genes, GO processes, and genes encoding for secretome during inflammation in adipose tissue compared to liver tissue, suggests that adipose tissue is the major organ contributing to the development of systemic inflammation observed in IR. The identified tissue-specific functional clusters and biomarkers might be used in a strategy for the development of tissue-targeted treatment of insulin resistance in patients.

Changes in gene expression caused by insect venom immunotherapy responsible for the long-term protection of insect venom-allergic patients.

BACKGROUND: Insect venom immunotherapy (VIT) is the only causative treatment of insect venom allergy (IVA). The immunological mechanism(s) responsible for long-term protection achieved by VIT are largely unknown. A better understanding is relevant for improving the diagnosis, prediction of anaphylaxis, and monitoring and simplifying treatment of IVA. OBJECTIVE: To find genes that are differentially expressed during the maintenance phase of VIT and after stopping, to get clues about the pathways involved in the long-term protective effect of immunotherapy. METHODS: Whole genome gene expression analysis was performed on RNA samples from 50 patients treated with VIT and 43 healthy controls. Patients were divided into three groups: (1) before the start of VIT; (2) on maintenance phase of VIT for at least 3 years still receiving injections; and (3) after VIT. RESULTS: Of all 48,804 probes present in the array, 48,773 transcripts had sufficient data for further analysis. The list of genes that were differentially expressed (at least log2 FC > 2; P < .05 corrected for multiple testing) during the maintenance phase of VIT as well as after successful VIT contains 89 entities. The function of these genes affects cell signaling, cell differentiation, and ion transport. CONCLUSION: This study shows that a group of genes is differentially expressed both during and after VIT in comparison with gene expression in patients before VIT. Although the results of this study should be confirmed prospectively, the relevance of these findings is supported by the fact that they are related to putative mechanisms of immunotherapy.

Human primary adipocytes exhibit immune cell function: adipocytes prime inflammation independent of macrophages.

BACKGROUND: Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT. METHODS AND RESULTS: Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-ß highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%. CONCLUSION: Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes.

Functional characterization of mutations in the myosin Vb gene associated with microvillus inclusion disease.

OBJECTIVES: Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in patients with MVID. Interestingly, several patients with MVID showed only a MYO5B mutation in 1 allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations. PATIENTS AND METHODS: The genomic DNA of 9 patients diagnosed as having MVID was screened for MYO5B mutations, and quantitative polymerase chain reaction and immunohistochemistry on the material of 2 patients was performed to investigate resultant cellular consequences. RESULTS: We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb messenger RNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb-controlled accumulation of Rab11a- and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative of altered myosin Vb function. Moreover, we report 8 novel MYO5B mutations in 9 patients of various ethnic backgrounds with MVID, including compound heterozygous mutations. CONCLUSIONS: Our functional analysis indicates that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein, and apical recycling endosomes, which, together with the additional compound heterozygous mutations, significantly strengthen the link between MYO5B and MVID.

Gene expression studies in cells from primary ciliary dyskinesia patients identify 208 potential ciliary genes.

Cilia are small cellular projections that either act as sensors (primary cilia) or propel fluid over the epithelia of various organs (motile cilia). The organellum has gained much attention lately because of its involvement in a group of human diseases called ciliopathies. Primary ciliary dyskinesia (PCD) is an autosomal recessive ciliopathy caused by mutations in cilia motility genes. The disease is characterized by recurrent respiratory tract infections due to the lack of an efficient mucociliary clearance. We performed whole-genome gene expression profiling in bronchial biopsies from PCD patients. We used the quality threshold clustering algorithm to identify groups of genes that revealed highly correlated RNA expression patterns in the biopsies. The largest cluster contained 372 genes and was significantly enriched for genes related to cilia. The database and literature search showed that 164 genes in this cluster were known cilia genes, strongly indicating that the remaining 208 genes were likely to be new cilia genes. The tissue expression pattern of the 208 new cilia genes and the 164 known genes was consistent with the presence of motile cilia in a given tissue. The analysis of the upstream promotor sequences revealed evidence for RFX transcription factors binding site motif in both subgroups. Based on the correlated expression patterns in PCD-affected tissues, we identified 208 genes that we predict to be involved in cilia biology. Our predictions are based directly on the human material and not on model organisms. This list of genes provides candidate genes for PCD and other ciliopathies.

Co-expressed immune and metabolic genes in visceral and subcutaneous adipose tissue from severely obese individuals are associated with plasma HDL and glucose levels: a microarray study.

BACKGROUND: Excessive accumulation of body fat, in particular in the visceral fat depot, is a major risk factor to develop a variety of diseases such as type 2 diabetes. The mechanisms underlying the increased risk of obese individuals to develop co-morbid diseases are largely unclear.We aimed to identify genes expressed in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) that are related to blood parameters involved in obesity co-morbidity, such as plasma lipid and glucose levels, and to compare gene expression between the fat depots. METHODS: Whole-transcriptome SAT and VAT gene expression levels were determined in 75 individuals with a BMI >35 kg/m2. Modules of co-expressed genes likely to be functionally related were identified and correlated with BMI, plasma levels of glucose, insulin, HbA1c, triglycerides, non-esterified fatty acids, ALAT, ASAT, C-reactive protein, and LDL- and HDL cholesterol. RESULTS: Of the approximately 70 modules identified in SAT and VAT, three SAT modules were inversely associated with plasma HDL-cholesterol levels, and a fourth module was inversely associated with both plasma glucose and plasma triglyceride levels (p < 5.33 x 10(-5)). These modules were markedly enriched in immune and metabolic genes. In VAT, one module was associated with both BMI and insulin, and another with plasma glucose (p < 4.64 x 10(-5)). This module was also enriched in inflammatory genes and showed a marked overlap in gene content with the SAT modules related to HDL. Several genes differentially expressed in SAT and VAT were identified. CONCLUSIONS: In obese subjects, groups of co-expressed genes were identified that correlated with lipid and glucose metabolism parameters; they were enriched with immune genes. A number of genes were identified of which the expression in SAT correlated with plasma HDL cholesterol, while their expression in VAT correlated with plasma glucose. This underlines both the singular importance of these genes for lipid and glucose metabolism and the specific roles of these two fat depots in this respect.

Gene expression analysis in predicting the effectiveness of insect venom immunotherapy.

BACKGROUND: Venom immunotherapy (VIT) enables longtime prevention of insect venom allergy in the majority of patients. However, in some, the risk of a resystemic reaction increases after completion of treatment. No reliable factors predicting individual lack of efficacy of VIT are currently available. OBJECTIVE: To determine the use of gene expression profiles to predict the long-term effect of VIT. METHODS: Whole genome gene expression analysis was performed on RNA samples from 46 patients treated with VIT divided into 3 groups: (1) patients who achieved and maintained long-term protection after VIT, (2) patients in whom insect venom allergy relapsed, and (3) patients still in the maintenance phase of VIT. RESULTS: Among the 48.071 transcripts analyzed, 1401 showed a >2 fold difference in gene expression (P < .05); 658 genes (47%) were upregulated and 743 (53%) downregulated. Forty-three transcripts still show significant differences in expression after correction for multiple testing; 12 of 43 genes (28%) were upregulated and 31 of 43 genes (72%) downregulated. A naive Bayes prediction model demonstrated a gene expression pattern characteristic of effective VIT that was present in all patients with successful VIT but absent in all subjects with failure of VIT. The same gene expression profile was present in 88% of patients in the maintenance phase of VIT. CONCLUSION: Gene expression profiling might be a useful tool to assess the long-term effectiveness of VIT. The analysis of differently expressed genes confirms the involvement of immunologic pathways described previously but also indicates novel factors that might be relevant for allergen tolerance.

Arginase 1 and arginase 2 variations associate with asthma, asthma severity and beta2 agonist and steroid response.

RATIONALE: Arginase probably plays an important role in asthma development, severity and progression. Polymorphisms in arginase 1 and arginase 2 genes have been associated with childhood asthma and FEV1 reversibility to beta2 agonists. OBJECTIVES: We investigated the association between arginase 1 and arginase 2 polymorphisms and adult asthma, asthma severity and treatment response in a longitudinal cohort of 200 asthma patients. METHODS: Patients were studied during 1962-1975 and reexamined during 1990-1999, together with their families. Longitudinal data on lung function and treatment were extracted from medical records. Associations between haplotype-tagging polymorphisms in arginase 1 (n=3) and arginase 2 (n=8) and asthma, asthma severity, acute response to bronchodilators and chronic response to inhaled corticosteroids were analyzed. MEASUREMENTS AND MAIN RESULTS: Two polymorphisms in arginase 2 (rs17249437 and rs3742879) were associated with asthma and with more severe airway obstruction. Increased airway hyperresponsiveness and lower beta2 agonist reversibility, but not anticholinergic reversibility, were associated with both arginase 1 and arginase 2. Inhaled corticosteroids slowed down the annual FEV1 decline, which was significantly less effective in homozygote carriers of the C-allele of the arginase 1 polymorphism, rs2781667. CONCLUSION: We show that previously reported associations between arginase polymorphisms and childhood asthma are also present in adult asthma and the previously found associations with lower reversibility are specific for beta2 agonists. Furthermore, we identified associations of arginase 1 and arginase 2 genes with asthma severity, as reflected by a lower lung function, more severe airway hyperresponsiveness, and less long-term response to inhaled corticosteroids. Studies on the functionality of the polymorphisms are warranted to further unravel the complex mechanisms underlying these observations.

Identification of PCDH1 as a novel susceptibility gene for bronchial hyperresponsiveness.

RATIONALE: Asthma is a chronic inflammatory airway disease that affects more than 300 million individuals worldwide. Asthma is caused by interaction of genetic and environmental factors. Bronchial hyperresponsiveness (BHR) is a hallmark of asthma and results from increased sensitivity of the airways to physical or chemical stimulants. BHR and asthma are linked to chromosome 5q31-q33. OBJECTIVES: To identify a gene for BHR on chromosome 5q31-q33. METHODS: In 200 Dutch families with asthma, linkage analysis and fine mapping were performed, and the Protocadherin 1 gene (PCDH1) was identified. PCDH1 was resequenced in 96 subjects from ethnically diverse populations to identify novel sequence variants. Subsequent replication studies were undertaken in seven populations from The Netherlands, the United Kingdom, and the United States, including two general population samples, two family samples, and three case-control samples. PCDH1 mRNA and protein expression was investigated using polymerase chain reaction, Western blotting, and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: In seven out of eight populations (n = 6,168) from The Netherlands, United Kingdom, and United States, PCHD1 gene variants were significantly associated with BHR (P values, 0.005-0.05) This association was present in both families with asthma and general populations. PCDH1 mRNA and protein were expressed in airway epithelial cells and in macrophages. CONCLUSIONS: PCDH1 is a novel gene for BHR in adults and children. The identification of PCDH1 as a BHR susceptibility gene may suggest that a structural defect in the integrity of the airway epithelium, the first line of defense against inhaled substances, contributes to the development of BHR.

Complex nature of SNP genotype effects on gene expression in primary human leucocytes.

BACKGROUND: Genome wide association studies have been hugely successful in identifying disease risk variants, yet most variants do not lead to coding changes and how variants influence biological function is usually unknown. METHODS: We correlated gene expression and genetic variation in untouched primary leucocytes (n = 110) from individuals with celiac disease - a common condition with multiple risk variants identified. We compared our observations with an EBV-transformed HapMap B cell line dataset (n = 90), and performed a meta-analysis to increase power to detect non-tissue specific effects. RESULTS: In celiac peripheral blood, 2,315 SNP variants influenced gene expression at 765 different transcripts (< 250 kb from SNP, at FDR = 0.05, cis expression quantitative trait loci, eQTLs). 135 of the detected SNP-probe effects (reflecting 51 unique probes) were also detected in a HapMap B cell line published dataset, all with effects in the same allelic direction. Overall gene expression differences within the two datasets predominantly explain the limited overlap in observed cis-eQTLs. Celiac associated risk variants from two regions, containing genes IL18RAP and CCR3, showed significant cis genotype-expression correlations in the peripheral blood but not in the B cell line datasets. We identified 14 genes where a SNP affected the expression of different probes within the same gene, but in opposite allelic directions. By incorporating genetic variation in co-expression analyses, functional relationships between genes can be more significantly detected. CONCLUSION: In conclusion, the complex nature of genotypic effects in human populations makes the use of a relevant tissue, large datasets, and analysis of different exons essential to enable the identification of the function for many genetic risk variants in common diseases.

Newly identified genetic risk variants for celiac disease related to the immune response.

Our genome-wide association study of celiac disease previously identified risk variants in the IL2-IL21 region. To identify additional risk variants, we genotyped 1,020 of the most strongly associated non-HLA markers in an additional 1,643 cases and 3,406 controls. Through joint analysis including the genome-wide association study data (767 cases, 1,422 controls), we identified seven previously unknown risk regions (P < 5 x 10(-7)). Six regions harbor genes controlling immune responses, including CCR3, IL12A, IL18RAP, RGS1, SH2B3 (nsSNP rs3184504) and TAGAP. Whole-blood IL18RAP mRNA expression correlated with IL18RAP genotype. Type 1 diabetes and celiac disease share HLA-DQ, IL2-IL21, CCR3 and SH2B3 risk regions. Thus, this extensive genome-wide association follow-up study has identified additional celiac disease risk variants in relevant biological pathways.