Cell Specific eQTL Analysis without Sorting Cells.

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

Fine mapping of the celiac disease-associated LPP locus reveals a potential functional variant.

Using the Immunochip for genotyping, we identified 39 non-human leukocyte antigen (non-HLA) loci associated to celiac disease (CeD), an immune-mediated disease with a worldwide frequency of ∼1%. The most significant non-HLA signal mapped to the intronic region of 70 kb in the LPP gene. Our aim was to fine map and identify possible functional variants in the LPP locus. We performed a meta-analysis in a cohort of 25 169 individuals from six different populations previously genotyped using Immunochip. Imputation using data from the Genome of the Netherlands and 1000 Genomes projects, followed by meta-analysis, confirmed the strong association signal on the LPP locus (rs2030519, P = 1.79 × 10(-49)), without any novel associations. The conditional analysis on this top SNP-indicated association to a single common haplotype. By performing haplotype analyses in each population separately, as well as in a combined group of the four populations that reach the significant threshold after correction (P < 0.008), we narrowed down the CeD-associated region from 70 to 2.8 kb (P = 1.35 × 10(-44)). By intersecting regulatory data from the ENCODE project, we found a functional SNP, rs4686484 (P = 3.12 × 10(-49)), that maps to several B-cell enhancer elements and a highly conserved region. This SNP was also predicted to change the binding motif of the transcription factors IRF4, IRF11, Nkx2.7 and Nkx2.9, suggesting its role in transcriptional regulation. We later found significantly low levels of LPP mRNA in CeD biopsies compared with controls, thus our results suggest that rs4686484 is the functional variant in this locus, while LPP expression is decreased in CeD.

Trans-eQTLs reveal that independent genetic variants associated with a complex phenotype converge on intermediate genes, with a major role for the HLA.

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10(-16)). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.

Common and different genetic background for rheumatoid arthritis and coeliac disease.

Recent genome-wide association studies (GWAS) have revealed genetic risk factors in autoimmune and inflammatory disorders. Several of the associated genes and underlying pathways are shared by various autoimmune diseases. Rheumatoid arthritis (RA) and coeliac disease (CD) are two autoimmune disorders which have commonalities in their pathogenesis. We aimed to replicate known RA loci in a Dutch RA population, and to investigate whether the effect of known RA and CD risk factors generalize across the two diseases. We selected all loci associated to either RA or CD in a GWAS and confirmed in an independent cohort, with a combined P-value cut-off P < 5 x 10(-6). We genotyped 11 RA and 11 CD loci in 1368 RA patients, 795 CD patients and 1683 Dutch controls. We combined our results in a meta-analysis with UK GWAS on RA (1860 cases; 2938 controls) and CD (767 cases; 1422 controls). In the Dutch RA cohort, the PTPN22 and IL2/IL21 variants showed convincing association (P = 3.4 x 10(-12) and P = 2.8 x 10(-4), respectively). Association of RA with the known CD risk variant in the SH2B3 was also observed, predominantly in the subgroup of rheumatoid factor-positive RA patients (P = 0.0055). In a meta-analysis of Dutch and UK data sets, shared association with six loci (TNFAIP3, IL2/IL21, SH2B3, LPP, MMEL1/TNFRSF14 and PFKFB3/PRKCQ) was observed in both RA and CD cohorts. We confirmed two known loci and identified four novel ones for shared CD-RA genetic risk. Most of the shared loci further emphasize a role for adaptive and innate immunity in these diseases.

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.

Immunochip meta-analysis in European and Argentinian populations identifies two novel genetic loci associated with celiac disease.

Celiac disease (CeD) is a common immune-mediated disease of the small intestine that is triggered by exposure to dietary gluten. While the HLA locus plays a major role in disease susceptibility, 39 non-HLA loci were also identified in a study of 24,269 individuals. We now build on this earlier study by adding 4125 additional Caucasian samples including an Argentinian cohort. In doing so, we not only confirm the previous associations, we also identify two novel independent genome-wide significant associations at loci: 12p13.31 and 22q13.1. By applying a genomics approach and differential expression analysis in CeD intestinal biopsies, we prioritize potential causal genes at these novel loci, including LTBR, CYTH4, and RAC2. Nineteen prioritized causal genes are overlapping known drug targets. Pathway enrichment analysis and expression of these genes in CeD biopsies suggest that they have roles in regulating multiple pathways such as the tumor necrosis factor (TNF) mediated signaling pathway and positive regulation of I-κB kinase/NF-κB signaling.

PROS1 analysis in 87 pedigrees with hereditary protein S deficiency demonstrates striking genotype-phenotype associations.

Hereditary protein S (PS) deficiency predisposes to venous thrombosis. Previously, we demonstrated a difference in risk of venous thrombosis between PS deficiency type I and type III. We used direct sequencing, multiplex ligation-dependent probe amplification (MLPA), and linkage analysis to study whether this difference could be explained by molecular heterogeneity. The study contained two sets of families with PS deficiency type I (cohort 1; 35 probands, 155 relatives) or type III (cohort 2; 52 probands, 241 relatives). In cohort 1, a mixed type I/type III PS-deficient phenotype was observed in 66% of the pedigrees. A total of 34 probands carried a mutant PROS1 allele, compared to one proband in cohort 2 (P<10(-10)). The proband's mutation was identified in all type I, but only in 57% of type III PS deficient relatives. MLPA-analysis in the mutation negative families did not reveal PROS1 deletions or insertions. Linkage analysis in 16 families showed cosegregation of PROS1 markers in the family with type I deficiency, but not in the 15 families with type III deficiency. The genotype-phenotype associations point to differences in genetic architecture. Whereas PS deficiency type I is a monogenic disease due to PROS1 allelic heterozygosity, PS deficiency type III is most likely a more complex or heterogeneous disorder.

A locus at 7p14.3 predisposes to refractory celiac disease progression from celiac disease.

BACKGROUND: Approximately 5% of patients with celiac disease (CeD) do not respond to a gluten-free diet and progress to refractory celiac disease (RCD), a severe progression that is characterized by infiltration of intraepithelial T lymphocytes. Patients with RCD type II (RCDII) show clonal expansions of intraepithelial T lymphocytes that result in a poor prognosis and a high mortality rate through development of aggressive enteropathy-associated T-cell lymphoma. It is not known whether genetic variations play a role in severe progression of CeD to RCDII. PATIENTS AND METHODS: We performed the first genome-wide association study to identify the causal genes for RCDII and the molecular pathways perturbed in RCDII. The genome-wide association study was performed in 38 Dutch patients with RCDII, and the 15 independent top-associated single nucleotide polymorphism (SNP) variants (P<5×10) were replicated in 56 independent French and Dutch patients with RCDII. RESULTS: After replication, SNP rs2041570 on chromosome 7 was significantly associated with progression to RCDII (P=2.37×10, odds ratio=2.36) but not with CeD susceptibility. SNP rs2041570 risk allele A was associated with lower levels of FAM188B expression in blood and small intestinal biopsies. Stratification of RCDII biopsies based on rs2041570 genotype showed differential expression of innate immune and antibacterial genes that are expressed in Paneth cells. CONCLUSION: We have identified a novel SNP associated with the severe progression of CeD to RCDII. Our data suggest that genetic susceptibility to CeD might be distinct from the progression to RCDII and suggest a role for Paneth cells in RCDII progression.

Identification of a novel PROS1 c.1113T-->GG frameshift mutation in a family with mixed type I/type III protein S deficiency.

We report a family with type I and type III protein S (PS) deficiency, which showed to be phenotypic variants of the same genetic disease. Direct sequencing analysis of the PROS1 gene was performed to establish the genotype. The ratio of protein C antigen and total PS antigen levels (protein C/S ratio) was used to classify subjects at risk of venous thromboembolism. All PS deficient subjects had increased protein C/S ratios as well as a novel PROS1 c.1113T-->GG frameshift mutation.

Serum lipid levels, body mass index, and their role in coronary artery calcification: a polygenic analysis.

BACKGROUND: Coronary artery calcification (CAC) is widely regarded as a cumulative lifetime measure of atherosclerosis, but it remains unclear what is the relationship between calcification and traditional risk factors for coronary artery disease (CAD) and myocardial infarction (MI). This study characterizes the genetic architecture of CAC by evaluating the overall impact of common alleles associated with CAD/MI and its traditional risk factors. METHODS AND RESULTS: On the basis of summary-association results from the CARDIoGRAMplusC4D study of CAD/MI, we calculated polygenic risk scores in 2599 participants of the Dutch and Belgian Lung Cancer Screening (NELSON) trial, in whom quantitative CAC levels (Agatston scores) were determined from chest computerized tomographic imaging data. The most significant polygenic model explained ≈14% of the observed CAC variance (P=1.6×10(-11)), which points to a residual effect because of many as yet unknown loci that overlap between CAD/MI and CAC. In addition, we constructed risk scores based on published single-nucleotide polymorphism associations for traditional cardiovascular risk factors and tested these scores for association with CAC. We found nominally significant associations for genetic risk scores of low-density lipoprotein-cholesterol, total cholesterol, and body mass index, which were successfully replicated in 2182 individuals of the Heinz Nixdorf Recall Study. CONCLUSIONS: Pervasive polygenic sharing between CAC and CAD/MI suggests that a substantial fraction of the heritable risk for CAD/MI is mediated through arterial calcification. We also provide evidence that genetic variants associated with serum lipid levels and body mass index influence CAC levels.

Susceptibility to chronic mucus hypersecretion, a genome wide association study.

BACKGROUND: Chronic mucus hypersecretion (CMH) is associated with an increased frequency of respiratory infections, excess lung function decline, and increased hospitalisation and mortality rates in the general population. It is associated with smoking, but it is unknown why only a minority of smokers develops CMH. A plausible explanation for this phenomenon is a predisposing genetic constitution. Therefore, we performed a genome wide association (GWA) study of CMH in Caucasian populations. METHODS: GWA analysis was performed in the NELSON-study using the Illumina 610 array, followed by replication and meta-analysis in 11 additional cohorts. In total 2,704 subjects with, and 7,624 subjects without CMH were included, all current or former heavy smokers (≥20 pack-years). Additional studies were performed to test the functional relevance of the most significant single nucleotide polymorphism (SNP). RESULTS: A strong association with CMH, consistent across all cohorts, was observed with rs6577641 (p = 4.25×10(-6), OR = 1.17), located in intron 9 of the special AT-rich sequence-binding protein 1 locus (SATB1) on chromosome 3. The risk allele (G) was associated with higher mRNA expression of SATB1 (4.3×10(-9)) in lung tissue. Presence of CMH was associated with increased SATB1 mRNA expression in bronchial biopsies from COPD patients. SATB1 expression was induced during differentiation of primary human bronchial epithelial cells in culture. CONCLUSIONS: Our findings, that SNP rs6577641 is associated with CMH in multiple cohorts and is a cis-eQTL for SATB1, together with our additional observation that SATB1 expression increases during epithelial differentiation provide suggestive evidence that SATB1 is a gene that affects CMH.

Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease.

Using variants from the 1000 Genomes Project pilot European CEU dataset and data from additional resequencing studies, we densely genotyped 183 non-HLA risk loci previously associated with immune-mediated diseases in 12,041 individuals with celiac disease (cases) and 12,228 controls. We identified 13 new celiac disease risk loci reaching genome-wide significance, bringing the number of known loci (including the HLA locus) to 40. We found multiple independent association signals at over one-third of these loci, a finding that is attributable to a combination of common, low-frequency and rare genetic variants. Compared to previously available data such as those from HapMap3, our dense genotyping in a large sample collection provided a higher resolution of the pattern of linkage disequilibrium and suggested localization of many signals to finer scale regions. In particular, 29 of the 54 fine-mapped signals seemed to be localized to single genes and, in some instances, to gene regulatory elements. Altogether, we define the complex genetic architecture of the risk regions of and refine the risk signals for celiac disease, providing the next step toward uncovering the causal mechanisms of the disease.

Multiple common variants for celiac disease influencing immune gene expression.

We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with P(GWAS) < 10(-4) and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome-wide significance (P(combined) < 5 x 10(-8)); most contain genes with immune functions (BACH2, CCR4, CD80, CIITA-SOCS1-CLEC16A, ICOSLG and ZMIZ1), with ETS1, RUNX3, THEMIS and TNFRSF14 having key roles in thymic T-cell selection. There was evidence to suggest associations for a further 13 regions. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P < 0.0028, FDR 5%) with cis gene expression.

HLA-A*02 is associated with a reduced risk and HLA-A*01 with an increased risk of developing EBV+ Hodgkin lymphoma.

Previous studies showed that the HLA class I region is associated with Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL) and that HLA-A is the most likely candidate gene in this region. This suggests that antigenic presentation of EBV-derived peptides in the context of HLA-A is involved in the pathogenesis of EBV+ HL by precluding efficient immune responses. We genotyped exons 2 and 3, encoding the peptide-binding groove of HLA-A, for 32 single nucleotide polymorphisms in 70 patients with EBV+ HL, 31 patients with EBV- HL, and 59 control participants. HLA-A*01 was significantly overrepresented and HLA-A*02 was significantly underrepresented in patients with EBV+ HL versus controls and patients with EBV- HL. In addition, HLA-A*02 status was determined by immunohistochemistry or HLA-A*02-specific polymerase chain reaction (PCR) on 152 patients with EBV+ HL and 322 patients with EBV- HL. The percentage of HLA-A*02+ patients in the EBV+ HL group (35.5%) was significantly lower than in 6107 general control participants (53.0%) and the EBV- HL group (50.9%). Our results indicate that individuals carrying the HLA-A*02 allele have a reduced risk of developing EBV+ HL, while individuals carrying the HLA-A*01 allele have an increased risk. It is known that HLA-A*02 can present EBV-derived peptides and can evoke an effective immune response, which may explain the protective phenotype.