High genetic risk for depression as an independent risk factor for mortality in patients referred for coronary angiography.

Background: Different observations have suggested that patients with depression have a higher risk for a number of comorbidities and mortality. The underlying causes have not been fully understood yet. Aims: The aim of our study was to investigate the association of a genetic depression risk score (GDRS) with mortality [all-cause and cardiovascular (CV)] and markers of depression (including intake of antidepressants and a history of depression) in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study involving 3,316 patients who had been referred for coronary angiography. Methods and results: The GDRS was calculated in 3,061 LURIC participants according to a previously published method and was found to be associated with all-cause (p = 0.016) and CV mortality (p = 0.0023). In Cox regression models adjusted for age, sex, body mass index, LDL-cholesterol, HDL-cholesterol, triglycerides, hypertension, smoking, and diabetes mellitus, the GDRS remained significantly associated with all-cause [1.18 (1.04-1.34, p = 0.013)] and CV [1.31 (1.11-1.55, p = 0.001)] mortality. The GDRS was not associated with the intake of antidepressants or a history of depression. However, this cohort of CV patients had not specifically been assessed for depression, leading to marked underreporting. We were unable to identify any specific biomarkers correlated with the GDRS in LURIC participants. Conclusion: A genetic predisposition for depression estimated by a GDRS was independently associated with all-cause and CV mortality in our cohort of patients who had been referred for coronary angiography. No biomarker correlating with the GDRS could be identified.

Sunlight exposure exerts immunomodulatory effects to reduce multiple sclerosis severity.

Multiple sclerosis (MS) disease risk is associated with reduced sun-exposure. This study assessed the relationship between measures of sun exposure (vitamin D [vitD], latitude) and MS severity in the setting of two multicenter cohort studies (nNationMS = 946, nBIONAT = 990). Additionally, effect-modification by medication and photosensitivity-associated MC1R variants was assessed. High serum vitD was associated with a reduced MS severity score (MSSS), reduced risk for relapses, and lower disability accumulation over time. Low latitude was associated with higher vitD, lower MSSS, fewer gadolinium-enhancing lesions, and lower disability accumulation. The association of latitude with disability was lacking in IFN-ß-treated patients. In carriers of MC1R:rs1805008(T), who reported increased sensitivity toward sunlight, lower latitude was associated with higher MRI activity, whereas for noncarriers there was less MRI activity at lower latitudes. In a further exploratory approach, the effect of ultraviolet (UV)-phototherapy on the transcriptome of immune cells of MS patients was assessed using samples from an earlier study. Phototherapy induced a vitD and type I IFN signature that was most apparent in monocytes but that could also be detected in B and T cells. In summary, our study suggests beneficial effects of sun exposure on established MS, as demonstrated by a correlative network between the three factors: Latitude, vitD, and disease severity. However, sun exposure might be detrimental for photosensitive patients. Furthermore, a direct induction of type I IFNs through sun exposure could be another mechanism of UV-mediated immune-modulation in MS.

CSF parameters associated with early MRI activity in patients with MS.

Objective: To identify CSF parameters at diagnosis of clinically isolated syndrome (CIS) and MS that are associated with early inflammatory disease activity as measured by standardized cerebral MRI (cMRI). Methods: One hundred forty-nine patients with newly diagnosed CIS and MS were included in the retrospective study. cMRI at onset and after 12 months was analyzed for T2 and gadolinium-enhancing lesions. CSF was tested for oligoclonal bands and intrathecal synthesis of immunoglobulin G (IgG), A (IgA), and M (IgM) before initiation of disease-modifying therapy (DMT). In a subgroup of patients, CSF and serum samples were analyzed for sCD27, neurofilament light chain, and IgG subclasses 1 and 3. Association between CSF parameters and cMRI activity was investigated by univariable and multivariable regression analysis in all patients, DMT-treated patients, and untreated patients. Results: IgG index, sCD27 levels in CSF, and to a lesser extent IgM index were associated with the occurrence of new cMRI lesions. IgG index and sCD27 levels in CSF were highly correlated. In a multivariable analysis, IgG index and to a lesser extent IgM index together with DMT treatment status and gender were strongest predictors of future cMRI activity. Conclusions: CSF parameters such as IgG and IgM index are independently associated with future MRI activity and thus might be helpful to support early treatment decisions in patients newly diagnosed with CIS and MS.

Gene set enrichment analysis and expression pattern exploration implicate an involvement of neurodevelopmental processes in bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is a common and highly heritable disorder of mood. Genome-wide association studies (GWAS) have identified several independent susceptibility loci. In order to extract more biological information from GWAS data, multi-locus approaches represent powerful tools since they utilize knowledge about biological processes to integrate functional sets of genes at strongly to moderately associated loci. METHODS: We conducted gene set enrichment analyses (GSEA) using 2.3 million single-nucleotide polymorphisms, 397 Reactome pathways and 24,025 patients with BD and controls. RNA expression of implicated individual genes and gene sets were examined in post-mortem brains across lifespan. RESULTS: Two pathways showed a significant enrichment after correction for multiple comparisons in the GSEA: GRB2 events in ERBB2 signaling, for which 6 of 21 genes were BD associated (PFDR = 0.0377), and NCAM signaling for neurite out-growth, for which 11 out of 62 genes were BD associated (PFDR = 0.0451). Most pathway genes showed peaks of RNA co-expression during fetal development and infancy and mapped to neocortical areas and parts of the limbic system. LIMITATIONS: Pathway associations were technically reproduced by two methods, although they were not formally replicated in independent samples. Gene expression was explored in controls but not in patients. CONCLUSIONS: Pathway analysis in large GWAS data of BD and follow-up of gene expression patterns in healthy brains provide support for an involvement of neurodevelopmental processes in the etiology of this neuropsychiatric disease. Future studies are required to further evaluate the relevance of the implicated genes on pathway functioning and clinical aspects of BD.

Supportive evidence for FOXP1, BARX1, and FOXF1 as genetic risk loci for the development of esophageal adenocarcinoma.

The Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) recently performed a genome-wide association study (GWAS) on esophageal adenocarcinoma (EAC) and Barrett's esophagus. They identified genome-wide significant association for variants at three genes, namely CRTC1, FOXP1, and BARX1. Furthermore, they replicated an association at the FOXF1 gene that has been previously found in a GWAS on Barrett's esophagus. We aimed at further replicating the association at these and other loci that showed suggestive association with P < 10(-4) in the BEACON sample. In total, we tested 88 SNPs in an independent sample consisting of 1065 EAC cases and 1019 controls of German descent. We could replicate the association at FOXP1, BARX1, and FOXF1 with nominal significance and thereby confirm that genetic variants at these genes confer EAC risk. In addition, we found association of variants near the genes XRCC2 and GATA6 that were strongly (P < 10(-5) ) although not genome-wide significantly associated with the BEACON GWAS. Therefore, both variants and corresponding genes represent promising candidates for future EAC association studies on independent samples.

MicroRNA related polymorphisms and breast cancer risk.

Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88-0.96), rs1052532 (OR 0.97; 95% CI: 0.95-0.99), rs10719 (OR 0.97; 95% CI: 0.94-0.99), rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05) located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects.

Characterizing the genetic basis of innate immune response in TLR4-activated human monocytes.

Toll-like receptors (TLRs) play a key role in innate immunity. Apart from their function in host defense, dysregulation in TLR signalling can confer risk to autoimmune diseases, septic shock or cancer. Here we report genetic variants and transcripts that are active only during TLR signalling and contribute to interindividual differences in immune response. Comparing unstimulated versus TLR4-stimulated monocytes reveals 1,471 expression quantitative trait loci (eQTLs) that are unique to TLR4 stimulation. Among these we find functional SNPs for the expression of NEU4, CCL14, CBX3 and IRF5 on TLR4 activation. Furthermore, we show that SNPs conferring risk to primary biliary cirrhosis (PBC), inflammatory bowel disease (IBD) and celiac disease are immune response eQTLs for PDGFB and IL18R1. Thus, PDGFB and IL18R1 represent plausible candidates for studying the pathophysiology of these disorders in the context of TLR4 activation. In summary, this study presents novel insights into the genetic basis of the innate immune response and exemplifies the value of eQTL studies in the context of exogenous cell stimulation.

Targeted resequencing and systematic in vivo functional testing identifies rare variants in MEIS1 as significant contributors to restless legs syndrome.

Restless legs syndrome (RLS) is a common neurologic condition characterized by nocturnal dysesthesias and an urge to move, affecting the legs. RLS is a complex trait, for which genome-wide association studies (GWASs) have identified common susceptibility alleles of modest (OR 1.2-1.7) risk at six genomic loci. Among these, variants in MEIS1 have emerged as the largest risk factors for RLS, suggesting that perturbations in this transcription factor might be causally related to RLS susceptibility. To establish this causality, direction of effect, and total genetic burden of MEIS1, we interrogated 188 case subjects and 182 control subjects for rare alleles not captured by previous GWASs, followed by genotyping of ∼3,000 case subjects and 3,000 control subjects, and concluded with systematic functionalization of all discovered variants using a previously established in vivo model of neurogenesis. We observed a significant excess of rare MEIS1 variants in individuals with RLS. Subsequent assessment of all nonsynonymous variants by in vivo complementation revealed an excess of loss-of-function alleles in individuals with RLS. Strikingly, these alleles compromised the function of the canonical MEIS1 splice isoform but were irrelevant to an isoform known to utilize an alternative 3' sequence. Our data link MEIS1 loss of function to the etiopathology of RLS, highlight how combined sequencing and systematic functional annotation of rare variation at GWAS loci can detect risk burden, and offer a plausible explanation for the specificity of phenotypic expressivity of loss-of-function alleles at a locus broadly necessary for neurogenesis and neurodevelopment.

Clinically apparent and occult metastasized seminoma: almost indistinguishable on the transcriptional level.

PURPOSE: The aim of the present study was to examine the biological differences between seminomas with occult and clinically apparent metastases at the time of diagnosis of the primary tumor to gain insight into the biology of these tumors and facilitate the identification of novel predictors of seminoma metastasis. MATERIALS AND METHODS: Total RNA including small RNAs was isolated from testicular tumors of patients with pure seminoma presenting with lymphogenic metastasis (n = 5, clinical stage IIb/c) and occult metastasis (n = 5, clinical stage I). The regulation of biological processes was examined (1) throughout the mRNA transcriptome (whole genome microarrays, 8×60 K Array, Agilent with 4 samples/group) and (2) the miRNA transcriptome employing small RNA next generation sequencing (SOLID, Life Technologies with 5 samples/group). Protein coding genes (mRNAs) and small RNAs showing a significant (≥2-fold) difference between the groups were identified. Finally (3), we examined 95 candidate miRNAs in 36 apparent metastasized and another 5 occult metastasized seminoma using logistic regression analysis. RESULTS: Among 19,596 genes, on average 12,894 mRNAs appeared expressed (65.8%, SD+/-2.4; range, 62.0-69.3%) and 16.99×106/13.94×106 small RNA reads were identified for apparent/occult metastasized seminoma. These reads on average convert into 9,901/9,675 small RNAs including 422/404 mature microRNAs. None of these mRNAs/small RNAs met our selection criteria for candidate genes. From 95 candidate miRNAs 44 appeared expressed, with 3 of them showing weak but significant (p = 0.05) differences among both groups. CONCLUSIONS: Occult and apparent metastasized seminomas are biologically almost indistinguishable and probably represent no separate tumor entities. These findings may simplify future research on seminoma metastasis.

Small RNAs in the peripheral blood discriminate metastasized from non-metastasized seminoma.

BACKGROUND: We aimed to better discriminate metastasized (lymphogen/occult/both combined) from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood. METHODS: Total RNAs including small RNAs were isolated from the peripheral blood of patients suffering from metastasized testicular tumours (lymphogen, n = 5, clinical stage IIb/c; occult, n = 5, clinical stage I) and non-metastasized patients (n = 5, clinical stage I). Small RNA next generation sequencing (SOLID, Life Technologies) was employed to examine post-transcriptional changes. We searched for small RNAs showing at least 50 reads and a significant ≥ 2-fold difference using peripheral blood small RNAs of non-metastasized tumours as the reference group. Candidate small RNAs were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines. RESULTS: On average 1.3 x 10(7), 1.2 x 10(7) and 1.2 x 10(7) small RNA reads were detectable in non-metastasized, lymphogen and occult metastasized seminoma, respectively of which 73-76% remained after trimming. From these between 80-82% represented annotated reads and 7.2-7.8% (1.6-1.7 x 10(4)) were annotated small RNA tags. Of them 137 small RNAs showed > 50 reads and a ≥ two-fold difference to the reference. In univariate analysis we detected 33-35 different small RNAs which significantly discriminated lymphogen/occult/combined metastasized from non-metastasized seminoma and among these different comparisons it were the same small RNAs in 44-79%. Many combinations of two of these small RNAs completely discriminated metastasized from non-metastasized seminoma irrespective of the metastasis subtype. CONCLUSIONS: Metastasized (either lymphogen or occult) seminoma can be completely discriminated from non-metastasized seminoma with a combination of two small RNAs measured in the peripheral blood.

Restless legs syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon.

Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.

A genome-wide association study of early-onset breast cancer identifies PFKM as a novel breast cancer gene and supports a common genetic spectrum for breast cancer at any age.

Early-onset breast cancer (EOBC) causes substantial loss of life and productivity, creating a major burden among women worldwide. We analyzed 1,265,548 Hapmap3 single-nucleotide polymorphisms (SNP) among a discovery set of 3,523 EOBC incident cases and 2,702 population control women ages ≤ 51 years. The SNPs with smallest P values were examined in a replication set of 3,470 EOBC cases and 5,475 control women. We also tested EOBC association with 19,684 genes by annotating each gene with putative functional SNPs, and then combining their P values to obtain a gene-based P value. We examined the gene with smallest P value for replication in 1,145 breast cancer cases and 1,142 control women. The combined discovery and replication sets identified 72 new SNPs associated with EOBC (P < 4 × 10(-8)) located in six genomic regions previously reported to contain SNPs associated largely with later-onset breast cancer (LOBC). SNP rs2229882 and 10 other SNPs on chromosome 5q11.2 remained associated (P < 6 × 10(-4)) after adjustment for the strongest published SNPs in the region. Thirty-two of the 82 currently known LOBC SNPs were associated with EOBC (P < 0.05). Low power is likely responsible for the remaining 50 unassociated known LOBC SNPs. The gene-based analysis identified an association between breast cancer and the phosphofructokinase-muscle (PFKM) gene on chromosome 12q13.11 that met the genome-wide gene-based threshold of 2.5 × 10(-6). In conclusion, EOBC and LOBC seem to have similar genetic etiologies; the 5q11.2 region may contain multiple distinct breast cancer loci; and the PFKM gene region is worthy of further investigation. These findings should enhance our understanding of the etiology of breast cancer.

Integrating gene expression and epidemiological data for the discovery of genetic interactions associated with cancer risk.

Dozens of common genetic variants associated with cancer risk have been identified through genome-wide association studies (GWASs). However, these variants only explain a modest fraction of the heritability of disease. The missing heritability has been attributed to several factors, among them the existence of genetic interactions (G × G). Systematic screens for G × G in model organisms have revealed their fundamental influence in complex phenotypes. In this scenario, G × G overlap significantly with other types of gene and/or protein relationships. Here, by integrating predicted G × G from GWAS data and complex- and context-defined gene coexpression profiles, we provide evidence for G × G associated with cancer risk. G × G predicted from a breast cancer GWAS dataset identified significant overlaps [relative enrichments (REs) of 8-36%, empirical P values < 0.05 to 10(-4)] with complex (non-linear) gene coexpression in breast tumors. The use of gene or protein data not specific for breast cancer did not reveal overlaps. According to the predicted G × G, experimental assays demonstrated functional interplay between lipoma-preferred partner and transforming growth factor-ß signaling in the MCF10A non-tumorigenic mammary epithelial cell model. Next, integration of pancreatic tumor gene expression profiles with pancreatic cancer G × G predicted from a GWAS corroborated the observations made for breast cancer risk (REs of 25-59%). The method presented here can potentially support the identification of genetic interactions associated with cancer risk, providing novel mechanistic hypotheses for carcinogenesis.

Large-scale genotyping identifies 41 new loci associated with breast cancer risk.

Breast cancer is the most common cancer among women. Common variants at 27 loci have been identified as associated with susceptibility to breast cancer, and these account for ∼9% of the familial risk of the disease. We report here a meta-analysis of 9 genome-wide association studies, including 10,052 breast cancer cases and 12,575 controls of European ancestry, from which we selected 29,807 SNPs for further genotyping. These SNPs were genotyped in 45,290 cases and 41,880 controls of European ancestry from 41 studies in the Breast Cancer Association Consortium (BCAC). The SNPs were genotyped as part of a collaborative genotyping experiment involving four consortia (Collaborative Oncological Gene-environment Study, COGS) and used a custom Illumina iSelect genotyping array, iCOGS, comprising more than 200,000 SNPs. We identified SNPs at 41 new breast cancer susceptibility loci at genome-wide significance (P < 5 × 10(-8)). Further analyses suggest that more than 1,000 additional loci are involved in breast cancer susceptibility.

Dilution of candidates: the case of iron-related genes in restless legs syndrome.

Restless legs syndrome (RLS) is a common multifactorial disease. Some genetic risk factors have been identified. RLS susceptibility also has been related to iron. We therefore asked whether known iron-related genes are candidates for association with RLS and, vice versa, whether known RLS-associated loci influence iron parameters in serum. RLS/control samples (n = 954/1814 in the discovery step, 735/736 in replication 1, and 736/735 in replication 2) were tested for association with SNPs located within 4 Mb intervals surrounding each gene from a list of 111 iron-related genes using a discovery threshold of P = 5 × 10(-4). Two population cohorts (KORA F3 and F4 with together n = 3447) were tested for association of six known RLS loci with iron, ferritin, transferrin, transferrin-saturation, and soluble transferrin receptor. Results were negative. None of the candidate SNPs at the iron-related gene loci was confirmed significantly. An intronic SNP, rs2576036, of KATNAL2 at 18q21.1 was significant in the first (P = 0.00085) but not in the second replication step (joint nominal P-value = 0.044). Especially, rs1800652 (C282Y) in the HFE gene did not associate with RLS. Moreover, SNPs at the known RLS loci did not significantly affect serum iron parameters in the KORA cohorts. In conclusion, the correlation between RLS and iron parameters in serum may be weaker than assumed. Moreover, in a general power analysis, we show that genetic effects are diluted if they are transmitted via an intermediate trait to an end-phenotype. Sample size formulas are provided for small effect sizes.

The aromatase gene CYP19A1: several genetic and functional lines of evidence supporting a role in reading, speech and language.

Inspired by the localization, on 15q21.2 of the CYP19A1 gene in the linkage region of speech and language disorders, and a rare translocation in a dyslexic individual that was brought to our attention, we conducted a series of studies on the properties of CYP19A1 as a candidate gene for dyslexia and related conditions. The aromatase enzyme is a member of the cytochrome P450 super family, and it serves several key functions: it catalyzes the conversion of androgens into estrogens; during early mammalian development it controls the differentiation of specific brain areas (e.g. local estrogen synthesis in the hippocampus regulates synaptic plasticity and axonal growth); it is involved in sexual differentiation of the brain; and in songbirds and teleost fishes, it regulates vocalization. Our results suggest that variations in CYP19A1 are associated with dyslexia as a categorical trait and with quantitative measures of language and speech, such as reading, vocabulary, phonological processing and oral motor skills. Variations near the vicinity of its brain promoter region altered transcription factor binding, suggesting a regulatory role in CYP19A1 expression. CYP19A1 expression in human brain correlated with the expression of dyslexia susceptibility genes such as DYX1C1 and ROBO1. Aromatase-deficient mice displayed increased cortical neuronal density and occasional cortical heterotopias, also observed in Robo1-/- mice and human dyslexic brains, respectively. An aromatase inhibitor reduced dendritic growth in cultured rat neurons. From this broad set of evidence, we propose CYP19A1 as a candidate gene for human cognitive functions implicated in reading, speech and language.

Genome-wide association analysis identifies three new breast cancer susceptibility loci.

Breast cancer is the most common cancer among women. To date, 22 common breast cancer susceptibility loci have been identified accounting for ∼8% of the heritability of the disease. We attempted to replicate 72 promising associations from two independent genome-wide association studies (GWAS) in ∼70,000 cases and ∼68,000 controls from 41 case-control studies and 9 breast cancer GWAS. We identified three new breast cancer risk loci at 12p11 (rs10771399; P = 2.7 × 10(-35)), 12q24 (rs1292011; P = 4.3 × 10(-19)) and 21q21 (rs2823093; P = 1.1 × 10(-12)). rs10771399 was associated with similar relative risks for both estrogen receptor (ER)-negative and ER-positive breast cancer, whereas the other two loci were associated only with ER-positive disease. Two of the loci lie in regions that contain strong plausible candidate genes: PTHLH (12p11) has a crucial role in mammary gland development and the establishment of bone metastasis in breast cancer, and NRIP1 (21q21) encodes an ER cofactor and has a role in the regulation of breast cancer cell growth.

PTGER4 expression-modulating polymorphisms in the 5p13.1 region predispose to Crohn's disease and affect NF-κB and XBP1 binding sites.

BACKGROUND: Genome-wide association studies identified a PTGER4 expression-modulating region on chromosome 5p13.1 as Crohn's disease (CD) susceptibility region. The study aim was to test this association in a large cohort of patients with inflammatory bowel disease (IBD) and to elucidate genotypic and phenotypic interactions with other IBD genes. METHODOLOGY/PRINCIPAL FINDINGS: A total of 7073 patients and controls were genotyped: 844 CD and 471 patients with ulcerative colitis and 1488 controls were analyzed for the single nucleotide polymorphisms (SNPs) rs4495224 and rs7720838 on chromosome 5p13.1. The study included two replication cohorts of North American (CD: n = 684; controls: n = 1440) and of German origin (CD: n = 1098; controls: n = 1048). Genotype-phenotype, epistasis and transcription factor binding analyses were performed. In the discovery cohort, an association of rs4495224 (p = 4.10×10⁻5; 0.76 [0.67-0.87]) and of rs7720838 (p = 6.91×10⁻4; 0.81 [0.71-0.91]) with susceptibility to CD was demonstrated. These associations were confirmed in both replication cohorts. In silico analysis predicted rs4495224 and rs7720838 as essential parts of binding sites for the transcription factors NF-κB and XBP1 with higher binding scores for carriers of the CD risk alleles, providing an explanation of how these SNPs might contribute to increased PTGER4 expression. There was no association of the PTGER4 SNPs with IBD phenotypes. Epistasis detected between 5p13.1 and ATG16L1 for CD susceptibility in the discovery cohort (p = 5.99×10⁻7 for rs7720838 and rs2241880) could not be replicated in both replication cohorts arguing against a major role of this gene-gene interaction in the susceptibility to CD. CONCLUSIONS/SIGNIFICANCE: We confirmed 5p13.1 as a major CD susceptibility locus and demonstrate by in silico analysis rs4495224 and rs7720838 as part of binding sites for NF-κB and XBP1. Further functional studies are necessary to confirm the results of our in silico analysis and to analyze if changes in PTGER4 expression modulate CD susceptibility.

Pregnane X receptor (PXR/NR1I2) gene haplotypes modulate susceptibility to inflammatory bowel disease.

BACKGROUND: The pregnane X receptor (PXR/NR1I2) is an important regulator of xenobiotic metabolism and intestinal integrity. However, there are controversial studies on the role of PXR/NR1I2 in inflammatory bowel disease (IBD). We therefore initiated the largest analysis to date on PXR/NR1I2 gene variants in IBD patients. METHODS: Genomic DNA from 2823 individuals of Caucasian origin including 859 patients with Crohn's disease (CD), 464 patients with ulcerative colitis (UC), and 1500 healthy, unrelated controls was analyzed for eight PXR/NR1I2 single nucleotide polymorphisms (SNPs) (rs12721602 (-25564), rs3814055 (-25385), rs1523128 (-24756), rs1523127 (-24381), rs45610735 = p.Gly36Arg (+106), rs6785049 (+7635), rs2276707 (+8055), and rs3814057 (+11156)). In addition, detailed haplotype and genotype-phenotype analyses were performed. RESULTS: The PXR/NR1I2 SNP rs2276707 was weakly associated with UC susceptibility (P = 0.01; odds ratio [OR] 1.27 [1.06-1.52]). None of the other PXR/NR1I2 SNPs were associated with UC or CD susceptibility. However, several rare PXR/NR1I2 haplotypes were highly associated with CD susceptibility. In CD, the strongest disease association was found for a haplotype consisting of the SNPs rs12721602-rs3814055-rs1523128-rs1523127-rs12721607-rs6785049-rs2276707-rs3814057 (omnibus P-value: 6.50 × 10(-15)) which was found in two separate cohorts (cohort I = discovery cohort: CD: n = 492, controls: n = 793; P = 4.51 × 10(-17); Bonferroni corrected: P = 1.27 × 10(-15); cohort II = replication cohort: CD: n = 367, controls: n = 707; P = 7.12 × 10(-4); P(corr) = 1.99 × 10(-2)). CONCLUSIONS: Several PXR/NR1I2 haplotypes contribute to CD susceptibility, suggesting a role for PXR in the IBD pathogenesis of a certain patient subcohort. Given the accumulating evidence for an important role of PXR in intestinal inflammation, further analyses are required to investigate the functional and pharmacogenetic implications of these PXR/NR1I2 gene variants in IBD.

CEACAM6 gene variants in inflammatory bowel disease.

BACKGROUND: The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) acts as a receptor for adherent-invasive E. coli (AIEC) and its ileal expression is increased in patients with Crohn's disease (CD). Given its contribution to the pathogenesis of CD, we aimed to investigate the role of genetic variants in the CEACAM6 region in patients with inflammatory bowel diseases (IBD). METHODOLOGY: In this study, a total of 2,683 genomic DNA samples (including DNA from 858 CD patients, 475 patients with ulcerative colitis (UC), and 1,350 healthy, unrelated controls) was analyzed for eight CEACAM6 SNPs (rs10415946, rs1805223 = p.Pro42Pro, rs4803507, rs4803508, rs11548735 = p.Gly239Val, rs7246116 = pHis260His, rs2701, rs10416839). In addition, a detailed haplotype analysis and genotype-phenotype analysis were performed. Overall, our genotype analysis did not reveal any significant association of the investigated CEACAM6 SNPs and haplotypes with CD or UC susceptibility, although certain CEACAM6 SNPs modulated CEACAM6 expression in intestinal epithelial cell lines. Despite its function as receptor of AIEC in ileal CD, we found no association of the CEACAM6 SNPs with ileal or ileocolonic CD. Moreover, there was no evidence of epistasis between the analyzed CEACAM6 variants and the main CD-associated NOD2, IL23R and ATG16L1 variants. CONCLUSIONS: This study represents the first detailed analysis of CEACAM6 variants in IBD patients. Despite its important role in bacterial attachment in ileal CD, we could not demonstrate a role for CEACAM6 variants in IBD susceptibility or regarding an ileal CD phenotype. Further functional studies are required to analyze if these gene variants modulate ileal bacterial attachment.