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Genome-wide association studies (GWAS) have identified hundreds of loci associated with Crohns disease (CD), however, as with all complex diseases, deriving pathogenic mechanisms from these non-coding GWAS discoveries has been challenging. To complement GWAS and better define actionable biological targets, we analysed sequenced data from more than 30,000 CD patients and 80,000 population controls. We observe rare coding variants in established CD susceptibility genes as well as ten genes where coding variation directly implicates the gene in disease risk for the first time.

Background and Aims Differential responsiveness to interleukin [IL]-2 between effector CD4+ T cells [Teff] and regulatory T cells [Treg] is a fundamental mechanism of immunoregulation. The single nucleotide polymorphism [SNP] rs61839660, located within IL2RA [CD25], has been associated with the development of Crohn’s disease [CD]. We sought to identify the T cell immune phenotype of IBD patients who carry this SNP. Methods Teff and Treg were isolated from individuals homozygous [TT], heterozygous [CT], or wild-type [CC] for the minor allele at rs61839660, and used for phenotyping [flow cytometry, Cytometry Time Of Flight] functional assays or T cell receptor [TCR] sequencing. Phosphorylation of signal transducer and activator of transcription 5 [STAT5] was assessed in response to IL-2, IL-7, and in the presence of basiliximab, a monoclonal antibody directed against CD25. Teff pro-inflammatory cytokine expression levels were assessed by reverse transcription quantitative polymerase chain reaction after IL-2 and/or TCR stimulation. Results Presence of the minor T allele enhances CD25 expression, leading to increased STAT5 phosphorylation and pro-inflammatory cytokine transcript expression by Teff in response to IL-2 stimulation in vitro. Teff from TT individuals demonstrate a more activated gut homing phenotype. TCR sequencing analysis suggests that TT patients may have a reduced clonal capacity to mount an optimal regulatory T cell response. Conclusions rs61839660 regulates the responsiveness of T cells to IL-2 signalling by modulating CD25 expression. As low-dose IL-2 is being trialled as a selective Treg modulator in CD, these findings highlight the potential for adverse effects in patients with this genotype., Graphical Abstract Graphical Abstract

A. Palotie on työryhmän Int IBD Genetics Consortium jäsen. One goal of human genetics is to understand the genetic basis of disease, a challenge for diseases of complex inheritance because risk alleles are few relative to the vast set of benign variants. Risk variants are often sought by association studies in which allele frequencies in case subjects are contrasted with those from population-based samples used as control subjects. In an ideal world we would know population-level allele frequencies, releasing researchers to focus on case subjects. We argue this ideal is possible, at least theoretically, and we outline a path to achieving it in reality. If such a resource were to exist, it would yield ample savings and would facilitate the effective use of data repositories by removing administrative and technical barriers. We call this concept the Universal Control Repository Network (UNICORN), a means to perform association analyses without necessitating direct access to individual-level control data. Our approach to UNICORN uses existing genetic resources and various statistical tools to analyze these data, including hierarchical clustering with spectral analysis of ancestry; and empirical Bayesian analysis along with Gaussian spatial processes to estimate ancestry-specific allele frequencies. We demonstrate our approach using tens of thousands of control subjects from studies of Crohn disease, showing how it controls false positives, provides power similar to that achieved when all control data are directly accessible, and enhances power when control data are limiting or even imperfectly matched ancestrally. These results highlight how UNICORN can enable reliable, powerful, and convenient genetic association analyses without access to the individual-level data.

BACKGROUND & AIMS: Relatives of individuals with Crohn's disease (CD) carry CD-associated genetic variants and are often exposed to environmental factors that increase their risk for this disease. We aimed to estimate the utility of genotype, smoking status, family history, and other biomarkers can be used to calculate risk in asymptomatic first-degree relatives of patients with CD.METHODS: We recruited 480 healthy first-degree relatives (full siblings, offspring or parents) of patients with CD through the Guy's and St Thomas' NHS Foundation Trust and from members of Crohn's and Colitis, United Kingdom. DNA samples were genotyped using the Immunochip. We calculated a risk score for 454 participants, based on 72 genetic variants associated with CD, family history, and smoking history. Participants were assigned to highest and lowest risk score quartiles. We assessed pre-symptomatic inflammation by capsule endoscopy and measured 22 markers of inflammation in stool and serum samples (reference standard). Two machine-learning classifiers (elastic net and random forest) were used to assess the ability of the risk factors and biomarkers to identify participants with small intestinal inflammation in the same dataset.RESULTS: The machine-learning classifiers identified participants with pre-symptomatic intestinal inflammation: elastic net (area under the curve, 0.80; 95% CI, 0.62-0.98) and random forest (area under the curve, 0.87; 95% CI, 0.75-1.00). The elastic net method identified 3 variables that can be used to calculate odds for intestinal inflammation: combined family history of CD (odds ratio, 1.31), genetic risk score (odds ratio, 1.14), and fecal level of calprotectin (odds ratio, 1.04). These same 3 variables were among the 5 factors associated with intestinal inflammation in the random forest model.CONCLUSION: Using machine learning classifiers, we found that genetic variants associated with CD, family history, and fecal level of calprotectin together identify individuals with pre-symptomatic intestinal inflammation who are therefore at risk for CD. A tool for detecting people at risk for CD before they develop symptoms would help identify the individuals most likely to benefit from early intervention.

ObjectiveOrofacial granulomatosis (OFG) is a rare disease characterised by chronic, non-caseating, granulomatous inflammation primarily affecting the oral cavity. Histologically, it is similar to Crohn’s disease (CD) and a proportion of patients have both OFG and CD. The cause of OFG remains elusive but it has been suggested that microbial interactions may be involved. The aim of this study was to compare the salivary microbial composition of subjects with OFG and/or CD and healthy controls.Design261 subjects were recruited, of whom 78 had OFG only, 40 had both OFG and CD, 97 had CD only with no oral symptoms and 46 were healthy controls. Bacterial community profiles were obtained by sequencing the V1-V3 region of the 16S rRNA gene.ResultsThere were no differences in richness or diversity of the salivary bacterial communities between patient groups and controls. The relative abundance of the Streptococcus salivarius-group were raised in patients with OFG or CD only compared to controls while that of the Streptococcus mitis -group was lower in CD compared to both OFG and controls. One S. salivarius oligotype made the major contribution to the increased proportions seen in patients with OFG and CD.ConclusionThe salivary microbiome of individuals with OFG and CD was similar to that found in health although the proportions of S. salivarius, a common oral Streptococcus were raised. One specific strain-level oligotype was found to be primarily responsible for the increased levels seen.

Oesophageal squamous cell carcinoma (OSCC) has a high incidence in southern Africa and a poor prognosis. Limited information is available on the contribution of genetic variants in susceptibility to OSCC in this region. However, recent genome-wide association studies have identified multiple susceptibility loci in Asian and European populations. In this study, we investigated genetic variants from seven OSCC risk loci identified in non-African populations for association with OSCC in the South African Black population. We performed association studies in a total of 1471 cases and 1791 controls from two study sample groups, which included 591 cases and 852 controls from the Western Cape and 880 cases and 939 controls from the Johannesburg region in the Gauteng province. Thereafter, we performed a meta-analysis for 11 variants which had been genotyped in both studies. A single nucleotide polymorphism in the CHEK2 gene, rs1033667, was significantly associated with OSCC [P = 0.002; odds ratio (OR) = 1.176; 95% confidence interval (CI): 1.06–1.30]. However, single nucleotide polymorphisms in the CASP8/ALS2CR12, TMEM173, PLCE1, ALDH2, ATP1B2/TP53 and RUNX1 loci were not associated with the disease (P > 0.05). The lack of association of six of these loci with OSCC in South African populations may reflect different genetic risk factors in non-African and African populations or differences in the genetic architecture of African genomes. The association at CHEK2, a gene with key roles in cell cycle regulation and DNA repair, in an African population provides further support for the contribution of common genetic variants at this locus to the risk of oesophageal cancer., Single nucleotide polymorphisms (SNPs) associated with oesophageal squamous cell carcinoma (OSCC) in Asian populations were genotyped in OSCC cases and controls from the South African Black population. A SNP in CHEK2 was associated with an increased risk of OSCC.

Background and aimsAlthough genome-wide association studies (GWAS) in inflammatory bowel disease (IBD) have identified a large number of common disease susceptibility alleles for both Crohn’s disease (CD) and ulcerative colitis (UC), a substantial fraction of IBD heritability remains unexplained, suggesting that rare coding genetic variants may also have a role in pathogenesis. We used high-throughput sequencing in families with multiple cases of IBD, followed by genotyping of cases and controls, to investigate whether rare protein altering genetic variants are associated with susceptibility to IBD.MethodsWhole exome sequencing was carried out in 10 families in which 3 or more individuals were affected with IBD. A stepwise filtering approach was applied to exome variants to identify potential causal variants. Follow-up genotyping was performed in 6,025 IBD cases (2,948 CD; 3,077 UC) and 7,238 controls.ResultsOur exome variant analysis revealed coding variants in the NLRP7 gene that were present in affected individuals in two distinct families. Genotyping of the two variants, p.S361L and p.R801H, in IBD cases and controls showed that the p.S361L variant was significantly associated with an increased risk of ulcerative colitis (odds ratio 4.79, p=0.0039) and IBD (odds ratio 3.17, p=0.037). A combined analysis of both variants showed suggestive association with an increased risk of IBD (odds ratio 2.77, p=0.018).ConclusionsThe results suggest that NLRP7 signalling and inflammasome formation may be a significant component in the pathogenesis of IBD.

Background and Aim Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. Methods Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). Results Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. Conclusions CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

ObjectiveRelatives of individuals with Crohn’s disease (CD) carry an increased number of CD-associated genetic variants and are at increased risk of developing the disease. Multiple environmental and genetic factors contribute to this increased risk. We aimed to estimate the utility of genotype, smoking, family history, and a panel of biomarkers to predict risk in asymptomatic first-degree relatives (FDRs) of CD patients.DesignWe calculated a combined genotype (72 CD-associated genetic markers) and smoking relative risk score in 454 FDRs, and performed capsule endoscopy and collected 22 biomarkers in individuals from the highest and lowest risk quartiles. We then predicted small intestinal inflammation using genetic risk score, smoking status, number of relatives with CD, capsule transit time, and the panel of biomarkers in 124 individuals with complete data. Our principal analysis was to calculate the predictive utility from two machine learning classifiers: an elastic net and a random forest.ResultsBoth classifiers successfully predicted FDRs with intestinal inflammation: elastic net (AUC=0.80, 95% CI: 0.62-0.98), random forest (AUC=0.87, 95% CI: 0.75-1.00). The elastic net selected a 3-predictor solution: CD family history (OR=1.31), genetic risk score (OR=1.14), and faecal calprotectin (OR=1.04). The same 3 variables were among the top 5 most important predictors as ranked by the random forest.ConclusionA readily collectable panel of genetic risk variants, added to family history and faecal calprotectin, predicts those at greatest risk for developing CD with a good degree of accuracy.

Background and Aims Although genome-wide association studies [GWAS] in inflammatory bowel disease [IBD] have identified a large number of common disease susceptibility alleles for both Crohn’s disease [CD] and ulcerative colitis [UC], a substantial fraction of IBD heritability remains unexplained, suggesting that rare coding genetic variants may also have a role in pathogenesis. We used high-throughput sequencing in families with multiple cases of IBD, followed by genotyping of cases and controls, to investigate whether rare protein-altering genetic variants are associated with susceptibility to IBD. Methods Whole-exome sequencing was carried out in 10 families in whom three or more individuals were affected with IBD. A stepwise filtering approach was applied to exome variants, to identify potential causal variants. Follow-up genotyping was performed in 6025 IBD cases [2948 CD; 3077 UC] and 7238 controls. Results Our exome variant analysis revealed coding variants in the NLRP7 gene that were present in affected individuals in two distinct families. Genotyping of the two variants, p.S361L and p.R801H, in IBD cases and controls showed that the p.S361L variant was significantly associated with an increased risk of ulcerative colitis [odds ratio 4.79, p = 0.0039] and IBD [odds ratio 3.17, p = 0.037]. A combined analysis of both variants showed suggestive association with an increased risk of IBD [odds ratio 2.77, p = 0.018]. Conclusions The results suggest that NLRP7 signalling and inflammasome formation may be a significant component in the pathogenesis of IBD.

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Genetic association studies have identified 215 risk loci for inflammatory bowel disease, thereby uncovering fundamental aspects of its molecular biology. We performed a genome-wide association study of 25,305 individuals and conducted a meta-analysis with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 25 new susceptibility loci, 3 of which contain integrin genes that encode proteins in pathways that have been identified as important therapeutic targets in inflammatory bowel disease. The associated variants are correlated with expression changes in response to immune stimulus at two of these genes (ITGA4 and ITGB8) and at previously implicated loci (ITGAL and ICAM1). In all four cases, the expression-increasing allele also increases disease risk. We also identified likely causal missense variants in a gene implicated in primary immune deficiency, PLCG2, and a negative regulator of inflammation, SLAMF8. Our results demonstrate that new associations at common variants continue to identify genes relevant to therapeutic target identification and prioritization.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access. Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10(-7), odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain. National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) DK064869 DK062432 National Human Genome Research Institute (NHGRI) DK064869 DK043351 HG005923 Crohns and Colitis Foundation 3765 Leona M. & Harry B. Helmsley Charitable Trust 2015PG-IBD001 Amgen 2013583217 CCFA 3765 Cedars-Sinai F. Widjaja Foundation, info:eu-repo/grantAgreement/EC/FP7/305479, European Union DK062413 AI067068 U54DE023789-01 Leona M. and Harry B. Helmsley Charitable Trust Crohn's and Colitis Foundation of America NIH DK062431 U01 DK062429 U01 DK062422 R01 DK092235 U01 DK062420 Medical Research Council, UK MR/J00314X/1 Wellcome Trust WT091310 098051 Inflammatory Bowel Disease Genetic Research Chair at the University of Pittsburgh PO1DK046763

Fraser syndrome (OMIM 219000) is a multisystem malformation usually comprising cryptophthalmos, syndactyly and renal defects(1). Here we report autozygosity mapping and show that the locus FS1 at chromosome 4q21 is associated with Fraser syndrome, although the condition is genetically heterogeneous. Mutation analysis identified five frameshift mutations in FRAS1, which encodes one member of a family of novel proteins related to an extracellular matrix (ECM) blastocoelar protein found in sea urchin. The FRAS1 protein contains a series of N-terminal cysteine-rich repeat motifs previously implicated in BMP metabolism, suggesting that it has a role in both structure and signal propagation in the ECM. It has been speculated that Fraser syndrome is a human equivalent of the blebbed phenotype in the mouse(2), which has been associated with mutations in at least five loci including bl(3). As mapping data were consistent with homology of FRAS1 and bl, we screened DNA from bl/bl mice and identified a premature termination of mouse Fras1. Thus, the bl mouse is a model for Fraser syndrome in humans, a disorder caused by disrupted epithelial integrity in utero.

Several risk factors for Crohn's disease have been identified in recent genome-wide association studies. To advance gene discovery further, we combined data from three studies on Crohn's disease ( a total of 3,230 cases and 4,829 controls) and carried out replication in 3,664 independent cases with a mixture of population-based and family-based controls. The results strongly confirm 11 previously reported loci and provide genome-wide significant evidence for 21 additional loci, including the regions containing STAT3, JAK2, ICOSLG, CDKAL1 and ITLN1. The expanded molecular understanding of the basis of this disease offers promise for informed therapeutic development.

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with diseaseIRGM for Crohns disease, HLA for Crohns disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetesalthough in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases. © 2010 Macmillan Publishers Limited. All rights reserved.

Genetic association studies have identified 210 risk loci for inflammatory bowel disease1–7, which have revealed fundamental aspects of the molecular biology of the disease, including the roles of autophagy and Th17 cell signaling and development. We performed a genome-wide association study of 25,305 individuals, and meta-analyzed with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 26 new genome-wide significant loci, three of which contain integrin genes that encode molecules in pathways identified as important therapeutic targets in inflammatory bowel disease. The associated variants are also correlated with expression changes in response to immune stimulus at two of these genes (ITGA4, ITGB8) and at two previously implicated integrin loci (ITGAL, ICAM1). In all four cases, the stimulus-dependent expression increasing allele also increases disease risk. We applied summary statistic fine-mapping and identified likely causal missense variants in the primary immune deficiency genePLCG2and the negative regulator of inflammation,SLAMF8. Our results demonstrate that new common variant associations continue to identify genes and pathways of relevance to therapeutic target identification and prioritization.

Oesophageal squamous cell carcinoma (OSCC) has a high prevalence in the Black and Mixed Ancestry populations of South Africa. Recently, three genome-wide association studies in Chinese populations identified five new OSCC susceptibility loci, including variants at PLCE1, C20orf54, PDE4D, RUNX1 and UNC5CL, but their contribution to disease risk in other populations is unknown. In this study, we report testing variants from these five loci for association with OSCC in the South African Black (407 cases and 849 controls) and Mixed Ancestry (257 cases and 860 controls) populations. The RUNX1 variant rs2014300, which reduced risk in the Chinese population, was associated with an increased risk of OSCC in the Mixed Ancestry population [odds ratio (OR) = 1.33, 95% confidence interval (CI) = 1.09-1.63, P = 0.0055], and none of the five loci were associated in the Black population. Since PLCE1 variants increased the risk of OSCC in all three Chinese studies, this gene was investigated further by sequencing in 46 Black South Africans. This revealed 48 variants, 10 of which resulted in amino acid substitutions, and much lower linkage disequilibrium across the PLCE1 locus than in the Chinese population. We genotyped five PLCE1 variants in cases and controls, and found association of Arg548Leu (rs17417407) with a reduced risk of OSCC (OR = 0.74, 95% CI = 0.60-0.93, P = 0.008) in the Black population. These findings indicate several differences in the genetic contribution to OSCC between the South African and Chinese populations that may be related to differences in their genetic architecture.

Patients with Crohn's disease (CD) have an intestinal dysbiosis with components of the microbiota exerting differential immune effects. Smoking is associated with an increased incidence of CD, more frequent relapse, and greater burden of surgery. This study aimed to investigate the association between smoking and the intestinal microbiota in patients with active CD.Patients with active CD (n = 103) and healthy controls (n = 66) were recruited and demographic and clinical data recorded including current smoking behavior. Fecal samples were collected and analyzed by fluorescent in situ hybridization using probes targeting 16S rRNA of bacteria previously shown to be altered in active CD (bifidobacteria, bacteroides, Clostridium coccoides-Eubacterium rectale, Escherichia coli, and Faecalibacterium prausnitzii).In total, 29/101 (29%) patients with CD and 8/58 (14%) controls were current smokers (P = 0.032). Following multivariate analysis, smoking was found to have a significant and independent effect on the microbiota of patients with CD, with higher Bacteroides-Prevotella in smokers (38.4%) compared with nonsmokers (28.1%) (F((1,93)) = 12.6, P = 0.001). Healthy controls who smoked also had higher Bacteroides-Prevotella (34.8%) than nonsmokers (24.1%) (F((1,55)) = 4.5, P = 0.038). In the pooled multivariate analysis, patients with CD had higher bifidobacteria (F((1,156)) = 30.5, P0.001), higher Bacteroides-Prevotella (F((1,156)) = 6.5, P = 0.012), and lower F. prausnitzii (F((1,156)) = 3.8, P = 0.052) compared with healthy controls.Smokers have luminal microbiota that consist of significantly higher bacteroides. Investigation of whether this is one mechanism through which the negative effects of smoking on CD are mediated is warranted.

Genetic variants in multiple cellular pathways have been associated with an altered risk of oesophageal cancer. In this study, eight genes previously associated with an altered risk of oesophageal squamous cell carcinoma (OSCC) in European or Asian populations were investigated in two South African populations. We genotyped 12 single-nucleotide polymorphisms and one insertion/deletion variant in 1463 individuals from the Black and Mixed Ancestry populations. No polymorphisms were associated with OSCC in the Black population. In the Mixed Ancestry population, ALDH2 +82 G > A (rs886205) was significantly associated with a reduced risk of OSCC (odds ratio = 0.70, 95% confidence interval = 0.55–0.89; P = 0.0038). Several other polymorphisms showed a suggestive association (P < 0.05), including ADH1B Arg48His (rs1229984), COX-2 −1195G > A (rs689466), CASP8 Asp302His (rs1045485) and MGMT Leu84Phe (rs12917). Haplotype analysis indicated that the FAS polymorphisms −670 A > G (rs1800682) and −1377 G > A (rs2234767) were both associated with OSCC in the Mixed Ancestry population (P = 0.006 and P = 0.004, respectively), as well as the CASP8 (−652 6Ndel:302His) haplotype (P = 0.0013). This study indicates several instances of population-specific differences in the genetic etiology of OSCC between these two South African populations and between them and other high-risk populations, which may reflect differences in their ancestry and environmental exposures.

Genome-wide association studies and candidate gene studies in ulcerative colitis have identified 18 susceptibility loci. We conducted a meta-analysis of six ulcerative colitis genome-wide association study datasets, comprising 6,687 cases and 19,718 controls, and followed up the top association signals in 9,628 cases and 12,917 controls. We identified 29 additional risk loci (P < 5 x 10(-8)), increasing the number of ulcerative colitis-associated loci to 47. After annotating associated regions using GRAIL, expression quantitative trait loci data and correlations with non-synonymous SNPs, we identified many candidate genes that provide potentially important insights into disease pathogenesis, including IL1R2, IL8RA-IL8RB, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1. The total number of confirmed inflammatory bowel disease risk loci is now 99, including a minimum of 28 shared association signals between Crohn's disease and ulcerative colitis.

Esophageal squamous cell carcinoma (ESCC) is common in many Black populations of sub-Saharan Africa, with high incidence regions in Eastern and Southern Africa. Clinical presentation in Africa is late, and treatment is mainly palliative with a very poor prognosis. Various environmental risk factors have been identified, but the possible contribution of inherited genetic variants to disease risk is unresolved. Genetic association studies of African ESCC have been limited to the analysis of small numbers of single nucleotide polymorphisms (SNPs) in candidate genes, and carried out only in the Black and Mixed Ancestry populations of the Western Cape of South Africa. Thus far no genetic associations with ESCC have been replicated in African populations. We tested SNPs which have been associated with ESCC in genome-wide association studies (GWAS) from Asian and European populations for association with ESCC in the South African Black population. Black cases with a histologically confirmed diagnosis of ESCC and matching population controls were recruited, after informed consent and institutional ethical approval, from the Western Cape and Gauteng provinces of South Africa. SNPs were genotyped either by individual TaqMan assays (Applied Biosystems) or in a multiplex MassARRAY (Agena Bioscience) and genotypes were tested for association. We genotyped 26 SNPs from OSCC risk loci and 10 ancestry informative markers in 880 Black South African (SAB) OSCC cases and 939 controls recruited by the Johannesburg Cancer Study. No significant associations were detected at the CASP8, ALS2CR12, ADH1C TMEM173, PLCE1, ALDH2, ATP1B2/TP53 or CHEK2 loci, although some SNPs showed the same direction of effect observed in other populations. 8 SNPs from these loci were genotyped in a further 493 cases and 820 controls from the Western Cape, and a combined test for association done in a total of 1373 cases and 1759 controls. The only SNP that was significantly associated with ESCC in the combined analysis after correction for multiple testing was rs1033667 in CHEK2 (OR = 1.18, P = 0.002). The lack of transference of most loci from Asian and European populations in an African population could result from smaller or absent effects of these loci in Africa. Alternatively, since we are likely to be genotyping tagging SNPs rather than the actual causal variants in these studies and linkage disequilibrium (LD) is lower in African genomes, our power to detect association with tagging SNPs will be reduced. This question could be resolved by carrying out a well-powered GWAS in African ESCC using a SNP array with sufficient African content to identify known or novel associations. We are currently conducting a GWAS for ESCC by genotyping 1700 ESCC cases and 6000 controls using a 2.4M SNP array designed by the H3 Africa Consortium. Citation Format: Christopher G. Mathew, Wenlong C. Chen, Hannah Bye, Natalie J. Prescott, Marco Matejcic, Robyn Kerr, Elvira Singh, Cathryn M. Lewis, Chantal Babb de Villiers, Mohamed I. Parker. The genetic architecture of African esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 237.

DNA polymorphisms in a region on chromosome 5q33.1 which contains two genes, immunity related GTPase related family, M (IRGM) and zinc finger protein 300 (ZNF300), are associated with Crohn's disease (CD). The deleted allele of a 20 kb copy number variation (CNV) upstream of IRGM was recently shown to be in strong linkage disequilibrium (LD) with the CD-associated single nucleotide polymorphisms and is itself associated with CD (P < 0.01). The deletion was correlated with increased or reduced expression of IRGM in transformed cells in a cell line-dependent manner, and has been proposed as a likely causal variant. We report here that small insertion/deletion polymorphisms in the promoter and 5' untranslated region of IRGM are, together with the CNV, strongly associated with CD (P = 1.37 x 10(-5) to 1.40 x 10(-9)), and that the CNV and the 5'-untranslated region variant -308(GTTT)(5) contribute independently to CD susceptibility (P = 2.6 x 10(-7) and P = 2 x 10(-5), respectively). We also show that the CD risk haplotype is associated with a significant decrease in IRGM expression (P < 10(-12)) in untransformed lymphocytes from CD patients. Further analysis of these variants in a Japanese CD case-control sample and of IRGM expression in HapMap populations revealed that neither the IRGM insertion/deletion polymorphisms nor the CNV was associated with CD or with altered IRGM expression in the Asian population. This suggests that the involvement of the IRGM risk haplotype in the pathogenesis of CD requires gene-gene or gene-environment interactions which are absent in Asian populations, or that none of the variants analysed are causal, and that the true causal variants arose after the European-Asian split.

There has been considerable recent success in the detection of gene-disease associations. We consider here the development of tools that facilitate the more detailed characterization of the effect of a genetic variant on disease. We replace the simplistic classification of individuals according to a single binary disease indicator with classification according to a number of subphenotypes. This more accurately reflects the underlying biological complexity of the disease process, but it poses additional analytical difficulties. Notably, the subphenotypes that make up a particular disease are typically highly associated, and it becomes difficult to distinguish which genes might be causing which subphenotypes. Such problems arise in many complex diseases. Here, we concentrate on an application to Crohn disease (CD). We consider this problem as one of model selection based upon log-linear models, fitted in a Bayesian framework via reversible-jump Metropolis-Hastings approach. We evaluate the performance of our suggested approach with a simple simulation study and then apply the method to a real data example in CD, revealing a sparse disease structure. Most notably, the associated NOD2.908G--R mutation appears to be directly related to more severe disease behaviors, whereas the other two associated NOD2 variants, 1007L--FS and 702R--W, are more generally related to disease in the small bowel (ileum and jejenum). The ATG16L1.300T--A variant appears to be directly associated with only disease of the small bowel.

CARD8 (TUCAN) is implicated in the regulation of apoptosis and inflammation, and is a positional and functional candidate gene for inflammatory bowel disease (IBD). Recent investigations have reported conflicting results of association between a CARD8 nonsynonymous SNP, rs2043211, and IBD. SNP rs2043211 results in an A>T transversion in the CARD8 template strand, which introduces a stop codon polymorphism (Cys10Stop), and genotyping of the Cys10Stop variant revealed that 9% of the control population was homozygous for the 'Stop' allele. The effect of the Stop allele on mRNA and protein expression of the two known isoforms of this gene was investigated. IBD patients homozygous for the Stop allele showed somewhat reduced expression of CARD8 mRNA, but, contrary to expectation, expressed a 48 kDa protein isoform. A search of the EST database and reverse transcription-PCR analysis revealed a novel coding exon and three novel CARD8 mRNA isoforms that are conserved in primates. The isoforms of CARD8 differ in their N-termini, resulting in diverse predicted molecular weights (47, 48, 51, 54 and 60 kDa) and multiple outcomes for the variant including Cys10Stop, Cys34Stop, Phe52Ile and Phe102Ile; one isoform may arise through transcription and translation initiated downstream of rs2043211 to yield a novel protein isoform of approximately 47 kDa. The multiple isoforms and differing consequences for a predicted stop codon polymorphism underline the importance of detailed analysis of the effects of proposed functional variants on gene expression.

OBJECTIVES: Genetic variants at the CARD15 and IBD5 loci are strongly associated with Crohn's disease (CD), but evidence of the effect of these variants on the clinical expression of CD is conflicting and has often been hampered by small sample sizes. We studied 630 well-characterized patients to clarify the genotype/phenotype relationship in CD. METHODS: Patients and healthy controls were genotyped for three common mutations in CARD15 and a marker of the IBD5 risk haplotype. Allele frequencies were compared between phenotypic subgroups using chi or Fisher's exact tests. Genotype/phenotype analysis was carried out using multinomial logistic regression modelling allowing for adjustment for correlated or confounding factors. RESULTS: The mean age at diagnosis was significantly lower in carriers of the CARD15 or IBD5 risk alleles. After correction for age and smoking, CARD15 mutations were strongly associated with both ileal disease (P=8.8x10) and stenotic disease (P=0.003), but the association with stenotic disease appeared to be due to a confounding effect with ileal disease. CARD15 mutations were also associated with the presence of granulomas (P=5.7x10), which remained significant after adjustment for age at diagnosis and disease location (P=0.0047). The IBD5 risk haplotype frequency was significantly elevated in cases with perianal disease (P=0.028) and axial spondyloarthropathy (P=0.012). CONCLUSION: Genetic variants at the CARD15 and IBD5 loci have diverse effects on clinical expression in CD. CARD15 mutations are significantly correlated with the presence of granulomas.

Previous work has shown that the gene DMBT1, which encodes a large secreted epithelial glycoprotein known as salivary agglutinin, gp340, hensin or muclin, is an innate immune defence protein that binds bacteria. A deletion variant of DMBT1 has been previously associated with Crohn's disease, and a DMBT1(-/-) knockout mouse has increased levels of colitis induced by dextran sulphate. DMBT1 has a complex copy number variable structure, with two, independent, rapidly mutating copy number variable regions, called CNV1 and CNV2. Because the copy number variable regions are predicted to affect the number of bacteria-binding domains, different alleles may alter host-microbe interactions in the gut. Our aim was to investigate the role of this complex variation in susceptibility to Crohn's disease by assessing the previously reported association. We analysed the association of both copy number variable regions with presence of Crohn's disease, and its severity, on three case-control cohorts. We also reanalysed array comparative genomic hybridisation data (aCGH) from a large case-control cohort study for both copy number variable regions. We found no association with a linear increase in copy number, nor when the CNV1 is regarded as presence or absence of a deletion allele. Taken together, we show that the DMBT1 CNV does not affect susceptibility to Crohn's disease, at least in Northern Europeans.European Journal of Human Genetics advance online publication, 27 January 2016; doi:10.1038/ejhg.2015.280.

Background & Aims: A genome-wide association scan of nonsynonymous DNA polymorphisms identified association of a threonine-to-alanine substitution (T300A) in the autophagy-related 16-like gene ATG16L1 with Crohn's disease. We investigated this association in independent U.K. cohorts of Crohn's disease and ulcerative colitis. Methods: The T300A variant (rs2241880) was genotyped in an independent sample of 727 Crohn's disease and 877 ulcerative colitis cases, and in 579 controls. We then performed an extension analysis combining these data with the U.K. data from the initial study to give a total of 1236 U.K. Crohn's disease cases and 1235 controls to estimate disease risk and test for interaction with the CARD15 and IBD5 risk loci and for association with disease subtypes. Results: The association of T300A was replicated in the independent sample of 727 Crohn's disease cases ( P = .001), and was strongly associated in the extended analysis of 1236 Crohn's cases ( P = 2.4 × 10 −6 ). The 300A/A genotype conferred a 1.65-fold risk of Crohn's disease, with a 2.2-fold risk of ileal disease. Analysis of the interaction of ATG16L1 with CARD15 and IBD5 indicated that all 3 loci contribute independently to disease risk. Homozygosity for the risk allele at all 3 loci conferred a combined risk of 20.4 (95% confidence interval: 8.71, 47.7) for Crohn's disease. The ATG16L1 risk genotype showed a modest but significant association with ulcerative colitis ( P = .026). Conclusions: The association of ATG16L1 with Crohn's disease and possibly with ulcerative colitis supports a role for autophagy in the pathogenesis of inflammatory bowel disease.

Background Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn’s disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage. Methods Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR. Results E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls. Conclusion In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Innate lymphoid cells (ILCs) are a family of immune cells that selectively accumulate in mucosal tissues serving as sentinels at the vanguard of host protective immunity. However, they are also implicated as cellular mediators of immune-mediated diseases, most notably IBD. ILCs are subdivided into distinct lineages based on the expression of effector cytokines and master transcription factors that programme their differentiation and inflammatory behaviour. Strikingly, these subsets closely resemble CD4(+) T-cell lineages, including T helper (TH)1, TH2 and TH17 cells that are similarly implicated in immune-mediated diseases. However, ILCs that promote the maintenance of intestinal epithelial cells, mostly through production of IL-22, also exist. ILCs rapidly respond to environmental cues, including cytokines, metabolic signals and luminal bacteria. They are potent and immediate producers of key cytokines linked to IBD pathogenesis, including TNF, IL-17, IL-22 and IFN-γ. Some subsets are implicated as mediators of chronic intestinal inflammation, whereas others might provide protective functions. They are present in the gut of patients with IBD and, intriguingly, closer scrutiny of IBD susceptibility loci shows that many of these genes are either expressed by, or are intimately linked to, ILC function. Looking forward, targeting ILCs could represent a new IBD treatment paradigm.

Background & Aims: Common germline genetic variation in the 3′ region of myosin IXB ( MYO9B ) has been associated recently with susceptibility to celiac disease, with a hypothesis that MYO9B variants might influence intestinal permeability. These findings suggested the current study investigating a possible further role for MYO9B variation in inflammatory bowel disease. Methods: Eight single-nucleotide polymorphisms (SNPs) were selected to tag common haplotypes from the 35-kb 3′ region of MYO9B . These included the strongest celiac disease–associated variants reported in a Dutch cohort. These SNPs were studied in 3 independently collected and genotyped case-control cohorts of European descent (UK, Dutch, and Canadian/Italian), comprising in total 2717 inflammatory bowel disease patients (1197 with Crohn's disease, 1520 with ulcerative colitis) and 4440 controls. Results: Common variation in MYO9B was associated with susceptibility to inflammatory bowel disease in all 3 cohorts examined (most associated SNP, rs1545620; meta-analysis P = 1.9 × 10 -6 ; odds ratio, 1.2), with the same alleles showing association as reported for celiac disease. Conclusions: MYO9B genetic variants predispose to inflammatory bowel disease. Interestingly, rs1545620 is a nonsynonymous variant leading to an amino acid change (Ala1011Ser) in the third calmodulin binding IQ domain of MYO9B. Unlike previous variants (in other genes) reported to predispose to inflammatory bowel disease, the association at MYO9B was considerably stronger with ulcerative colitis, although weaker association with Crohn's disease also was observed. These data imply shared causal mechanisms underlying intestinal inflammatory diseases.

Esophageal squamous cell carcinoma (ESCC) is common in Black populations of the sub-Saharan Africa, with high incidence regions in East and South Africa. Clinical presentation in Africa is late, and treatment is mainly palliative with a very poor prognosis. Various environmental risk factors have been identified, but the possible contribution of genetics to disease risk is an important question, which is unresolved. The aim of this project is to test the hypothesis that genetic variation in the South African Black population contributes significantly to the risk of ESCC. We have begun by testing genetic risk factors for ESCC which were identified by genome-wide association scans in other populations for association with ESCC in the South African Black population. Cases with a histologically confirmed diagnosis of ESCC and population controls were recruited, with informed consent and institutional ethical approval. All individuals were from the Black population of the Western Cape, with 98.2% being from the Xhosa ethnic group. A total of 513 ESCC cases and 820 controls were genotyped for 15 single nucleotide polymorphisms (SNPs) by validated TaqMan assays on a 7900HT (Applied Biosystems). SNPs were tested for association using an allelic chi-square test; odds ratios (OR) and 95% confidence intervals (CI) used the common allele as reference. A significance threshold of p=0.0033 was applied, from a Bonferroni multiple testing correction for 15 SNPs. No significant evidence of association was observed for the index SNPs at the previously reported loci ATP1B2, CASP8, c20orf54, HEATR3, PDE4D, PLCE1, PTPN2, RUNX1, SMG6, ST6GAL1, TMEM173 and UNC5L loci. However, the SNP rs2239815 at XBP1 on chromosome 22q12 was associated with ESCC (OR =1.41, P = 0.00087), as were 2 SNPs in CHEK2, rs4822983 (OR = 1.31, P = 0.0013) and rs1033667 (OR = 1.29, P = 0.0025). These 3 SNPs are within 70kb of each other and are in partial linkage disequilibrium in African populations. Fine-mapping of this locus will be required to determine the causal gene and variant/s driving this association. The lack of association with ESCC in the Black South African population at most of the loci previously associated in Asian GWAS suggests that at least some of these may not contribute to susceptibility to ESCC in the South African population. However, expansion of samples sizes and thus statistical power is required for more definitive exclusions, and the lower level of linkage disequilibrium (LD) across African genomes may produce a negative result if the SNP which is tagging the causal variant in Asian or European populations is in weak LD with the causal SNP in African populations. In order to address these issues we are now expanding the power and density of this study by genotyping 36 SNPs from loci of interest in 1000 South African ESCC cases and 940 controls from the Johannesburg Cancer Study by mass spectrometry using the MassArray genotyping system (Agena Bioscience). Citation Format: Wenlong Carl Chen, Hannah Bye, Marco Matejcic, Robyn Kerr, Elvira Singh, Natalie J. Prescott, Cathryn M. Lewis, Chantal Babb de Villers, Iqbal Parker, Christopher G. Mathew. The genetic etiology of esophageal cancer in South African Black populations [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A34.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a complex disorder of multigenic origin involving between two and ten loci. Linkage and association studies of CL/P have implicated a number of candidate genes and regions but have often proved difficult to replicate. Here, we report the findings from a two-stage genome-wide scan of 92 affected sib-pairs to identify susceptibility loci to CL/P. An initial set of 400 microsatellite markers was used, with an average spacing of 10 cM throughout the genome. Eleven regions on eight chromosomes were found to have a P-value smaller than 0.05. These eight chromosomes were then further mapped with a second set of markers to increase the average map density to 5 cM. In seven out of eleven areas densely mapped, significance was markedly increased by decreasing the marker interval. Excessive allele sharing was found at 1p (NPL=2.35, P=0.009, MLS=1.51), 2p (NPL=1.77, P= 0.04, MLS=0.66), 6p (NPL=2.35, P=0.009, MLS=1.34), 8q (NPL=2.15, P=0.015, MLS= 1.51) 11 cen (NPL=2.70, P=0.003, MLS=2.10), 12q (NPL=2.08, P=0.02, MLS= 1.5), 16p (NPL=2.1, P=0.018, MLS=0.97) and Xcen-q (NPL=2.40, P=0.008, MLS=2.68). Although none reached the level required for significant susceptibility loci, two of these areas have previously been implicated in CL/P, viz. 2p13, an area harbouring the TGFA gene, and 6p23-24. We also demonstrate highly suggestive linkage to a susceptibility locus for nonsyndromic clefting on the X chromosome. Further studies are currently underway to replicate these findings in a larger cohort of affected sib-pairs.

Background: Psoriasis is an immune-mediated skin disorder that is inherited as a multifactorial trait. Linkage analyses have clearly mapped a primary disease susceptibility locus to the major histocompatibility complex (MHC) region on chromosome 6p21. More recently, whole-genome association studies have identified two non-MHC disease genes ( IL12B and IL23R ), both of which also confer susceptibility to Crohn disease (CD). Objective and methods: To ascertain the genetic overlap between these two inflammatory conditions further, we investigated 15 CD-associated loci in a psoriasis case–control dataset. Results: The analysis of 1256 patients and 2938 unrelated controls found significant associations for loci mapping to chromosomes 1q24 (rs12035082, p = 0.009), 6p22 (rs6908425, p = 0.00015) and 21q22 (rs2836754, p = 0.0003). Notably, the marker showing the strongest phenotypic effect (rs6908425) maps to CDKAL1 , a gene also associated with type 2 diabetes. Conclusions: These results substantiate emerging evidence for a pleiotropic role for s genes that contribute to the pathogenesis of immune-mediated disorders.

A genome- wide association scan in individuals with Crohn's disease by the Wellcome Trust Case Control Consortium detected strong association at four novel loci. We tested 37 SNPs from these and other loci for association in an independent case- control sample. We obtained replication for the autophagy- inducing IRGM gene on chromosome 5q33.1 ( replication P = 6.6 x 10(-4), combined P = 2.1 x 10(-10)) and for nine other loci, including NKX2- 3, PTPN2 and gene deserts on chromosomes 1q and 5p13.

Crohn's disease (CD) is associated with intestinal dysbiosis, altered blood T cell populations, elevated faecal calprotectin (FC) and increased intestinal permeability (IP). CD-associated features present in siblings (increased risk of CD) but not in healthy controls, provide insight into early CD pathogenesis. We aimed to (1) Delineate the genetic, immune and microbiological profile of patients with CD, their siblings and controls and (2) Determine which factors discriminate between groups.Faecal microbiology was analysed by quantitative PCR targeting 16S ribosomal RNA, FC by ELISA, blood T cell phenotype by flow cytometry and IP by differential lactulose-rhamnose absorption in 22 patients with inactive CD, 21 of their healthy siblings and 25 controls. Subject's genotype relative risk was determined by Illumina Immuno BeadChip.Strikingly, siblings shared aspects of intestinal dysbiosis with patients with CD (lower concentrations of Faecalibacterium prausnitzii (p=0.048), Clostridia cluster IV (p=0.003) and Roseburia spp. (p=0.09) compared with controls). As in CD, siblings demonstrated a predominance of memory T cells (p=0.002) and elevated naïve CD4 T cell β7 integrin expression (p=0.01) compared with controls. FC was elevated (50 μg/g) in 8/21 (38%) siblings compared with 2/25 (8%) controls (p=0.028); whereas IP did not differ between siblings and controls. Discriminant function analysis determined that combinations of these factors significantly discriminated between groups (χ(2)=80.4, df=20, p0.001). Siblings were separated from controls by immunological and microbiological variables.Healthy siblings of patients with CD manifest immune and microbiological abnormalities associated with CD distinct from their genotype-related risk and provide an excellent model in which to investigate early CD pathogenesis.

A single-nucleotide polymorphism (C1858T) causing an amino acid substitution (R620W) in the lymphoid protein tyrosine phosphatase gene PTPN22 has been implicated in type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, juvenile idiopathic arthritis and Hashimoto's thyroiditis, thus revealing a general role for this gene in autoimmune disease. We investigated the association of the C1858T variant in an additional autoimmune disease population by performing a case-control study of 514 British individuals with inflammatory bowel disease (IBD) [294 with Crohn's disease (CD) and 220 with ulcerative colitis (UC)] and 374 normal controls. No significant differences in genotype or allele frequencies were observed between IBD, CD or UC and controls, indicating that PTPN22 does not influence risk of IBD.

Introduction Inflammatory Bowel Disease (IBD) is a complex genetic disease characterised by chronic inflammation of the gastro-intestinal tract. Genome-wide association study (GWAS) meta-analysis and replication has identified over 200 genomic regions involved in IBD susceptibility although only a few of the associated single nucleotide polymorphisms (SNPs) directly alter the coding sequence of a gene. In addition, >92 of the IBD-susceptibility loci are known to co-localise with active regulatory elements, suggesting the majority of IBD associated SNPs alter gene expression in some way. Here we employ whole transcriptome sequencing to investigate the role of altered gene expression in intestinal tissue. Methods Patients and non-IBD control individuals were recruited after informed consent through endoscopy clinics at Guy’s and St Thomas’ Hospital, London. Whole RNA was extracted from 2–4 colonic biopsies from 104 IBD patients (76 CD, 28 UC) and 24 non-IBD controls and ribo-depleted (Ribo-zero, Illumina). We performed whole RNA sequencing using Illumina TruSeq and HiSeq2500 on pools of 4 indexed RNA libraries across 1–2 lanes. Quality control of sequence reads was performed via FastQC. Subsequent alignment and assembly, quantification and differential expression (DE) analysis of each transcript was performed using Tophat2, Cufflinks, Cuffmerge and EdgeR. Results So far a detailed bioinformatics analysis has been carried out on the first 32 whole RNA-seq libraries. We have observed expression above background (FPKM > 1) for approximately 46% of all known transcripts located within known IBD susceptibility loci. DE analysis identified 249 transcripts with significant difference in expression (q Conclusion We have generated high coverage whole RNAseq data on intestinal biopsy samples from 128 individuals. We have shown Disclosure of Interest None Declared

Objective To test the hypothesis that communicating risk of developing Crohn’s disease based on genotype and that stopping smoking can reduce this risk, motivates behaviour change among smokers at familial risk. Design Parallel group, cluster randomised controlled trial. Setting Families with Crohn’s disease in the United Kingdom. Participants 497 smokers (mean age 42.6 (SD 14.4) years) who were first degree relatives of probands with Crohn’s disease, with outcomes assessed on 209/251 (based on DNA analysis) and 217/246 (standard risk assessment). Intervention Communication of risk assessment for Crohn’s disease by postal booklet based on family history of the disease and smoking status alone, or with additional DNA analysis for the NOD2 genotype. Participants were then telephoned by a National Health Service Stop Smoking counsellor to review the booklet and deliver brief standard smoking cessation intervention. Calls were tape recorded and a random subsample selected to assess fidelity to the clinical protocol. Main outcome measure The primary outcome was smoking cessation for 24 hours or longer, assessed at six months. Results The proportion of participants stopping smoking for 24 hours or longer did not differ between arms: 35% (73/209) in the DNA arm versus 36% (78/217) in the non-DNA arm (difference −1%, 95% confidence interval −10% to 8%, P=0.83). The proportion making a quit attempt within the DNA arm did not differ between those who were told they had mutations putting them at increased risk (36%), those told they had none (35%), and those in the non-DNA arm (36%). Conclusion Among relatives of patients with Crohn’s disease, feedback of DNA based risk assessments does not motivate behaviour change to reduce risk any more or less than standard risk assessment. These findings accord with those across a range of populations and behaviours. They do not support the promulgation of commercial DNA based tests nor the search for gene variants that confer increased risk of common complex diseases on the basis that they effectively motivate health related behaviour change. Trial registration Current Controlled Trials ISRCTN21633644.

Background Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. Results We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Conclusions Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion.