The genetics of non-monogenic IBD.

Inflammatory bowel disease (IBD), with Crohn's disease and ulcerative colitis as main subtypes, is a prototypical multifactorial disease with both genetic and environmental factors involved. Genetically, IBD covers a wide spectrum from monogenic to polygenic forms. In polygenic disease, many genetic variants each contribute a small amount to disease risk. With the advent of genome-wide association studies (GWAS), it became possible to find these variants and corresponding genes, leading so far to the discovery of ca 240 loci associated with IBD. Together, these however explain only 20-25% of the heritability of IBD, leaving a large portion unaccounted for. This missing heritability might be hidden in common variants with even lower effect than the ones currently found through GWAS, but also in rare variants which can be found through large-scale sequencing studies or potentially in multiplex families. In this review, we will give an overview of the current knowledge about the genetics of non-monogenic IBD and how it differs from the monogenic form(s), and future perspectives. The history of IBD genetic studies from twin studies over linkage studies to GWAS, and finally large-scale sequencing studies and the revisiting of multiplex families will be discussed.

Fine-mapping inflammatory bowel disease loci to single-variant resolution.

Inflammatory bowel diseases are chronic gastrointestinal inflammatory disorders that affect millions of people worldwide. Genome-wide association studies have identified 200 inflammatory bowel disease-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 inflammatory bowel disease loci using high-density genotyping in 67,852 individuals. We pinpoint 18 associations to a single causal variant with greater than 95% certainty, and an additional 27 associations to a single variant with greater than 50% certainty. These 45 variants are significantly enriched for protein-coding changes (n = 13), direct disruption of transcription-factor binding sites (n = 3), and tissue-specific epigenetic marks (n = 10), with the last category showing enrichment in specific immune cells among associations stronger in Crohn's disease and in gut mucosa among associations stronger in ulcerative colitis. The results of this study suggest that high-resolution fine-mapping in large samples can convert many discoveries from genome-wide association studies into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms.

Genetic contributors to risk of schizophrenia in the presence of a 22q11.2 deletion.

Schizophrenia occurs in about one in four individuals with 22q11.2 deletion syndrome (22q11.2DS). The aim of this International Brain and Behavior 22q11.2DS Consortium (IBBC) study was to identify genetic factors that contribute to schizophrenia, in addition to the ~20-fold increased risk conveyed by the 22q11.2 deletion. Using whole-genome sequencing data from 519 unrelated individuals with 22q11.2DS, we conducted genome-wide comparisons of common and rare variants between those with schizophrenia and those with no psychotic disorder at age ≥25 years. Available microarray data enabled direct comparison of polygenic risk for schizophrenia between 22q11.2DS and independent population samples with no 22q11.2 deletion, with and without schizophrenia (total n = 35,182). Polygenic risk for schizophrenia within 22q11.2DS was significantly greater for those with schizophrenia (padj = 6.73 × 10-6). Novel reciprocal case-control comparisons between the 22q11.2DS and population-based cohorts showed that polygenic risk score was significantly greater in individuals with psychotic illness, regardless of the presence of the 22q11.2 deletion. Within the 22q11.2DS cohort, results of gene-set analyses showed some support for rare variants affecting synaptic genes. No common or rare variants within the 22q11.2 deletion region were significantly associated with schizophrenia. These findings suggest that in addition to the deletion conferring a greatly increased risk to schizophrenia, the risk is higher when the 22q11.2 deletion and common polygenic risk factors that contribute to schizophrenia in the general population are both present.

Inflammatory bowel disease and Parkinson's disease: common pathophysiological links.

Inflammatory bowel disease and Parkinson's disease are chronic progressive disorders that mainly affect different organs: the gut and brain, respectively. Accumulating evidence has suggested a bidirectional link between gastrointestinal inflammation and neurodegeneration, in accordance with the concept of the 'gut-brain axis'. Moreover, recent population-based studies have shown that inflammatory bowel disease might increase the risk of Parkinson's disease. Although the precise mechanisms underlying gut-brain interactions remain elusive, some of the latest findings have begun to explain the link. Several genetic loci are shared between both disorders with a similar direction of effect on the risk of both diseases. The most interesting example is LRRK2 (leucine-rich repeat kinase 2), initially identified as a causal gene in Parkinson's disease, and recently also implicated in Crohn's disease. In this review, we highlight recent findings on the link between these seemingly unrelated diseases with shared genetic susceptibility. We discuss supporting and conflicting data obtained from epidemiological and genetic studies along with remaining questions and concerns. In addition, we discuss possible biological links including the gut-brain axis, microbiota, autoimmunity, mitochondrial function and autophagy.

Prostaglandin E2 receptor PTGER4-expressing macrophages promote intestinal epithelial barrier regeneration upon inflammation.

OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.

Effect of vedolizumab (anti-α4ß7-integrin) therapy on histological healing and mucosal gene expression in patients with UC.

OBJECTIVE: Lymphocyte recruitment to the inflamed gut is increased in UC. Inhibition of this cell trafficking by vedolizumab (VDZ) was successful in inducing and maintaining remission and in induction of endoscopic mucosal healing. There are no data on histological healing with VDZ. We studied histological changes following VDZ therapy and compared gene expression in patients with UC before and after therapy. DESIGN: Forty-one patients with UC from GEMINI I and LTS were studied before and at three time points (weeks 6/12/52) following VDZ therapy. Colonic biopsies were scored using the Geboes index and correlated with Mayo endoscopic subscore. Gene expression was analysed using Affymetrix gene arrays. RESULTS: Fifty-five per cent of patients achieving endoscopic healing (= Mayo endoscopic subscore 0-1) with VDZ at the studied time points also had histological healing (= Geboes grade 0-1). In most healers, some residual histological changes (eg, disturbed architecture and increased mononuclear cell infiltrate) were still observed, although this was less at week 52. VDZ restored expression of many inflammatory genes in patients with endoscopic healing only at week 52 and not before. In VDZ healers, the expression of many genes remained dysregulated at weeks 6/12/52 compared with controls. CONCLUSIONS: VDZ induces histological healing in >50% of patients with endoscopic healing, with maximal effect at week 52. VDZ also restored, although incompletely, the colonic expression of many immune-related genes in patients with UC achieving endoscopic healing at week 52. However, persistent histological and gene dysregulations did remain even in healers, suggesting that maintenance therapy will be necessary to control the intestinal inflammation. TRIAL REGISTRATION NUMBERS: NCT00783718 and NCT00790933; post-results.

Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.

Crohn's disease and ulcerative colitis, the two common forms of inflammatory bowel disease (IBD), affect over 2.5 million people of European ancestry, with rising prevalence in other populations. Genome-wide association studies and subsequent meta-analyses of these two diseases as separate phenotypes have implicated previously unsuspected mechanisms, such as autophagy, in their pathogenesis and showed that some IBD loci are shared with other inflammatory diseases. Here we expand on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn's disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Most loci contribute to both phenotypes, and both directional (consistently favouring one allele over the course of human history) and balancing (favouring the retention of both alleles within populations) selection effects are evident. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. Gene co-expression network analysis emphasizes this relationship, with pathways shared between host responses to mycobacteria and those predisposing to IBD.

Inherited determinants of Crohn's disease and ulcerative colitis phenotypes: a genetic association study.

BACKGROUND: Crohn's disease and ulcerative colitis are the two major forms of inflammatory bowel disease; treatment strategies have historically been determined by this binary categorisation. Genetic studies have identified 163 susceptibility loci for inflammatory bowel disease, mostly shared between Crohn's disease and ulcerative colitis. We undertook the largest genotype association study, to date, in widely used clinical subphenotypes of inflammatory bowel disease with the goal of further understanding the biological relations between diseases. METHODS: This study included patients from 49 centres in 16 countries in Europe, North America, and Australasia. We applied the Montreal classification system of inflammatory bowel disease subphenotypes to 34,819 patients (19,713 with Crohn's disease, 14,683 with ulcerative colitis) genotyped on the Immunochip array. We tested for genotype-phenotype associations across 156,154 genetic variants. We generated genetic risk scores by combining information from all known inflammatory bowel disease associations to summarise the total load of genetic risk for a particular phenotype. We used these risk scores to test the hypothesis that colonic Crohn's disease, ileal Crohn's disease, and ulcerative colitis are all genetically distinct from each other, and to attempt to identify patients with a mismatch between clinical diagnosis and genetic risk profile. FINDINGS: After quality control, the primary analysis included 29,838 patients (16,902 with Crohn's disease, 12,597 with ulcerative colitis). Three loci (NOD2, MHC, and MST1 3p21) were associated with subphenotypes of inflammatory bowel disease, mainly disease location (essentially fixed over time; median follow-up of 10·5 years). Little or no genetic association with disease behaviour (which changed dramatically over time) remained after conditioning on disease location and age at onset. The genetic risk score representing all known risk alleles for inflammatory bowel disease showed strong association with disease subphenotype (p=1·65 × 10(-78)), even after exclusion of NOD2, MHC, and 3p21 (p=9·23 × 10(-18)). Predictive models based on the genetic risk score strongly distinguished colonic from ileal Crohn's disease. Our genetic risk score could also identify a small number of patients with discrepant genetic risk profiles who were significantly more likely to have a revised diagnosis after follow-up (p=6·8 × 10(-4)). INTERPRETATION: Our data support a continuum of disorders within inflammatory bowel disease, much better explained by three groups (ileal Crohn's disease, colonic Crohn's disease, and ulcerative colitis) than by Crohn's disease and ulcerative colitis as currently defined. Disease location is an intrinsic aspect of a patient's disease, in part genetically determined, and the major driver to changes in disease behaviour over time. FUNDING: International Inflammatory Bowel Disease Genetics Consortium members funding sources (see Acknowledgments for full list).

Evolution of cytokines and inflammatory biomarkers during infliximab induction therapy and the impact of inflammatory burden on primary response in patients with Crohn's disease.

OBJECTIVE: Primary non-response to infliximab in Crohn's disease is still incompletely understood. Our aim was to further characterize the role of inflammatory burden during infliximab induction therapy. MATERIALS AND METHODS: We studied a well-characterized cohort of 201 anti-TNF naive Crohn's disease patients treated with infliximab 5mg/kg at week 0, 2, 6 and 14 who had serum samples drawn just before every infusion. All serum samples were analyzed for CRP, albumin, TNF, IFN-γ, IL-6, IL-8, IL-10, infliximab trough concentrations (in-house-developed ELISA) and antibodies to infliximab (HMSA, Prometheus Laboratories Inc., San Diego, CA). Primary non-response was defined as the absence of clinical improvement at week 14. RESULTS: The incidence of primary non-response to infliximab was 8% (n = 16). IL-8 concentrations at baseline were higher (p = .01) and albumin at week 6 was lower in primary non-responders (p = .01) compared to responders. During induction, IFN-γ and IL-6 concentrations decreased significantly at week 2 and week 6 in responders compared to primary non-responders (p < .05). Serum TNF increased significantly after each infliximab infusion and this increase from week 0 to week 14 was more pronounced in responders (p = .03). Multiple logistic regression identified TNF/CRP ratio at baseline as predictive for primary non-response to infliximab at week 14 (OR 2.8 (95% CI 1.4-5.5; p = .003)). CONCLUSIONS: In this intensively sampled cohort of Crohn's disease patients, we demonstrate that inflammatory burden is more determining for primary non-response than drug exposure or immunogenicity. Our findings furthermore suggest that the contribution of TNF in inflammation might be higher in primary non-response, contradicting the non-TNF-driven concept.

Characteristics of Skin Lesions Associated With Anti-Tumor Necrosis Factor Therapy in Patients With Inflammatory Bowel Disease: A Cohort Study.

BACKGROUND: A subgroup of patients with inflammatory bowel disease (IBD) treated with anti-tumor necrosis factor (TNF) antibodies develop skin lesions, but the lesions and their clinical course are not well-characterized. OBJECTIVE: To describe patients treated with anti-TNF antibodies who did and did not develop skin lesions. DESIGN: Retrospective cohort. SETTING: Single IBD tertiary referral center. PATIENTS: 917 consecutive patients with IBD who initiated anti-TNF therapy. MEASUREMENTS: Skin lesions, patient demographic characteristics, treatments, clinical course, and serologic and genetic markers. RESULTS: During a median follow-up of 3.5 years (interquartile range [IQR], 0.5 to 7.4 years), skin lesions associated with the use of anti-TNF therapy developed in 264 of 917 (29%) patients (psoriasiform eczema, 30.6%; eczema, 23.5%; xerosis cutis, 10.6%; palmoplantar pustulosis, 5.3%; psoriasis, 3.8%; other, 26.1%). Lesions typically developed at flexural regions, genitalia, and the scalp, especially the psoriasiform lesions. Thirty-one percent of women and 26% of men developed lesions. Median cumulative doses (2864 mg/y [IQR, 2203 to 3819 mg/y] and 2927 mg/y [IQR, 2377 to 3667 mg/y]) and trough levels (4.2 µg/mL [IQR, 2.6 to 5.8 µg/mL] and 4.0 µg/mL [IQR, 1.6 to 5.9 µg/mL]) of infliximab were similar in patients with and without lesions. All but 28 patients (11%) were successfully managed without needing to stop therapy because of lesions. LIMITATION: Retrospective nature and no matched control group of patients not receiving anti-TNF therapy. CONCLUSION: Skin lesions occur frequently in association with anti-TNF therapy but rarely require discontinuation of therapy. Close surveillance and early referral to a dedicated dermatologist are recommended. PRIMARY FUNDING SOURCE: Research Foundation Flanders (FWO), Belgium; Geconcerteerde Onderzoekacties of KU Leuven; and Janssen Biologics.

A Genetic Variation in the Neonatal Fc-Receptor Affects Anti-TNF Drug Concentrations in Inflammatory Bowel Disease.

OBJECTIVES: Ample evidence exists that Anti-tumor necrosis factor (TNF) concentrations during induction determine short and long-term outcome in inflammatory bowel disease (IBD). We investigated if a variable number of tandem repeats (VNTR) polymorphism in the neonatal Fc-receptor (FcRn), responsible for extending half-life of IgG, influences anti-TNF concentrations in patients with IBD. METHODS: Retrospective single-center study, including a cohort of 395 infliximab (IFX) naive IBD patients treated with IFX 5 mg/kg on weeks 0, 2, and 6 and a second cohort of 139 adalimumab naive patients, treated with adalimumab 160-80-40 mg on weeks 0, 2, and 4. Area under the serum anti-TNF concentration-time curve (AUC), from week 2 and 6 for IFX and week 2 and 4 for adalimumab, was used to identify factors influencing these drug concentrations. RESULTS: The VNTR2/VNTR3 genotype was associated with a 14% lower IFX AUC compared with patients homozygous for VNTR3/VNTR3 (P=0.03), although this effect became apparent only when immunogenicity (26% lower concentrations, P=9 × 10-5) was not present. Prior anti-TNF use predicted a 27% lower IFX AUC (P=0.002). Similarly, VNTR2/VNTR3 patients had a 24% predicted lower adalimumab AUC than VNTR3/VNTR3 patients (P=0.005). The combined presence of VNTR2/VNTR3 genotype, male gender, and prior IFX use predicted a 41% lower adalimumab AUC concentration (P=0.04). CONCLUSIONS: The VNTR2/3 genotype in the FcRn gene is associated with lower IFX but also lower adalimumab drug exposure during induction in patients with IBD. Previously identified pharmacokinetic modifying factors were confirmed. Identifying risk factors in patients is important as higher induction doses may be needed to ensure optimal disease outcome.

Withdrawal of immunomodulators after co-treatment does not reduce trough level of infliximab in patients with Crohn's disease.

BACKGROUND & AIMS: The addition of immunomodulators increases the efficacy of maintenance therapy with infliximab for up to 1 year in patients with Crohn's disease who have not been previously treated with immunomodulators. However, there are questions about the effect of withdrawing immunomodulator therapy from these patients. We studied the effects of treatment with infliximab and immunomodulators (co-treatment) and then immunomodulator withdrawal on long-term outcomes of patients, as well as trough levels of infliximab and formation of anti-infliximab antibodies (ATI). METHODS: In a retrospective study with the median follow-up period of 34 months (interquartile range, 19-58 months), we analyzed data from 223 patients treated for Crohn's disease between May 1999 and December 2010 at the University Hospitals, Leuven, Belgium (65 received infliximab monotherapy, 158 received infliximab and an immunomodulator). Trough levels of infliximab and levels of ATI were measured in blood samples collected from 117 patients throughout co-treatment, as well as the time of immunomodulator withdrawal and after withdrawal. RESULTS: Patients receiving co-treatment had higher trough levels of infliximab (adjusted mean increase, 1.44-fold) than those receiving infliximab monotherapy (95% confidence interval [CI], 1.07-1.92; P = .02). A smaller percentage of patients receiving co-treatment developed ATI (35 of 158, 22%) than those receiving infliximab monotherapy (25 of 65, 38%; P = .01). Among co-treated patients, levels of infliximab remained stable after immunomodulators were withdrawn (before: 3.2 µg/mL; 95% CI, 1.6-5.8 µg/mL and after: 3.7 µg/mL; 95% CI, 1.3-6.3 µg/mL; P = .70). After withdrawal of immunomodulators, 45 of 117 patients (38%) required increasing doses of infliximab, and 21 of 117 (18%) discontinued infliximab. At the time of immunomodulator withdrawal, trough levels of infliximab and C-reactive protein were most strongly associated with response to infliximab thereafter. CONCLUSIONS: In a retrospective analysis, we confirmed that withdrawal of immunomodulators after at least 6 months (median, 13 months) of co-treatment with infliximab does not reduce the trough levels of infliximab in patients with Crohn's disease. Detectable trough levels of infliximab at the time of immunomodulator withdrawal are associated with long-term response.

The genetic architecture of inflammatory bowel disease: past, present and future.

PURPOSE OF REVIEW: Genome-wide association studies (GWAS) have helped to understand the genetic basis and pathogenesis of inflammatory bowel disease (IBD). However, understanding the functional and clinical consequences of the associated alleles has not followed the same pace. In this review, we discuss how studying the genetic predisposition to IBD has increased our understanding about IBD pathogenesis and how epigenetics is becoming more and more important. We describe the potential clinical applications of genetics, and provide important challenges in the field and the future steps to be taken. RECENT FINDINGS: GWAS and meta-analyses have identified 163 loci associated with IBD, and have implicated key pathways in IBD pathogenesis. Only few of the association signals correspond to nonsynonymous coding variation with a clear effect on protein function. The majority of signals involve noncoding genetic variation, of which a large part is related to gene expression changes. More recently, expression and epigenetic studies in IBD are increasingly being reported, and have shown that many effects seem to be highly cell-type specific.Predictive genetic testing will not be for the immediate future for the majority of IBD patients. However, for the subset of patients with very-early onset IBD, several causal mutations have been found. Predictive genetic panels for these adolescents presenting with a very severe disease course, and/or families with high penetrance of disease will be of benefit. SUMMARY: Genetic, transcriptomic and epigenetic studies have offered exciting clues about IBD pathogenesis but are unlikely to provide all answers. To fully grasp the function of disease-associated genetic variants, identifying causal genes and translating this knowledge into predictive biomarkers and new treatments, we should now integrate all these disciplines.

Genetic variants in CDC42 and NXPH1 as susceptibility factors for constipation and diarrhoea predominant irritable bowel syndrome.

OBJECTIVE: The complex genetic aetiology underlying irritable bowel syndrome (IBS) needs to be assessed in large-scale genetic studies. Two independent IBS cohorts were genotyped to assess whether genetic variability in immune, neuronal and barrier integrity genes is associated with IBS. DESIGN: 384 single nucleotide polymorphisms (SNPs) covering 270 genes were genotyped in an exploratory cohort (935 IBS patients, 639 controls). 33 SNPs with Puncorrected<0.05 were validated in an independent set of 497 patients and 887 controls. Genotype distributions of single SNPs were assessed using an additive genetic model in IBS and clinical subtypes, IBS-C and IBS-D, both in individual and combined cohorts. Trait anxiety (N=614 patients, 533 controls), lifetime depression (N=654 patients, 533 controls) and mRNA expression in rectal biopsies (N=22 patients, 29 controls) were correlated with SNP genotypes. RESULTS: Two SNPs associated independently in the exploratory and validation cohort: rs17837965-CDC42 with IBS-C (ORexploratory=1.59 (1.05 to 1.76); ORvalidation=1.76 (1.03 to 3.01)) and rs2349775-NXPH1 with IBS-D (ORexploratory=1.28 (1.06 to 1.56); ORvalidation=1.42 (1.08 to 1.88)). When combining both cohorts, the association of rs2349775 withstood post hoc correction for multiple testing in the IBS-D subgroup. Additionally, three SNPs in immune-related genes (rs1464510-LPP, rs1881457-IL13, rs2104286-IL2RA), one SNP in a neuronal gene (rs2349775-NXPH1) and two SNPs in epithelial genes (rs245051-SLC26A2, rs17837965-CDC42) were weakly associated with total-IBS (Puncorrected<0.05). At the functional level, rs1881457 increased IL13 mRNA levels, whereas anxiety and depression scores did not correlate with rs2349775-NXPH1. CONCLUSIONS: Rs2349775 (NXPH1) and rs17837965 (CDC42) were associated with IBS-D and IBS-C, respectively, in two independent cohorts. Further studies are warranted to validate our findings and to determine the mechanisms underlying IBS pathophysiology.

Genetic variation in the lymphotoxin-α (LTA)/tumour necrosis factor-α (TNFα) locus as a risk factor for idiopathic achalasia.

BACKGROUND: Idiopathic achalasia is a rare motor disorder of the oesophagus characterised by neuronal loss at the lower oesophageal sphincter. Achalasia is generally accepted as a multifactorial disorder with various genetic and environmental factors being risk-associated. Since genetic factors predisposing to achalasia have been poorly documented, we assessed whether single nucleotide polymorphisms (SNPs) in genes mediating immune response and neuronal function contribute to achalasia susceptibility. METHODS: 391 SNPs covering 190 immune and 67 neuronal genes were genotyped in an exploratory cohort from Central Europe (589 achalasia patients, 794 healthy volunteers (HVs)). 24 SNPs (p<0.05) were validated in an Italian (160 achalasia patients, 278 HVs) and Spanish cohort (281 achalasia patients, 296 HVs). 16 SNPs in linkage disequilibrium (LD) with rs1799724 (r(2)>0.2) were genotyped in the exploratory cohort. Genotype distributions of patients (1030) and HVs (1368) were compared using Cochran-Armitage trend test. RESULTS: The rs1799724 SNP located between the lymphotoxin-α (LTA) and tumour necrosis factor-α (TNFα) genes was significantly associated with achalasia and withstood correction for testing multiple SNPs (p=1.17E-4, OR=1.41 (1.18 to 1.67)). SNPs in high LD with rs1799724 were associated with achalasia. Three SNPs located in myosin-5B, adrenergic receptor-ß-2 and interleukin-13 (IL13) showed nominally significant association to achalasia that was strengthened by replication. CONCLUSIONS: Our study provides evidence for rs1799724 at the LTA/TNFα locus as a susceptibility factor for idiopathic achalasia. Additional studies are needed to dissect which genetic variants in the LTA/TNFα locus are disease-causing and confirm other variants as potential susceptibility factors for achalasia.

Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease.

OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (p(FDR)=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.

Genetic factors conferring an increased susceptibility to develop Crohn's disease also influence disease phenotype: results from the IBDchip European Project.

OBJECTIVE: Through genome-wide association scans and meta-analyses thereof, over 70 genetic loci (Crohn's disease (CD) single nucleotide polymorphisms (SNPs)) are significantly associated with CD. We aimed to investigate the influence of CD-SNPs and basic patient characteristics on CD clinical course, and develop statistical models to predict CD clinical course. DESIGN: This retrospective study included 1528 patients with CD with more than 10 years of follow-up from eight European referral hospitals. CD outcomes of interest were ileal (L1), colonic (L2) and ileocolonic disease location (L3); stenosing (B2) or penetrating behaviour (B3); perianal disease; extraintestinal manifestations; and bowel resection. A complicated disease course was defined as stenosing or penetrating behaviour, perianal disease and/or bowel resection. Association between CD-SNPs or patient characteristics and specified outcomes was studied. RESULTS: Several CD-SNPs and clinical characteristics were statistically associated with outcomes of interest. The NOD2 gene was the most important genetic factor, being an independent predictive factor for ileal location (p=2.02 × 10(-06), OR=1.90), stenosing (p=3.16 × 10(-06), OR=1.82) and penetrating (p=1.26 × 10(-02), OR=1.25) CD behaviours, and need for surgery (p=2.28 × e-05, OR=1.73), and as such was also the strongest factor associated with a complicated disease course (p=6.86 × 10(-06), OR=2.96). Immunomodulator (azathioprine/6-mercaptopurine and methotrexate) use within 3 years after diagnosis led to a reduction in bowel stenoses (p=1.48 × 10(-06), OR=0.35) and surgical rate (p=1.71 × 10(-07), OR=0.34). Association between each outcome and genetic scores, created using significant SNPs in the univariate analysis, revealed large differences in the probability of developing fistulising disease (IL23R, LOC441108, PRDM1, NOD2; p=9.64e-4, HR=1.43), need for surgery (IRGM, TNFSF15, C13ORF31, NOD2; p=7.12 × 10(-03), HR=1.35), and stenosing disease (NOD2, JAK2, ATG16L1; p=3.01 × 10(-02), HR=1.29) among patients with low and high score. CONCLUSIONS: This large multicentre cohort study has found several genetic and clinical factors influencing the clinical course of CD. NOD2 and early immunomodulator use are the clinically most meaningful predictors for its clinical course.

Clinical and genetic determinants of vitamin D receptor expression in cutaneous melanoma patients.

Decrease of vitamin D receptor (VDR) expression is observed in melanocytic naevi and melanoma compared to normal skin. Little is known about factors influencing VDR expression in cutaneous melanoma (CM). We investigated the correlation of VDR expression in CM with 25-hydroxy vitamin D (25OHD) levels, demographic/clinical parameters, genetic variants of VDR and pathology of the primary tumor. Demographic/clinical parameters were recorded in 407 prospectively recruited CM patients of a multi-center controlled study (ViDMe trial). We determined VDR expression both in the nucleus and in the cytoplasm by semi-quantitative assessment in CM tissue using histochemistry in 279 patients, expressed in percentages and histoscore (H-score). Genomic DNA from 332 patients was extracted to genotype thirteen VDR single nucleotide polymorphisms (SNPs) using TaqMan. VDR expression in CM tissue from 279 patients was correlated with clinical/demographic parameters and 25OHD levels (univariable and multivariable analysis), VDR SNPs (univariable analysis) and pathology parameters of primary CM tissue (univariable analysis). Cytoplasmic VDR expression was increased in patients who stated to have a high sun exposure during their life compared to patients with low sun exposure (p H-score,univariable : 0.001, p H-score,multivariable : 0.004). The A allele of the genetic VDR polymorphism Fok1 was associated with a higher expression of the VDR in the cytoplasm (p cytoplasmic, univariable : 0.001 and p H-score, univariable : 0.02). In the primary tumor, presence of mitosis (p nucleus,%, univariable : 0.002) and perineural invasion (p nucleus,%,univariable : 0.03) were significantly associated with low nuclear VDR expression. ClinicalTrials.gov Identifier: NCT01748448.

Plasma pTau181 and pTau217 predict asymptomatic amyloid accumulation equally well as amyloid PET.

The dynamic phase of preclinical Alzheimer's disease, as characterized by accumulating cortical amyloid-ß, is a window of opportunity for amyloid-ß-lowering therapies to have greater efficacy. Biomarkers that accurately predict amyloid-ß accumulation may be of critical importance for participant inclusion in secondary prevention trials and thus enhance development of early Alzheimer's disease therapies. We compared the abilities of baseline plasma pTau181, pTau217 and amyloid-ß PET load to predict future amyloid-ß accumulation in asymptomatic elderly. In this longitudinal cohort study, baseline plasma pTau181 and pTau217 were quantified using single molecule array assays in cognitively unimpaired elderly selected from the community-recruited F-PACK cohort based on the availability of baseline plasma samples and longitudinal amyloid-ß PET data (median time interval = 5 years, range 2-10 years). The predictive abilities of pTau181, pTau217 and PET-based amyloid-ß measures for PET-based amyloid-ß accumulation were investigated using receiver operating characteristic analyses, correlations and stepwise regression analyses. We included 75 F-PACK subjects (mean age = 70 years, 48% female), of which 16 were classified as amyloid-ß accumulators [median (interquartile range) Centiloid rate of change = 3.42 (1.60) Centiloids/year). Plasma pTau181 [area under the curve (95% confidence interval) = 0.72 (0.59-0.86)] distinguished amyloid-ß accumulators from non-accumulators with similar accuracy as pTau217 [area under the curve (95% confidence interval) = 0.75 (0.62-0.88) and amyloid-ß PET [area under the curve (95% confidence interval) = 0.72 (0.56-0.87)]. Plasma pTau181 and pTau217 strongly correlated with each other (r = 0.93, Pfalse discovery rate < 0.001) and, together with amyloid-ß PET, similarly correlated with amyloid-ß rate of change (r pTau181 = 0.33, r pTau217 = 0.36, r amyloid-ß PET = 0.35, all Pfalse discovery rate ≤ 0.01). Addition of plasma pTau181, plasma pTau217 or amyloid-ß PET to a linear demographic model including age, sex and APOE-ε4 carriership similarly improved the prediction of amyloid-ß accumulation (ΔAkaike information criterion ≤ 4.1). In a multimodal biomarker model including all three biomarkers, each biomarker lost their individual predictive ability. These findings indicate that plasma pTau181, plasma pTau217 and amyloid-ß PET convey overlapping information and therefore predict the dynamic phase of asymptomatic amyloid-ß accumulation with comparable performances. In clinical trial recruitment, confirmatory PET scans following blood-based prescreening might thus not provide additional value for detecting participants in these early disease stages who are destined to accumulate cortical amyloid-ß. Given the moderate performances, future studies should investigate whether integrating plasma pTau species with other factors can improve performance and thus enhance clinical and research utility.