Association of a functional variant in the Wnt co-receptor LRP6 with early onset ileal Crohn's disease.

Ileal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD-associated genes including NOD2, ATG16L1, and recently the ß-catenin-dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort (Oxford: n = 1,893) and prospectively tested 2 additional European sample sets (Leuven: n = 688, Vienna: n = 1,628). We revealed an association of a non-synonymous SNP (rs2302685; Ile1062Val) with early onset ileal CD (OR 1.8; p = 0.00034; for homozygous carriers: OR 4.1; p = 0.00004) and additionally with penetrating ileal CD behaviour (OR 1.3; p = 0.00917). In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals (15 controls, 32 ileal, and 12 exclusively colonic CD), we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent pathophysiological hallmarks in small intestinal inflammation and should therefore be considered as interesting targets for new therapeutic approaches.

Innate antimicrobial host defense in small intestinal Crohn's disease.

Paneth cells (PCs) are specialized epithelial cells predominantly found in the small intestinal crypts of Lieberkuehn. They produce different broad spectrum antimicrobial peptides most abundantly the alpha-defensins HD-5 and -6 (DEFA5 und DEFA6). Both these PC products show a specific reduction in small intestinal Crohn's disease (CD) - a form of inflammatory bowel disease (IBD). Their decrease is independent of current inflammation and an association with a NOD2 frameshift mutation has been demonstrated. More recently, another independent and even more frequent mechanism has been found which is linked to diminished levels of the Wnt pathway transcription factor TCF7L2 (also known as TCF4). Besides regulating the expression of HD-5 and HD-6 as TCF4 target genes, the Wnt pathway also orchestrates Paneth cell differentiation and maturation and controls stem cell maintenance in the small intestine. Besides NOD2 (which is predominantly expressed in PC) and ATG16L1 (inter alia important in the exocytosis of PC products), TCF4 is the third gene which is associated with small intestinal CD and Paneth cell antimicrobial function. Thus, Paneth cells seem to be key player emphazising a paramount importance of antimicrobial host defense in small intestinal CD pathogenesis.

Genetic variants of Wnt transcription factor TCF-4 (TCF7L2) putative promoter region are associated with small intestinal Crohn's disease.

Reduced expression of Paneth cell antimicrobial alpha-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathway transcription factor, orchestrates Paneth cell differentiation, directly regulates the expression of HD-5 and -6, and was previously associated with the decrease of these antimicrobial peptides in a subset of ileal CD. To investigate a potential genetic association of TCF-4 with ileal CD, we sequenced 2.1 kb of the 5' flanking region of TCF-4 in a small group of ileal CD patients and controls (n = 10 each). We identified eight single nucleotide polymorphisms (SNPs), of which three (rs3814570, rs10885394, rs10885395) were in linkage disequilibrium and found more frequently in patients; one (rs3814570) was thereby located in a predicted regulatory region. We carried out high-throughput analysis of this SNP in three cohorts of inflammatory bowel disease (IBD) patients and controls. Overall 1399 healthy individuals, 785 ulcerative colitis (UC) patients, 225 CD patients with colonic disease only and 784 CD patients with ileal involvement were used to determine frequency distributions. We found an association of rs3814570 with ileal CD but neither with colonic CD or UC, in a combined analysis (allele positivity: OR 1.27, 95% CI 1.07 to 1.52, p = 0.00737), which was the strongest in ileal CD patients with stricturing behaviour (allele frequency: OR 1.32, 95% CI 1.08 to1.62, p = 0.00686) or an additional involvement of the upper GIT (allele frequency: OR 1.38, 95% CI 1.03 to1.84, p = 0.02882). The newly identified genetic association of TCF-4 with ileal CD provides evidence that the decrease in Paneth cell alpha-defensins is a primary factor in disease pathogenesis.