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 β-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

Increased activity of β-catenin–dependent Wnt via overexpression of LRP6 transactivates human α-defensin 5 promoter activity in HEK-293 cells.

<p>As demonstrated with immunohistochemistry staining A), the Wnt co-receptor LRP6 is widely expressed in the cells of the small intestinal epithelium and also found at the base of the crypts where Paneth and stem cells are located. Additionally, LRP6 is sporadically found in infiltrating immune cells. As previously demonstrated, overexpression of LRP6 in HEK293 cells leads to an activation of β-catenin dependent Wnt signalling B) as monitored by TopFlash activity. The transcriptional activity of a luciferase reporter under the control of an 1 kb HD-5 promoter was also increased after LRP6 mediated activation of Wnt C). Both effects were not seen using a non-functional version of the LRP6 Co-receptor which is lacking cytoplasmatic parts that are necessary for signal transduction. Values are the average of triple determinations with the SEM indicated by error bars.</p

Age of included individuals.

<p>Provided are the mean age ± standard deviation. For controls we listed the age at time of DNA sampling, for CD patients we provide the mean age at onset.</p

Risk for rs2302685 C-allele carriers in the different disease subgroups and the influence of detailed phenotyping.

<p>A) Odds ratios and confidence intervals for the different comparisons are shown. The frequency distribution of the minor rs2302685 allele was analysed in different cohorts and combined samples: odds ratios and 95% confidence interval for the allele frequency are shown for patients with specific disease subtypes of ileal CD (classified as either L1 (solely ileal) or L3 (ileal and colonic involvement)) compared to healthy control individuals. The early onset as well as the penetrating behaviour subgroup displays an significantly increased Minor allele frequencies (MAF). B) An especially high risk for early onset ileal CD is found for homozygous minor C - allele carriers. This becomes apparent in the high odds ratios for such individuals in the different cohorts and combined samples (left panel) and an highly increased CC genotype frequency compared to the control samples and other disease groups (right panel). C) The MAF distribution was analysed in different cohorts and combined samples: odds ratios and 95% confidence interval for the MAF are shown for patients with ulcerative colitis (UC) (left panel) and CD patients with solely colonic involvement (right panel) compared to healthy control individuals. The lack of an increase in the odds ratios for the different comparisons allows excluding major effects of the variant in the colonic IBD subgroups.</p

Known exonic SNPs in LRP6.

<p>(A) LRP6 gene card with locations of exonic SNPs published in the NCBI SNPdb. Variants which lead to an amino acid exchange are marked in red. A deletion mutation causing a different protein chain is marked in blue, silent variants are marked in green. (B) Linkage disequilibria (LD) of the exonic SNPs in CD patients of the Oxford cohort. 7 of the genotyped SNPs were not present. We conclude that there is no significant LD between the sole coding variant (rs2302685) and any of the synonymous SNPs.</p

Correlation of HD-5 and LRP6.

<p>Linear regression and Spearman rank analysis were used on all samples from the mRNA analysis. The samples were grouped according to A) their disease state (diseased mucosa or non-inflamed mucosa of patients) as well as in all samples B) according to the LRP6 genotype. Interestingly the two factors exhibit a correlating pattern in controls (independent of the genotype) as well as in all wild type samples but not in the C-allele carrying individuals.</p

Overall statistical analysis in controls versus early onset ileal CD.

<p>The genotypes for which the risk was calculated are marked in bold. The Bonferroni adjusted p-values were calculated with regards to all comparisons in the overall sample group (adjusted for 11 tests).</p

LRP6 rs2302685 frequency distribution in the separate cohort samples (Oxford, Leuven, Vienna).

<p>The different distribution of genotypes is demonstrated for each group and subgroup: controls, ulcerative colitis (UC), Crohn's disease (CD) with solely colonic involvement (colonic CD; L2) and CD with solely ileal as well as ileal and colonic involvement (ileal CD; L1+L3). The ileal CD group was further sub-grouped according to gender, disease behaviour (inflammatory B1; stricturing B2; penetrating B3) and age at diagnosis (older than 17: >17; 17 and younger: </p

HD-6 but not lysozyme is reduced in patients who carry the rare LRP6 variant.

<p>Linear regression and Spearman rank analysis were used on all samples from the mRNA analysis according to the LRP6 genotype. (A) The two Paneth cell α-defensins correlate in both subgroups fitting the also found genotype dependent decrease of HD-6 in ileal CD patients. This, as well as the independence from inflammation matches the pattern seen for HD-5. (B) Lysozyme on the other hand does not correlate with HD-5 and seems to be uninfluenced by the LRP6 genotype in ileal CD.</p