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

Koslowski MJ, Teltschik Z, Beisner J, Schaeffeler E, Wang G, Kübler I, Gersemann M, Cooney R, Jewell D, Reinisch W, Vermeire S, Rutgeerts P, Schwab M, Stange EF, Wehkamp J - PLoS Genet. (2012)

Bottom Line: 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.In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection.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.

View Article: PubMed Central - PubMed

Affiliation: Dr. Margarete-Fischer-Bosch Institute for Clinical Pharmacology and University of Tübingen, Stuttgart, Germany.

ABSTRACT
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.

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Risk for rs2302685 C-allele carriers in the different disease subgroups and the influence of detailed phenotyping.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.
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pgen-1002523-g002: Risk for rs2302685 C-allele carriers in the different disease subgroups and the influence of detailed phenotyping.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.

Mentions: After association of rs2302685 with early onset ileal CD in the Oxford patients, we prospectively tested if a higher frequency of this functional variant can also be found in other cohorts. Consistent with the first analysis in the Oxford cohort (Table 1, Oxford), subsequent analysis of two large sample sets (Table 1, Leuven and Vienna) showed the same overall result, whereas the frequency distributions among the control groups as well as the not further sub-grouped patients (IBD, CD, UC) were strikingly similar (MAFs between 18.47 and 20.59%, Table 1 and Table 2). Combining all tested samples, an association with early onset ileal CD (diagnosis at ages 17 and younger) (MAF: 29.57%; OR 1.797, 95% CI 1.298 to 2.486, p = 0.00034) and penetrating behaviour (internal fistulae; Montreal classification B3) (MAF: 23.24%; OR 1.296, 95% CI 1.066 to 1.575, p = 0.00917) suggests that Ile1062Val may influence both, disease onset and severity (Table 2, Figure 2), even though statistical significance of the latter association was lost after adjusting for multiple testing (Bonferroni adjustment for penetrating ileal CD behaviour: p = 0.10087). Gender on the other hand had no impact on the allele distribution (Table 2). The homozygous genotype of the minor allele displayed the highest risk for early onset ileal disease underlining a potential dose effect of the mutation (homozygous minor allele carriers: controls: 3,33%, early onset ileal CD: 10.75%; OR 4.093, 95% CI 1.981 to 8.455, p = 0.00004). Amongst the 237 analysed patients with exclusive colonic CD (L2) only 19 had a disease onset prior to age 18. None of these were homozygous for the risk variant and with a MAF of 13,64%, the SNP distribution showed no significant difference to controls (OR 0.803, 95% CI 0.334 to 1.926, p = 0.62172). We also compared early versus late onset in ileal CD patients and found a similar result as in the comparison with healthy controls (allele frequency: OR 1.760, 95% CI. 1.251 to 2.477, p = 0.00106; homozygous carriers: OR 4.484, 95% CI 1.995–10.077, p = 0.00009). The mean age of onset was similar between the CD patients in the different cohorts, as was the mean age of controls at the time of blood sampling for later DNA extraction (Table 3). In addition to testing for allele frequency differences and the increased risk of homozygous carriers, we also used additive, recessive and dominant models of inheritance to compare the genotype distribution between controls and early onset ileal CD as presented in Table 4.


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

Koslowski MJ, Teltschik Z, Beisner J, Schaeffeler E, Wang G, Kübler I, Gersemann M, Cooney R, Jewell D, Reinisch W, Vermeire S, Rutgeerts P, Schwab M, Stange EF, Wehkamp J - PLoS Genet. (2012)

Risk for rs2302685 C-allele carriers in the different disease subgroups and the influence of detailed phenotyping.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285585&req=5

pgen-1002523-g002: Risk for rs2302685 C-allele carriers in the different disease subgroups and the influence of detailed phenotyping.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.
Mentions: After association of rs2302685 with early onset ileal CD in the Oxford patients, we prospectively tested if a higher frequency of this functional variant can also be found in other cohorts. Consistent with the first analysis in the Oxford cohort (Table 1, Oxford), subsequent analysis of two large sample sets (Table 1, Leuven and Vienna) showed the same overall result, whereas the frequency distributions among the control groups as well as the not further sub-grouped patients (IBD, CD, UC) were strikingly similar (MAFs between 18.47 and 20.59%, Table 1 and Table 2). Combining all tested samples, an association with early onset ileal CD (diagnosis at ages 17 and younger) (MAF: 29.57%; OR 1.797, 95% CI 1.298 to 2.486, p = 0.00034) and penetrating behaviour (internal fistulae; Montreal classification B3) (MAF: 23.24%; OR 1.296, 95% CI 1.066 to 1.575, p = 0.00917) suggests that Ile1062Val may influence both, disease onset and severity (Table 2, Figure 2), even though statistical significance of the latter association was lost after adjusting for multiple testing (Bonferroni adjustment for penetrating ileal CD behaviour: p = 0.10087). Gender on the other hand had no impact on the allele distribution (Table 2). The homozygous genotype of the minor allele displayed the highest risk for early onset ileal disease underlining a potential dose effect of the mutation (homozygous minor allele carriers: controls: 3,33%, early onset ileal CD: 10.75%; OR 4.093, 95% CI 1.981 to 8.455, p = 0.00004). Amongst the 237 analysed patients with exclusive colonic CD (L2) only 19 had a disease onset prior to age 18. None of these were homozygous for the risk variant and with a MAF of 13,64%, the SNP distribution showed no significant difference to controls (OR 0.803, 95% CI 0.334 to 1.926, p = 0.62172). We also compared early versus late onset in ileal CD patients and found a similar result as in the comparison with healthy controls (allele frequency: OR 1.760, 95% CI. 1.251 to 2.477, p = 0.00106; homozygous carriers: OR 4.484, 95% CI 1.995–10.077, p = 0.00009). The mean age of onset was similar between the CD patients in the different cohorts, as was the mean age of controls at the time of blood sampling for later DNA extraction (Table 3). In addition to testing for allele frequency differences and the increased risk of homozygous carriers, we also used additive, recessive and dominant models of inheritance to compare the genotype distribution between controls and early onset ileal CD as presented in Table 4.

Bottom Line: 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.In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection.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.

View Article: PubMed Central - PubMed

Affiliation: Dr. Margarete-Fischer-Bosch Institute for Clinical Pharmacology and University of Tübingen, Stuttgart, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus