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Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

Diegelmann J, Czamara D, Le Bras E, Zimmermann E, Olszak T, Bedynek A, Göke B, Franke A, Glas J, Brand S - PLoS ONE (2013)

Bottom Line: Several DMBT1 SNPs were associated with CD susceptibility.All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)).Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine II - Grosshadern, Ludwig-Maximilians-University (LMU), Munich, Germany ; Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University (LMU), Munich, Germany.

ABSTRACT

Objectives: DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression.

Methods: Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays.

Results: IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7), OR 1.42; 95% CI 1.24-1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

Conclusion: We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.

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The CD risk allele of DMBT1 SNP rs2981804 is associated with lower colonic DMBT1 expression.(A) Colonic DMBT1 mRNA expression was determined by quantitative PCR and was normalized to β-actin expression in the respective samples (n = 75 biopsies from 27 CD patients). There was a trend towards higher DMBT1 expression in carriers of the G allele (GA+GG) in comparison to AA carriers (p = 0.06). Each dot represents one biopsy and the black horizontal bar is the median of each group. (B) When biopsies were subdivided according to inflammation status, homozygous carriers of the AA risk allele of SNP rs2981804 had lower basal and inflammation-induced DMBT1 expression levels in comparison to carriers of the G allele (p = 0.03 AA vs. GA+GG). (C) DMBT1 mRNA expression is independent of NOD2 genotype status. DMBT1 mRNA expression was determined in biopsies from CD patients irrespective of NOD2 genotype (n = 75; black bars) or excluding biopsies from NOD2 SNP carriers (n = 45 NOD2 wild-type carriers; grey bars). Data are presented as median DMBT1 expression in the respective groups. There were no significant differences in DMBT1 expression between all biopsies and NOD2 wild-type carriers for the different DMBT1 rs2981804 genotypes. n.s. = not significant; ‡: no standard deviation is given for GG NOD2 wild-type carriers as this group comprised only one biopsy.
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pone-0077773-g007: The CD risk allele of DMBT1 SNP rs2981804 is associated with lower colonic DMBT1 expression.(A) Colonic DMBT1 mRNA expression was determined by quantitative PCR and was normalized to β-actin expression in the respective samples (n = 75 biopsies from 27 CD patients). There was a trend towards higher DMBT1 expression in carriers of the G allele (GA+GG) in comparison to AA carriers (p = 0.06). Each dot represents one biopsy and the black horizontal bar is the median of each group. (B) When biopsies were subdivided according to inflammation status, homozygous carriers of the AA risk allele of SNP rs2981804 had lower basal and inflammation-induced DMBT1 expression levels in comparison to carriers of the G allele (p = 0.03 AA vs. GA+GG). (C) DMBT1 mRNA expression is independent of NOD2 genotype status. DMBT1 mRNA expression was determined in biopsies from CD patients irrespective of NOD2 genotype (n = 75; black bars) or excluding biopsies from NOD2 SNP carriers (n = 45 NOD2 wild-type carriers; grey bars). Data are presented as median DMBT1 expression in the respective groups. There were no significant differences in DMBT1 expression between all biopsies and NOD2 wild-type carriers for the different DMBT1 rs2981804 genotypes. n.s. = not significant; ‡: no standard deviation is given for GG NOD2 wild-type carriers as this group comprised only one biopsy.

Mentions: To determine whether rs2981804 is associated with differential DMBT1 expression in CD patients, DMBT1 mRNA expression levels measured in human intestinal biopsies (Fig. 3B) were subdivided according to the rs2981804 genotype [AA: n = 11 patients, 36 biopsies, 21 not inflamed, 15 inflamed; GA: n = 14 patients, 37 biopsies, 22 not inflamed, 15 inflamed; GG: n = 2 patients, 2 biopsies (both not inflamed)]. Overall, a trend towards higher DMBT1 expression in carriers of the protective G allele was observed (Fig. 7A, p = 0.06, AA vs. GA+GG). An inflammation-induced increase in DMBT1 expression was observed for both rs2981804 AA and GA carriers (Fig. 7B). However, basal DMBT1 expression was lower in homozygous carriers of the IBD risk allele (AA) than in GA and GG carriers (p = 0.052 AA vs. GA, p = 0.03 AA vs. GA+GG) (Fig. 7B). Similar results were obtained when NOD2 SNP carriers (see table 2) were excluded from analysis (Fig. 7C).


Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

Diegelmann J, Czamara D, Le Bras E, Zimmermann E, Olszak T, Bedynek A, Göke B, Franke A, Glas J, Brand S - PLoS ONE (2013)

The CD risk allele of DMBT1 SNP rs2981804 is associated with lower colonic DMBT1 expression.(A) Colonic DMBT1 mRNA expression was determined by quantitative PCR and was normalized to β-actin expression in the respective samples (n = 75 biopsies from 27 CD patients). There was a trend towards higher DMBT1 expression in carriers of the G allele (GA+GG) in comparison to AA carriers (p = 0.06). Each dot represents one biopsy and the black horizontal bar is the median of each group. (B) When biopsies were subdivided according to inflammation status, homozygous carriers of the AA risk allele of SNP rs2981804 had lower basal and inflammation-induced DMBT1 expression levels in comparison to carriers of the G allele (p = 0.03 AA vs. GA+GG). (C) DMBT1 mRNA expression is independent of NOD2 genotype status. DMBT1 mRNA expression was determined in biopsies from CD patients irrespective of NOD2 genotype (n = 75; black bars) or excluding biopsies from NOD2 SNP carriers (n = 45 NOD2 wild-type carriers; grey bars). Data are presented as median DMBT1 expression in the respective groups. There were no significant differences in DMBT1 expression between all biopsies and NOD2 wild-type carriers for the different DMBT1 rs2981804 genotypes. n.s. = not significant; ‡: no standard deviation is given for GG NOD2 wild-type carriers as this group comprised only one biopsy.
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pone-0077773-g007: The CD risk allele of DMBT1 SNP rs2981804 is associated with lower colonic DMBT1 expression.(A) Colonic DMBT1 mRNA expression was determined by quantitative PCR and was normalized to β-actin expression in the respective samples (n = 75 biopsies from 27 CD patients). There was a trend towards higher DMBT1 expression in carriers of the G allele (GA+GG) in comparison to AA carriers (p = 0.06). Each dot represents one biopsy and the black horizontal bar is the median of each group. (B) When biopsies were subdivided according to inflammation status, homozygous carriers of the AA risk allele of SNP rs2981804 had lower basal and inflammation-induced DMBT1 expression levels in comparison to carriers of the G allele (p = 0.03 AA vs. GA+GG). (C) DMBT1 mRNA expression is independent of NOD2 genotype status. DMBT1 mRNA expression was determined in biopsies from CD patients irrespective of NOD2 genotype (n = 75; black bars) or excluding biopsies from NOD2 SNP carriers (n = 45 NOD2 wild-type carriers; grey bars). Data are presented as median DMBT1 expression in the respective groups. There were no significant differences in DMBT1 expression between all biopsies and NOD2 wild-type carriers for the different DMBT1 rs2981804 genotypes. n.s. = not significant; ‡: no standard deviation is given for GG NOD2 wild-type carriers as this group comprised only one biopsy.
Mentions: To determine whether rs2981804 is associated with differential DMBT1 expression in CD patients, DMBT1 mRNA expression levels measured in human intestinal biopsies (Fig. 3B) were subdivided according to the rs2981804 genotype [AA: n = 11 patients, 36 biopsies, 21 not inflamed, 15 inflamed; GA: n = 14 patients, 37 biopsies, 22 not inflamed, 15 inflamed; GG: n = 2 patients, 2 biopsies (both not inflamed)]. Overall, a trend towards higher DMBT1 expression in carriers of the protective G allele was observed (Fig. 7A, p = 0.06, AA vs. GA+GG). An inflammation-induced increase in DMBT1 expression was observed for both rs2981804 AA and GA carriers (Fig. 7B). However, basal DMBT1 expression was lower in homozygous carriers of the IBD risk allele (AA) than in GA and GG carriers (p = 0.052 AA vs. GA, p = 0.03 AA vs. GA+GG) (Fig. 7B). Similar results were obtained when NOD2 SNP carriers (see table 2) were excluded from analysis (Fig. 7C).

Bottom Line: Several DMBT1 SNPs were associated with CD susceptibility.All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)).Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine II - Grosshadern, Ludwig-Maximilians-University (LMU), Munich, Germany ; Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University (LMU), Munich, Germany.

ABSTRACT

Objectives: DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression.

Methods: Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays.

Results: IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7), OR 1.42; 95% CI 1.24-1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

Conclusion: We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.

Show MeSH
Related in: MedlinePlus