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

Genetic Variants of Wnt Transcription Factor TCF-4
(TCF7L2) Putative Promoter Region Are Associated with
Small Intestinal Crohn’s Disease
Maureen J. Koslowski1, Irmgard Ku¨bler1,3, Mathias Chamaillard2, Elke Schaeffeler1, Walter Reinisch4,
Guoxing Wang1, Julia Beisner1, Alexander Teml1, Laurent Peyrin-Biroulet5, Stefan Winter1,6, Klaus R.
Herrlinger3, Paul Rutgeerts7, Se´verine Vermeire7, Rachel Cooney8, Klaus Fellermann3, Derek Jewell8,
Charles L. Bevins9, Matthias Schwab1,10, Eduard F. Stange3, Jan Wehkamp1,3*
1Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tu¨bingen, Stuttgart, Germany, 2 Inserm, U801, Institut Pasteure Lille, Universite´ Lille 2, Lille,
France, 3Department of Internal Medicine I, Robert Bosch Hospital, Stuttgart, Germany, 4Department of Internal Medicine IV, Medical University of Vienna, Vienna,
Austria, 5 Service d’He´pato-gastro-ente´rologie, CHU Nancy-Brabois alle´e du Morvan, Vandceuvre-le`s-Nancy, France, 6Division of Mathematics, Institute of Stochastics and
Applications, University of Stuttgart, Stuttgart, Germany, 7Division of Gastroenterology, University of Leuven Hospitals, Leuven, Belgium, 8Medical Science Division, John
Radcliffe Hospital, Oxford, Oxford, United Kingdom, 9Department of Microbiology and Immunology, School of Medicine, University of California Davis, Davis, California,
United States of America, 10Department of Clinical Pharmacology, University of Tu¨bingen, Tu¨bingen, Germany
Abstract
Reduced expression of Paneth cell antimicrobial a-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 59 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 a-defensins is a primary factor in disease pathogenesis.
Citation: Koslowski MJ, Ku¨bler I, Chamaillard M, Schaeffeler E, Reinisch W, et al. (2009) Genetic Variants of Wnt Transcription Factor TCF-4 (TCF7L2) Putative
Promoter Region Are Associated with Small Intestinal Crohn’s Disease. PLoS ONE 4(2): e4496. doi:10.1371/journal.pone.0004496
Editor: Amanda Ewart Toland, Ohio State University Medical Center, United States of America
Received June 19, 2008; Accepted December 5, 2008; Published February 16, 2009
Copyright: � 2009 Koslowski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: JW is an Emmy Noether scholar of Deutsche Forschungsgemeinschaft (DFG) who supported this work (WE 436/1-1). This work was also supported by
the Robert Bosch Foundation (Stuttgart, Germany), the Ernst Jung Foundation (Hamburg, Germany), the National Institute of Health (AI32738 and AI50843), as
well as the DCCV (German patient organization of Crohn’s disease and ulcerative colitis). S. V and P. R. are supported by the Funds for Scientific Research (FWO),
Flandres, Belgium.
Competing Interests: MJK, JB, EFS and JW have a pending patent application regarding TCF-4 SNP detection.
* E-mail: jan.wehkamp@ikp-stuttgart.de
Introduction
Inflammatory bowel disease (IBD), a chronic inflammation of
the intestine, is commonly classified into ulcerative colitis (UC) and
Crohn’s disease (CD) on the basis of clinical features and
histopathology [1]. Whereas UC is typically restricted to the
colon, CD can occur at many sites, predominantly in the small
intestinal ileum, the colon, or in both locations. Emerging details
of disease pathogenesis support the current concept that ongoing
immune activation in IBD is driven by bacterial microbiota,
possibly as a result to an attenuated antimicrobial barrier in
genetically predisposed individuals [1–3]. Both UC and CD have a
complex polygenic, multifactorial background, with a coincidence
of susceptibility genes and environmental factors involved in
pathogenesis. It is likely that different genetically affected factors
may explain the various clinical patterns of IBD, especially
location of disease in CD, which is stable over time [4–6].
Different explanations for disease location, including a central role
of small intestinal Paneth cells and other defects in intestinal innate
immunity, were the focus of recent discussion [2]. For ileal CD,
reduced expression of small intestinal Paneth cell a-defensins HD-
5 and -6 (DEFA5 and DEFA6) has been described in several
cohorts [7–12]. The defensin deficiency is proposed to attenuate
the antibacterial host defense capacity of the intestinal mucosa,
and may initiate and/or perpetuate the chronic inflammation
characterizing the disease at this site [7–12]. We recently reported
PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4496

one mechanism to explain, in part, the decrease of these
antimicrobial peptides [9,13]: A reduced expression of the Wnt
pathway transcription factor TCF-4 (also known as transcription
factor 7-like 2), which directly controls Paneth cell defensin
expression (HD-5, HD-6, and orthologous mouse cryptdin
peptides [9,13]).
Wnt proteins are a family of secreted morphogenes that play an
important role in regulating cell fate and differentiation during
embryogenesis [14]. The Wnt signalling pathway is induced by
binding of Wnt family proteins to cell surface receptors, leading to
stabilization of cytoplasmatic b-catenin, translocation of this
regulatory protein into the nucleus, formation of a complex with
transcription factors of the Tcf/Lef family and subsequently the
activation of various target genes [13]. In the small intestine, epithelial
cells transit through differentiation steps initiated in progenitor cells,
which reside adjacent to Paneth cells at the base of the crypts [15].
Wnt signalling helps to maintain an undifferentiated state of the
intestinal stem cells [16,17] and, paradoxically, also regulates
positioning, differentiation and maturation of Paneth cells [13,18].
The Paneth cell gene program is critically dependent on TCF-4 [13].
Using a rodent model, we observed that very small changes (a 50%
decrease of TCF-4 levels) are sufficient to compromise mouse Paneth
cell cryptidin expression as well as its corresponding antimicrobial
function against several bacterial species. We also reported that a
reduced level of TCF-4 expression and activity was associated with a
decrease of Paneth cell a-defensin levels in CD of the small intestine.
The decrease of TCF-4 expression was found to be independent of
inflammation in the tissue specimens, and also independent of the
1007fsinsC SNP in NOD2, a mutation in this pattern recognition
receptor which has previously been associated with ileal CD [9]. We
hypothesized that decreased TCF-4 expression might be the result of
primary genetic variances in TCF-4, at least in some patients with
ileal CD. Since there was a decrease in TCF-4 mRNA levels in these
studies, an aberration in the promoter region of TCF-4 could be a
possible explanation. Thus, the aim of this study was to sequence the
promoter region of the TCF-4 gene in a group of patients with ileal
CD to identify potential polymorphisms and to perform a subsequent
association study on candidate genetic variants in well-defined
cohorts of patients. We identified a total of 8 SNP variants, of which
three (rs3814570, rs10885394, rs10885395) were in linkage disequi-
librium and seemed to exhibit a higher frequency in ileal CD patients.
One of these SNPs was found to be located in a putative regulatory
region. We carried out high- throughput analysis of this SNP in three
IBD cohorts from Oxford, Leuven and Vienna [19–21]. Herein we
report an association of the SNP rs3814570 with ileal involvement of
CD, but not with colonic CD or UC.
Methods
Patients and human material
For genetic analysis, we obtained DNA samples from a patient
cohort of Caucasians with Crohn’s disease (N= 259) or ulcerative
colitis (N=149) from the University Hospital in Vienna, as well as
a control group of unrelated, healthy Caucasian blood donors in
Stuttgart (N= 833). For subsequent testing, we obtained DNA
samples from Caucasians with Crohn’s disease (N= 277), UC
(N=74) and healthy controls (N= 242) from the University of
Leuven, Belgium (3) as well as an additional third Caucasian
cohort from Oxford with DNA of Caucasian healthy individuals
(N= 324), UC (N=562) and CD (N=473) patients. In line with
the Montreal classification (4) three subgroups were defined: ileal
disease only (L1), colonic disease only (L2) and ileo colonic disease
(L3). A total of 1399 randomly recruited healthy control
individuals, 785 UC patients, 225 CD (L2) patients with disease
limited to the colon and 784 CD patients with ileal involvement
(L1+L3) were used to elucidate the frequency distribution of SNPs
[19–21]. The numbers of patient subgroups and controls in the
different cohorts are shown in Table 1 and detailed statistical
analyses are provided in Table 2. To exclude major differences
between the groups in age or gender, CD patients as well as
controls were sub grouped according to these criteria (Table 3).
Additional points of interest were the behaviour as well as the
aggressiveness of the disease. We therefore decided to separately
test for an association with inflammatory, stricturing and
penetrating behaviour as well as an association of the variant
with surgery for Crohn’s disease. Finally we checked patients with
an additional involvement of the upper gastrointestinal tract (L4).
The study was approved by the ethics committees of the Medical
University Vienna, Austria, the University Hospital Tu¨bingen,
Germany, the University of Leuven, Belgium and the Oxford
Radcliffe Hospital Trust. All patients gave informed and written
consent for their DNA to be analyzed for this study.
Sequencing of TCF-4 promoter and gene region
To determine possible genetic variants in the TCF-4 promoter,
we sequenced the 2.1 kb upstream region of randomly selected
healthy controls (n = 10) and patients with ileal CD (n= 10). In
addition, we sequenced the region of the TCF-4 gene in which
functional insertions and deletions have been reported in colonic
cancer [22]. Subsequently, a sequence analysis of known TCF-4
exons was carried out, including ,100 bp intron boundaries, to
identify additional potential variants of this gene in these regions.
Primers were designed using ENSG00000148737 of the Ensemble
genome browser database for the promoter and exon sequencing.
Sequencing was performed according to standard procedures and
the primers are provided upon request.
TCF-4 genotyping
Leukocyte DNA was isolated by standard procedures (QIAamp
DNA Blood Mini Kit, Qiagen, Hilden, Germany) from whole blood
samples. Genotyping of the samples from the cohorts from Vienna
and Leuven was performed using the matrix assisted laser
desorption/ionization time-of-flight (MALDI-TOF) based mass
spectrometry (MS) of allele specific primer extension products with
a system from Bruker (Daltonik,Leipzig, Germany). Presence of TCF-
4 SNPs detected by MALDI-TOF MS was confirmed by TaqManH
analysis and direct sequencing in a subset of samples. MALDI-TOF
MS based genotyping of the DNA samples obtained from Oxford
was carried out using a MassARRAYH Compact System from
Sequenom (San Diego, USA). Primers were designed using reference
sequence NT 030059 and will be provided on request.
NOD2 genotype analysis
Genotyping for the common NOD2 variants (SNP8, SNP12,
and SNP13) was performed in the Vienna patient samples using
Table 1. Overview of the origin of samples from IBD patients
and healthy controls.
Controls UC CD (L1) CD (L2) CD (L3)
Vienna 833* 149 54 55 150
Leuven 242 74 81 45 151
Oxford 324 562 94 125 254
*healthy blood donors from Stuttgart.
doi:10.1371/journal.pone.0004496.t001
TCF-4 Variants in Ileal CD
PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4496

TaqMan technology (Applied Biosystems, Foster City, California,
USA), as described previously [7].
Computer analysis and statistics
In silico screen of a 10 kb TCF-4 upstream region was performed
using ‘‘Promoter 2.0: for the recognition of PolII promoter
sequences.’’ TESS (Transcription Element Search System) data-
base software allowed assessing of potential binding sites for
certain transcription factors in the candidate sequence. Polymor-
phisms were tested for Hardy–Weinberg equilibrium using Finetti
specialized software (http://ihg2.helmholtz-muenchen.de/
cgi-bin/hw/hwa1.pl) using log likelihood ratio chi square test in
the three cohorts. For genetic analysis (comparing IBD subgroups
versus controls) we used this software to calculate odds ratios,
Confidence Intervals (C.I.) and to perform Pearson’s goodness-of-
fit chi-square tests. Differences in genotype frequencies were
subject to both t tests and Armitage’s trend tests. Values below
0.05 were considered significant. Linkage disequilibrium between
TCF-4 SNPs and haplotype blocks were calculated and identified
using Haploview. To exclude a coincidental association of the SNP
rs3814570, the significance of p- values,0.05 was verified using
Benjamini- Hochberg correction in the overall group.
Results
SNP selection and haplotypes
To investigate potential genetic linkage of TCF-4 to ileal CD, we
screened for SNPs by sequencing 2.1 kb of the 59 flanking region
of TCF-4 in a random group of 10 ileal CD patients and 10
healthy controls. We found eight SNPs in this putative promoter
region (Figure 1), of which three (rs3814570, rs10885394,
rs10885395) were in linkage disequilibrium (LD) in both the
patient and control groups. In the control group, two of ten
individuals carried the variants; in patients with ileal CD, six of ten
individuals were heterozygous for the SNPs. On the basis of these
findings, we studied a well-defined cohort of patients with CD and
healthy controls from Vienna, Austria. In both the control and CD
groups, we found LD between the 3 SNPs that defined a novel
haplotype block (Figure 2a).
An in silico promoter and transcription factor binding-site
analysis of the sequenced region revealed a potential regulatory
region close to the location of rs3814570. Because of (i) the
observed decreased expression of TCF-4 mRNA, (ii) the higher
frequency of the promoter variant in patients as well as (iii) the
presence of a putative regulatory locus, we tested the hypothesis
that rs3814570 exhibits an association with small intestinal
involvement of CD. To exclude additional major variants in the
gene region and possible LD of the identified promoter SNPs to
other potentially functional variants in the TCF-4 gene, we
sequenced known coding exons, with ,100 kb overlapping intron
boundaries in 10 randomly chosen controls (6 identical to
promoter analysis) as well as 25 patients with ileal CD (7 identical
to promoter analysis) (Figure S1). We found ten additional putative
SNPs, of which two were in LD, but none exhibited LD with the
described promoter SNPs (data not shown). A further search for
haplotypes in TCF-4 was conducted based on published data from
the HapMap project (Figure 2b), and no haplotype block including
rs3814570 or additional SNPs in the gene region were identified.
A TCF-4 promoter variant is associated with ileal CD
predisposition
Analysis of SNP rs3814570 frequency distribution was carried
out in a total of 1399 controls (T allele frequency = 25.59%), 785
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TCF-4 Variants in Ileal CD
PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4496

UC patients (T allele frequency= 25.22%), 225 CD patients with
L2 classification (T allele frequency = 24.67%), and 784 CD
patients with ileal involvement (L1+L3) (T allele frequen-
cy = 29.66%). In contrast to UC (OR 0.98, 95% CI 0.85 to
1.13, n.s.) which was similar to controls, the CD patients in
aggregate exhibited a weak association for the minor variant (T
allele positivity: OR 1.18, 95% CI 1.01 to1.39, p = 0.04358)
(Table 2). Consistent with our initial hypothesis, investigation of
the different CD subgroups revealed an association of the variant
(T) with ileal CD (OR 1.23, 95% CI 1.07 to 1.41, p = 0.00371),
Figure 1. Sequencing of TCF-4 (TCF7L2) 59upstream putative promoter region. Sequencing of a 2.1 kb upstream region was performed in 10
healthy controls and 10 patients with ileal Crohn’s disease. Putative regulatory regions were determined using promoter prediction software. Likely
and marginal prediction sites are depicted as red boxes (upper panel). Relative location of identified variants is marked via grey dashes (upper part)
and their allele frequency is demonstrated via bars for controls as well as patients (lower part).
doi:10.1371/journal.pone.0004496.g001
Table 3. Patients and controls sub grouped according to age and gender. Shown are percentages of individuals per group as well
as the TCF-4 (TCF7L2) rs3814570 T- allele frequency (minor allele frequency MAF). Differences in genotype distribution compared to
controls in general as well as the amount of all carriers (allele positivity) and the amount of homozygous carriers were subject to t-
tests in patients with ileal CD. Finally, results of the Armitage’s trend tests for verification of significant associations of the rare T-
variant are shown.
Age and gender
overall controls* L1 L2 L3 ileal CD CD controls ,. L1+L3
Age groups A1 (,16 Y) 2,42% 5,88% 6,02% 9,76% 8,64% 8,06% statistics for A3 C,.T CC,.CT+TT
MAF 34,85% 26,92% 23,08% 23,58% 24,24% 24,05% 1.37; p = 0.06312 1.32; p = 0.20576
A2(16–40 y) 58,22% 72,85% 68,06% 80,85% 78,53% 76,22% Armitage’s trend CC,.TT
MAF 25,28% 31,06% 24,15% 29,61% 30,00% 28,85% 1.37; p = 0.07315 2.02; p = 0.04347
A3(.40 y) 39,35% 21,27% 25,93% 9,39% 12,83% 15,71% statistics for A2 C,.T CC,.CT+TT
MAF 25,28% 27,66% 23,21% 35,29% 31,63% 28,57% 1.27; p = 0.00567 1.36; p = 0.00438
gender male/M 58,21% 50,88% 35,11% 40,43% 43,46% 41,59% Armitage’s trend CC,.TT
MAF 25,63% 33,91% 20,89% 28,57% 30,38% 28,59% 1.22; p = 0.00818 1.39; p = 0.08396
female/V 41,79% 49,12% 64,89% 59,57% 56,54% 58,41%
MAF 25,44% 26,13% 26,71% 30,15% 29,14% 28,53%
MAF=minor allele frequency.
*controls A1 only from Leuven.
doi:10.1371/journal.pone.0004496.t003
TCF-4 Variants in Ileal CD
PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4496

but not with colonic CD (OR 0.95, 95% CI 0.76 to 1.20, ns).
Testing for allele positivity by analyzing wildtype homozygous
individuals (CC) versus all carriers of the minor variant (CT+TT),
revealed the effect more clearly comparing healthy controls versus
ileal CD (OR 1.27, 95% CI 1.07–1.52, p = 0.007372). Odds ratios
and confidence intervals of the group analysis’ in the respective
cohorts as well as the combined analysis of all genotyping results
are shown in Figure 3.
Since there were differences in allele frequencies between the
cohorts (Tables S1, S2, and S3), we tested if those apparent
frequency differences were statistically significant. In general the
Oxford cohort exhibited a lower T allele frequency in controls
(23.30%) compared to Leuven (26.65%) as well as to Vienna
(26.17%) The same was true for CD patients (T allele frequency in
Oxford: 27.38%, Leuven: 30.14% and Vienna: 28.96%), but
could partly be explained by the different percentage of colonic
CD patients in the groups. For CD with ileal involvement only, the
frequency distributions in the cohorts were more similar (T allele
frequency in Oxford: 28.30%, Leuven: 30.82% and Vienna:
30.64%) and not significantly different. Even though we found a
possible change in frequency distribution between the Oxford
control group with both the Leuven (allele frequency: OR 1.20,
Figure 2. Distribution of haploblocks of TCF-4 (TCF7L2). Both colour schemes (a and b) illustrate the linkage disequilibria. The variants are listed
in the upper part of a and b, respectively. Haplotypes for TCF-4 (TCF7L2) rs3814570 (SNP1), rs10885394 (SNP2), rs10885395 (SNP3) and SNPs
associated with diabetes in the Vienna cohort are shown in a. A missing number for D9 or r2 equals 1. Figure 2b: HapMap data based haplotype blocks
and linkage disequilibria (LD) for TCF-4 (TCF7L2) polymorphisms. The intensity of red colouring in b is proportional to the extent of D9 or r2
respectively and a missing number for each of them equals. The observed SNP in the putative promoter region is not part of any significant
haplotype block.
doi:10.1371/journal.pone.0004496.g002
TCF-4 Variants in Ileal CD
PLoS ONE | www.plosone.org 5 February 2009 | Volume 4 | Issue 2 | e4496