Investigation of multiple susceptibility loci for inflammatory bowel disease in an Italian cohort of patients.

Abstract

Recent GWAs and meta-analyses have outlined about 100 susceptibility genes/loci for inflammatory bowel diseases (IBD). In this study we aimed to investigate the influence of SNPs tagging the genes/loci PTGER4, TNFSF15, NKX2-3, ZNF365, IFNG, PTPN2, PSMG1, and HLA in a large pediatric- and adult-onset IBD Italian cohort.
Eight SNPs were assessed in 1,070 Crohn's disease (CD), 1,213 ulcerative colitis (UC), 557 of whom being diagnosed at the age of ≤16 years, and 789 healthy controls. Correlations with sub-phenotypes and major variants of NOD2 gene were investigated.
The SNPs tagging the TNFSF15, NKX2-3, ZNF365, and PTPN2 genes were associated with CD (P values ranging from 0.037 to 7×10(-6)). The SNPs tagging the PTGER4, NKX2-3, ZNF365, IFNG, PSMG1, and HLA area were associated with UC (P values 0.047 to 4×10(-5)). In the pediatric cohort the associations of TNFSF15, NKX2-3 with CD, and PTGER4, NKX2-3, ZNF365, IFNG, PSMG1 with UC, were confirmed. Association with TNFSF15 and pediatric UC was also reported. A correlation with NKX2-3 and need for surgery (P  =  0.038), and with HLA and steroid-responsiveness (P  =  0.024) in UC patients was observed. Moreover, significant association in our CD cohort with TNFSF15 SNP and colonic involvement (P  =  0.021), and with ZNF365 and ileal location (P  =  0.024) was demonstrated.
We confirmed in a large Italian cohort the associations with CD and UC of newly identified genes, both in adult and pediatric cohort of patients, with some influence on sub-phenotypes.

Full text

Page 1

Investigation of Multiple Susceptibility Loci for
Inflammatory Bowel Disease in an Italian Cohort of
Patients
Anna Latiano1*, Orazio Palmieri1, Tiziana Latiano1, Giuseppe Corritore1, Fabrizio Bossa1, Giuseppina
Martino1, Giuseppe Biscaglia1, Daniela Scimeca1, Maria Rosa Valvano1, Maria Pastore2, Antonio
Marseglia2, Renata D’Inca `3, Angelo Andriulli1, Vito Annese4
1Gastroenterology Unit, IRCCS ‘‘Casa Sollievo della Sofferenza,’’ San Giovanni Rotondo, Italy, 2Division of Paediatrics, IRCCS ‘‘Casa Sollievo della Sofferenza,’’ San Giovanni
Rotondo, Italy, 3Department of Surgical and Gastroenterological Sciences, University of Padua, Padua, Italy, 4Division of Gastroenterology, University Hospital Careggi,
Florence, Italy
Abstract
Background: Recent GWAs and meta-analyses have outlined about 100 susceptibility genes/loci for inflammatory bowel
diseases (IBD). In this study we aimed to investigate the influence of SNPs tagging the genes/loci PTGER4, TNFSF15, NKX2-3,
ZNF365, IFNG, PTPN2, PSMG1, and HLA in a large pediatric- and adult-onset IBD Italian cohort.
Methods: Eight SNPs were assessed in 1,070 Crohn’s disease (CD), 1,213 ulcerative colitis (UC), 557 of whom being
diagnosed at the age of #16 years, and 789 healthy controls. Correlations with sub-phenotypes and major variants of NOD2
gene were investigated.
Results: The SNPs tagging the TNFSF15, NKX2-3, ZNF365, and PTPN2 genes were associated with CD (P values ranging from
0.037 to 761026). The SNPs tagging the PTGER4, NKX2-3, ZNF365, IFNG, PSMG1, and HLA area were associated with UC (P
values 0.047 to 461025). In the pediatric cohort the associations of TNFSF15, NKX2-3 with CD, and PTGER4, NKX2-3, ZNF365,
IFNG, PSMG1 with UC, were confirmed. Association with TNFSF15 and pediatric UC was also reported. A correlation with
NKX2-3 and need for surgery (P = 0.038), and with HLA and steroid-responsiveness (P = 0.024) in UC patients was observed.
Moreover, significant association in our CD cohort with TNFSF15 SNP and colonic involvement (P = 0.021), and with ZNF365
and ileal location (P = 0.024) was demonstrated.
Conclusions: We confirmed in a large Italian cohort the associations with CD and UC of newly identified genes, both in adult
and pediatric cohort of patients, with some influence on sub-phenotypes.
Citation: Latiano A, Palmieri O, Latiano T, Corritore G, Bossa F, et al. (2011) Investigation of Multiple Susceptibility Loci for Inflammatory Bowel Disease in an
Italian Cohort of Patients. PLoS ONE 6(7): e22688. doi:10.1371/journal.pone.0022688
Editor: Jan-Hendrik Niess, Ulm University, Germany
Received February 22, 2011; Accepted June 28, 2011; Published July 27, 2011
Copyright: ? 2011 Latiano 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: The study was supported by a grant from the Italian Minister of the Health (RC0902GA33 . The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: a.latiano @operapadrepio.it
Introduction
The pathogenesis of inflammatory bowel diseases (IBD), namely
Crohn’s disease (CD) and ulcerative colitis (UC), is still
incompletely understood, but it is widely accepted that the two
conditions result from an inappropriate and exaggerated mucosal
immune response to constituents of the intestinal flora in a
genetically susceptible host [1,2]. In the past year, genome-wide
association (GWA) studies have identified several genes involved in
the pathogenesis of IBD and, subsequently GWAS meta-analysis
has led to confirmation of more than 70 genes or loci that confer
susceptibility to CD and 47 to UC, mostly in adult populations
[3,4,5]. In particular, GWA and replications studies identified
gene variants, including protein tyrosine phosphathase nonrecep-
tor type 2 (PTPN2), NK2 transcription factor related locus 3
(NKX2-3), and tumor necrosis factor superfamily member 15
(TNFSF15) [6,7,8]. A subsequent North American GWA study
identified two additional loci for UC located on chromosome 1p36
and 12q15, each of them harboring multiple genes, including
several with a definite role in inflammation and immunity, like
group II secreted phospholipase A2 (PLA2G2E), interferon gamma
(IFNG), interleukin 26 (IL26) and interleukin 22 (IL22). In addition,
combined genome-wide significant evidence for association was
found at two additional loci, namely HLA on chromosome 6p21
and IL23R (interleukin-23 receptor) on chromosome 1p31 [9].
Finally, a GWA study carried out in pediatric-onset IBD patients
identified two novel IBD loci located on chromosome 20q13 and
21q22, close to the tumor necrosis factor receptor superfamily
member 6B (TNFRSF6B) and proteasome-assembling chaperone 1
(PSMG1) genes [10].
In the present study, we investigated whether potential loci
reported in the meta-analysis and GWA studies on chromosomes
PLoS ONE | www.plosone.org1 July 2011 | Volume 6 | Issue 7 | e22688
)

Page 2

5p13 (PTGER4), 12q15 (IFNG, IL22, IL26), 6p21 (HLA), 21q22
(PSMG1), and PTPN2, NKX2-3, TNFSF15, were associated in a
large and phenotypically well-characterized Italian cohort of IBD
patients, and we also attempted to elucidate their involvement in
early onset disease. In addition, we tested the potential epistasis
between these variants and IBD-associated NOD2/CARD15 SNPs,
as well as possible genotype-phenotype correlations.
Materials and Methods
Ethics statement
The IBD cohort and unaffected controls were recruited from
adult individuals referred the IRCCS, ‘‘Casa Sollievo della
Sofferenza ‘‘ Hospital in San Giovanni Rotondo, and from
pediatric centers of the Italian Society of Pediatric Gastroenter-
ology, Hepatology and Nutrition (SIGENP). Written informed
consent was obtained from all adult participants and, for patients
under age of 19 years, from related parents. Ethical approval was
acquired from the Ethics Review Board of ‘‘Casa Sollievo della
Sofferenza’’ Hospital, San Giovanni Rotondo, and each partici-
pating center approved the recruitment protocol. The study was
supported by a grant from the Italian Minister of the Health
(RC0902GA33).
Extensive clinical characterization was available for all patients.
The diagnosis of CD or UC was established by conventional
clinical, radiological, endoscopic, and histological findings [11].
The CD phenotype was classified based on age at disease onset
(A), maximal extent of disease (L), and behavior (B) according to
the Montreal classification [12,13]. In patients with UC the colon
location was also classified according to the Montreal classifica-
tion, by distinguishing ulcerative proctitis (E1), left-side colitis (E2),
and extensive colitis (E3). For all IBD patients further clinical
characteristics were analyzed, namely the occurrence of previous
resective surgery, IBD family history, smoking habits, extra-
intestinal manifestations and response to medical therapy. More
specifically the need for use of corticosteroids, immunosuppressors
(thiopurines and methotrexate) and anti-TNF agents were
evaluated. In addition, on the basis of review of medical records,
patients with the use of corticosteroid (CS) were classified as CS-
responder (at least one course of systemic steroids with clinical
remission reported in the medical history), or CS-refractory (when
an unsuccessful clinical response was achieved leading to
alternative therapies like surgery, use of anti-TNF or other
immunosuppressors drugs) [14,15]. Patients with incomplete or
unclear information were excluded from this analysis.
SNPs analysis and genotyping
We selected 8 polymorphisms for genotyping: three of them
(rs4613763, rs4263839, rs11190140) identified by the CD GWA
study meta-analysis [3], two (rs10761659, rs2542151) by the
WTCCC [6] as showing the strongest association signals, two
(rs2395185, rs1558744) by the first UC GWA study [9], and one
(rs2836878) by a GWA analysis for early-onset IBD [10]. The
genotypic variants in the NOD2/CARD15 gene (rs2066844,
rs2066845, rs2066847) had already been analyzed for all patients
and controls. Genomic DNA was extracted from peripheral blood
leukocytes by standard procedures using the DNA blood maxi kit
from Qiagen (Hilden, Germany) in accordance with the
manufacturer’s instructions. All of the genotyping was performed
at the Molecular Laboratory of the Gastroenterology Unit at the
San Giovanni Rotondo Hospital, Italy. Genotyping was per-
formed using Custom TaqmanH SNP assay (Applied Biosystems,
Foster City, CA), following manufacturer’s instructions. The
overall success rate of the genotyping assay was over 98%.
Statistical analysis
Statistical analysis was performed using Haploview Software
version 4.1 (http://www.broad.mit.edu/personal/mpg/haploview)
and SPSS software version 13.0 (Chicago, IL, USA). Hardy-
Weinberg Equilibrium (HWE) tests were performed for all
investigated polymorphisms independently among cases and
controls. For the case-control analysis, comparisons of genotypes
and allele frequencies was performed using X2or Fisher’s exact test,
where appropriate. Genotype-phenotype associations were first
analyzed by means of univariate analysis and subsequently
expressed as Odds Ratios (OR) with 95% confidence intervals
(95% CI) by means of stepwise logistic regression analysis. For
detecting gene-gene interactions we used a logistic regression based
on forward stepwise selection procedures using the number of risk
alleles as predictor variable. P values of less than 0.05 were
considered significant.
Results
Case-control analysis with IBD patients
A total of 2283 individuals with IBD, including 1070 with CD
and 1213 with UC, was analyzed. The control group consisted of
789 individuals from the same ethnicity who did not have IBD
(neither CD nor UC). Clinical and demographic features of the
IBD cases are listed in Table 1. There were 296 CD patients and
261 UC patients with the initial diagnosis before their 16th
birthday. The adult cohort was constituted of 774 patients with
CD and 952 with UC.
We compared single-marker allele frequencies using x2statistics
(Tables 2–3). Frequencies were in accordance with the Hardy-
Weinberg equilibrium. In the overall cohort of IBD patients, four
markers were above the threshold for significance for CD: the
TNFSF15 (rs4263839, P = 7.18361026), NKX2-3 (rs11190140, P
= 0.003), ZNF365 (rs10761659, P
(rs2542151, P = 0.037). Of these four markers, two, namely,
rs11190140 (P = 461025), and rs10761659 (P = 461025), were
also associated with UC. In this latter subset of patients, four more
markers,
PTGER4
rs4613763
rs2395185 (P= 0.001), IFNG-IL22-IL26 rs1558744 (P
0.007), and PSMG1 rs2836878 (P = 0.047) were exclusively
associated.
To determine whether the previously identified variants were
shared by both adult and pediatric individuals, we stratified all
IBD patients according to their age at initial diagnosis. For the
adult subset, associations were confirmed for all SNPs for either
CD and UC with two remarkable exceptions: the rs4613763
variant, which was associated with CD (P = 0.031)(Table 4), and
the rs2836878 variant which lost the association for UC (Table 5).
Concerning childhood-onset cohort, the genotype frequencies of
all considered polymorphisms remained significantly associated for
UC, with the exception of the rs2395185 variant (Table 5). In CD
pediatric patients the association was confirmed for rs4263839 (P
= 0.008) and rs11190140 (P = 0.007) variants (Table 4).
= 0.007), and PTPN2
(P= 0.012),
HLA-BTNL2
=
Genotype association with IBD phenotypes
We also assessed whether the investigated SNPs could bear an
influence on specific disease phenotypes, such as gender, disease
location and behavior, resective surgery, family history, smoking
habit, extra-intestinal manifestations, and medical therapies. At
logistic regression analysis (Table 6), using a custom/stepwise
model (forward entry) and after correction for all other covariates
(age at diagnosis, disease localization, duration of follow-up,
smoking status, etc), a significant association with the rs2395185
variant of the HLA gene and steroid-response (P = 0.024, OR =
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org2 July 2011 | Volume 6 | Issue 7 | e22688

Page 3

2.07, CI 95% 1.10–3.89), and positive family history (P , 0.01,
OR = 3.68, CI 95% 1.93–7.04) in UC patients was observed. In
the CD cohort, the ZNF365 (rs10761659) SNP was associated with
ileal location (L1) compared to patients with colonic (L2)
involvement (81.2% vs 72.1%, P = 0.024, OR = 1.67, CI
95%, 1.06–2.62). Moreover, colon involvement in CD was
significantly more frequent (45%) in patients carrying the minor
variant (AA+Aa) of TNFSF15 (rs4263839) SNP vs a 35%
frequency observed in those with only ileal involvement (P =
0.021, OR = 1.54, 5% CI, 1.06–2.22). A negative association was
found for the minor allele of the NKX2-3 (rs11190140) gene in UC
patients with needs for surgery (P = 0.038, OR = 0.64, 95% CI,
0.42–0.97). In CD, the same variant prevailed in patients with
smoking habit (P = 0.018, OR = 1.46, CI 95%, 1.06–2.00).
There was no effect of the other four SNPs (PTGER4-rs4613763,
PTPN2-rs2542151, IFNG-rs1558744, PSMG1-rs2836878) on all
the evaluated sub-phenotypes (data not shown).
We asked whether the identified genotype/phenotype associa-
tions would differ after stratifying the IBD population in respect to
age at diagnosis. For the HLA-rs2395185 SNP the association
persisted both in the adult- (P = 0.003) and in the pediatric-onset
subset of patients (P = 0.006) with a positive family history
(Table 6). Despite the large number of pediatric-onset IBD
patients investigated, a trend was found for the genotype/
phenotypefrequenciesof rs10761659,
rs11190140 SNPs, but did not reach statistical significance.
rs4263839,and
Gene-gene interactions
The possible interactions of tested variants (rs4613763,
rs2395185, rs4263839, rs11190140, rs10761659, rs1558744,
rs2542151, and rs2836878) with polymorphisms in the established
susceptibility gene NOD2/CARD15 (the three main polymorphisms
rs2066844, rs2066845, and rs2066847: at least 1 variant against
wild type) were evaluated. After correction for multiple testing,
there was no significant evidence for interaction among the
considered SNPs (P . 0.05) (data not shown), thus implying that
each gene independently contributes to the disease risk.
Discussion
Recent GWA studies have enhanced our understanding of the
complex genetic architecture of IBD. Most associations appear to
be common to both types of IBD, while some genes/loci may be
specific to adult- or pediatric-onset, and the factors that determine
age of onset are unknown at present.
The major aim of our study was to examine recently described
potential association of genes involved in the immune response
and inflammation (PTGER4, HLA, TNFSF15, NKX2-3, ZNF365,
IFNG, PTPN2, and PSMG1) in adult and pediatric-onset IBD in an
Italian population.
Table 1. Clinical and demographic characteristics of the
study population.
CDUC
n=1070 n=1213
Gender
F 468 (44)511 (42)
M 602 (56)702 (58)
Duration of follow-up (yr)
mean 6 SD867967
median (range) 6 (1–37) 7 (1–41)
Age at diagnosis (yr)
mean 6 SD27615 31616
median (range) 25 (1–79) 28 (1–83)
#16 (A1)296 (28)261 (21)
17–40 (A2) 586 (55)642 (53)
. 40 (A3) 188 (17) 310 (26)
Disease location CD, n (%)
Ileum (L16L4)331 (31)
Colon (L26L4) 265 (25)
leo-colon (L36L4) 465 (43)
Upper GI (L4)9 (1)
Disease extent UC, n (%)
Rectum (E1)139 (11)
Left colon (E2)554 (46)
Pancolitis (E3)520 (43)
Disease behavior CD, n (%)
Inflammatory677 (63)
Stricturing279 (26)
Penetrating 114 (11)
Perianal Disease
No882 (82)
yes 188 (18)
EIM
No 651 (61) 908 (75)
yes419 (39) 305 (25)
Family history
No 959 (90)1107 (91)
yes 111 (10)106 (9)
*Smoker
Yes292 (32) 164 (15)
No 490 (53)622 (57)
Ex 140 (15) 311 (28)
Resection
No749 (70)1082 (89)
yes 321 (30) 131 (11)
Steroids
Refractory83 (13) 101(13)
Responder569 (87) 673 (87)
IMS (AZT/6MP, Ciclo, MTX)
No 676 (42) 927(58)
Yes394 (58) 286 (42)
Infliximab
CD UC
n=1070n=1213
No 914 (85) 1168 (96)
Yes 156 (15) 45 (4)
CD: Crohn’s disease; UC: ulcerative colitis; EIM: extra-intestinal manifestations.
IMS: immunosuppressors.
*For 148 CD and 116 UC patients the information is missing.
doi:10.1371/journal.pone.0022688.t001
Table 1. Cont.
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org3 July 2011 | Volume 6 | Issue 7 | e22688

Page 4

Using a case-control design, we were able to replicate
associations between GWA reported SNPs with CD (TNFSF15
rs4263839, P = 7.18361026; NKX2-3 rs11190140, P = 0.003;
ZNF365 rs10761659, P = 0.007; PTPN2 rs2542151, P = 0.037),
and UC (NKX2-3 rs11190140, P = 461025; ZNF365 rs10761659,
P = 461025, PTGER4 rs4613763, P = 0.012; HLA-BTNL2
rs2395185, P = 0.001; IFNG-IL22-IL26 rs1558744, P = 0.007;
PSMG1 rs2836878, P = 0.047).
In addition, our analysis reveals significant association with
pediatric UC cohort for six out the eight investigated variants,
each of them with a P value , 0.016 (PTGER4, TNFSF15, NKX2-
3, ZNF365, IFNG, and PSMG1): this finding suggests that these
genes may also be involved in susceptibility to UC pediatric-onset.
To date, no studies on the tested polymorphisms have indicated
associations with specific sub-phenotype. We were able to
demonstrate, by stepwise logistic regression analysis, a correlation
with, NKX2-3 and need for surgery (P =0.038), and with HLA and
steroid-responsiveness (P = 0.024), and a positive family history in
UC patients. Moreover, significant association in our CD cohort
with TNFSF15 SNP and colonic involvement (P = 0.021), and
with ZNF365 and ileal location (P = 0.024) was demonstrated.
The clinical significance of these associations, if any, remains to be
investigated.
A summary of previously described allelic distributions of SNPs
of genes/loci analyzed is depicted in Supplementary Table S1.
Kugathasan et al [10] carried out a GWA analysis in a pediatric-
onset IBD and identified a significant association of both CD and
UC with an intergenic SNP, rs2836878, located on chromosome
21q22 in a small region of linkage disequilibrium that harbors no
genes but is close to the PSMG1 gene. The association was also
reiterated by the largest GWA study conducted so far in early-onset
IBD (UC: P=2.6561029)[16]. In keeping with previous finding is
the contributionof the PSMG1 locus to disease susceptibilityin adult
UC [17]. McGovern et al [18] confirmed the association after
combining data from two new GWA studies and performing a
meta-analysis with a published study. A trend for similar
associations at rs2836878 variant was observed in CD Canadian
cohort but did not achieve statistical significance [19](Table S1).
Ourresultsconfirmtheassociationinparticularwithpediatric-onset
UC, pinpointing to a significant relevance of the 21q22 for UC.
Larger studies that include functional data on the PSMG1 gene will
be required to confirm association at this locus.
Table 2. Genotype distribution of associated SNPs in Crohn’s disease (CD) patients and healthy controls.
No. of CD genotyped No. of control genotyped
Gene(s) or locusSNP AA* Aaaa AA* Aaaa P value OR (95% CI)
PTGER4rs4613763 7 (1.1%) 127 (19.3%) 523 (79.6%) 2 (0.4%)86 (15.7%) 460 (83.9%)0.0531 1.34 (0.99–1.80)
HLA, BTNL2rs2395185--- 22 (5.1%) 154 (35.7%)256 (59.2%)--
TNFSF15 rs426383957 (6.3%) 322 (35.2%)535 (58.5%)74 (9.4%) 339 (43.0%) 376 (47.6%)7.183E-060.65 (0.53–0.78)
NKX2-3rs11190140 163 (18.8%)437 (50.2%)270 (31.0%) 176 (22.5%) 414 (52.9%)193 (24.6%) 0.0039 0.73 (0.58–0.90)
ZNF365 rs10761659 240 (30.2%)379 (47.7%) 175 (22.1%) 131 (24.3%)255 (47.2%) 154 (28.5%)0.00701.41 (1.09–1.81)
IFNG, IL22, IL26rs1558744--- 77 (13.9%)262 (47.4%) 214 (38.7%)--
PTPN2 rs254215121 (2.4%) 229 (26.3%)622 (71.3%) 10 (1.3%) 178 (22.8%) 591 (75.9%)0.0371 1.26 (1.01-1.57)
PSMG1 rs283687810 (3.4%) 113 (38.3%)172 (58.3%) 43 (7.6%) 203 (35.9%) 320 (56.5%)nsns
A* denotes a risk allele.
OR: odds ratio; CI: confidence interval.
SNP: single nucleotide polymorphism.
doi:10.1371/journal.pone.0022688.t002
Table 3. Genotype distribution of associated SNPs in ulcerative colitis (UC) patients and healthy controls.
No. of UC genotyped No. of control genotyped
Gene(s) or locus SNP AA*Aa aa AA*Aa aaP valueOR (95% CI)
PTGER4rs46137638 (1.2%) 142 (20.5%) 542 (78.3%) 2 (0.4%)86 (15.7%) 460 (83.9%)0.0126 1.45 (1.08–1.93)
HLA, BTNL2rs2395185 29 (3.7%) 221 (28.1%)537 (68.2%)22 (5.1%) 154 (35.7%) 256 (59.2%)0.00170.68 (0.53–0.86)
TNFSF15rs4263839 66. (7.3%) 375 (41.6%)460 (51.1%)74 (9.4%) 339 (43.0%)376 (47.6%)ns ns
NKX2-3 rs11190140 162 (17.9%)438 (48.3%) 306 (33.8%)176 (22.5%)414 (52.9%)193 (24.6%)4E-05 0.64 (0.52–0.79)
ZNF365 rs10761659168 (28.8%) 309 (53.0%)106 (18.2%) 131 (24.3%) 255 (47.2%)154 (28.5%)4E-051.79 (1.36–2.38)
IFNG, IL22, IL26rs1558744 167 (20.1%) 401 (48.2%)264 (31.7%) 77 (13.9%)262 (47.4%) 214 (38.7%) 0.0076 1.36 (1.08–1.70)
PTPN2 rs254215110 (1.1%)232 (25.4%) 672 (73.5%) 10 (1.3%) 178 (22.8%)591 (75.9%)ns ns
PSMG1rs283687832 (3.7%) 302 (34.5%)540 (61.8%)43 (7.6%) 203 (35.9%)320 (56.5%)0.0473 0.80 (0.65–0.99)
A* denotes a risk allele.
OR: odds ratio; CI: confidence interval.
SNP: single nucleotide polymorphism.
doi:10.1371/journal.pone.0022688.t003
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org4July 2011 | Volume 6 | Issue 7 | e22688

Page 5

A significant association of geneticvariants of the TNFSF15 (TL1A)
geneonchromosome9q33withCDwasobservedinalargecohortof
Japanese patients, in several European cohorts [8,20,21], in US
Jewish patients [22], in the combined data from the NIDDK IBD
Consortium and the WTCCC [3], in Koreans patients [23], and in
UC GWA study [7]. The TNFSF15 is the only gene that has been
associated in either Asiatic and European IBD patients [8,19](Table
S1). TNFSF15 is a member of tumor necrosis factor (TNF)
superfamily that binds to death domain receptor 3 (DR3,
TNFRSF25) and is expressed in endothelial cells, lymphocytes,
plasma cells, monocytes, and dendritic cells [24,25]. Our analysis
confirms the TNFSF15 as a susceptibility locus in CD both in adult
and pediatric population (P = , 0.001, and P = 0.008 respectively),
and shows an association also with early onset UC (P = 0.016).
The WTCCC [6] reported on CD cases a novel association
involving a cluster of SNPs around the rs10883365 variant on
chromosome 10q24.2, which maps within NKX2-3 (NK2 tran-
scription factor related, locus 3) gene, a member of the NKX
family of homeodomain-containing transcription factors. The
results of Parkes et al [26] supported this findings with an
independent set of CD cases and controls of European descent. In
addition, a large-scale meta-analysis [3] on CD cohort and
replication study on IBD samples [7] found association with
rs11190140 polymorphism in complete linkage disequilibrium
with the risk allele at the SNP rs10883365 from the WTCCC
study (r2= 1.0). In addition, a modest association was also
reported with UC in a nonsynonymous SNP scan [27], in GWA
scan for UC [17], and in the UC GWA meta-analysis and
Table 4. Genotype distribution of associated SNPs in adult- and childhood-onset Crohn’s disease (CD) cohort.
No. of Adult CD genotypedNo. of Pediatric CD genotyped
SNPAA Aaaa AAAaaa
P value OR (95% CI)
rs4613763 6 (1.2%)99 (20.0%) 389 (78.8%)1 (0.6%)28 (17.2%) 134 (82.2%)
Adult
Pediatric
0.0310
ns
1.41 (1.03–1.93)
ns
rs2395185------
Adult
Pediatric
-
-
-
-
rs426383935 (5.5%) 223 (35.2%) 376 (59.3%)22 (7.8%) 99 (35.4%) 159 (56.8%)
Adult
Pediatric
,0.001
0.0087
0.62 (0.51–0.77)
0.69 (0.52–0.91)
rs11190140 116 (19.4%)301 (50.4%)180 (30.2%) 47 (17.2%) 136 (49.8%) 90 (33.0%)
Adult
Pediatric
0.0258
0.0075
0.84 (0.72–0.98)
0.66 (0.49–0.89)
rs10761659 187 (30.4%)301 (48.8%) 128 (20.8%)53 (29.8%)78 (43.8%) 47 (26.4%)
Adult
Pediatric
0.0022
ns
1.52 (1.16–1.99)
ns
rs1558744------
Adult
Pediatric
-
-
-
-
rs2542151 13 (2.1%)169 (27.8%) 426 (70.1%)8 (3.0%) 60 (22.8%)196 (74.2%)Adult
Pediatric
0.0153
ns
1.34 (1.06–1.70)
ns
rs2836878------
Adult
Pediatric
-
-
-
-
doi:10.1371/journal.pone.0022688.t004
Table 5. Genotype distribution of associated SNPs in adult- and childhood-onset ulcerative colitis (UC) cohort.
No. of Adult UC genotypedNo. of Pediatric UC genotyped
SNPAA Aa aaAA Aaaa
P valueOR (95% CI)
rs46137635 (5.2%) 78 (18.4%)341 (80.42%) 3 (1.1%)64 (23.9%) 201 (75.0%)
Adult
Pediatric
ns
0.0022
ns
1.74 (1.21–2.49)
rs2395185 19 (3.1%) 176 (28.4%)425 (68.5%)10 (6.0%) 45 (26.9%) 112 (67.1%)
Adult
Pediatric
0.0019
ns
0.67 (0.52–0.86)
ns
rs4263839 50 (7.5%) 289 (43.4%) 327 (49.1%)16 (6.8%)86(36.6%) 133 (56.6%)
Adult
Pediatric
ns
0.0161
ns
0.69 (0.52–0.94)
rs11190140132 (19.8%)313 (46.9%) 222 (33.3%)30 (12.6%) 125 (52.3%)84 (35.1%)
Adult
Pediatric
0.0003
0.0014
0.66 (0.52–0.82)
0.60 (0.44–0.82)
rs10761659 121 (27.7%)235 (53.8%)81 (18.5%)47 (32.2%) 74 (50.7%)25 (17.1%)
Adult
Pediatric
0.0003
0.0054
1.75 (1.29–2.38)
1.93 (1.21–3.09)
rs1558744127 (18.7%)329 (48.5%) 223 (32.8%)40 (26.1%)72 (47.1%) 41 (26.8%)
Adult
Pediatric
0.0326
0.0067
1.29 (1.02–1.63)
1.72 (1.16–2.56)
rs25421518 (1.2%) 169 (24.9%)500 (73.9%)2 (0.8%) 63 (26.6%) 172 (72.6%)
Adult
Pediatric
ns
ns
ns
ns
rs2836878 30 (4.2%)252 (35.6%) 426 (60.2%)2 (1.2%) 50 (30.1%)114 (68.7%)
Adult
Pediatric
ns
0.0051
ns
0.59 (0.41–0.86)
doi:10.1371/journal.pone.0022688.t005
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org5 July 2011 | Volume 6 | Issue 7 | e22688

Page 6

replication studies [18](Table S1). Similarly, in the present study
the rs11190140 variant was associated with an increased risk for
both CD and UC (P = 0.003 and P = 461025respectively), both
in the adult and early-onset cohort. Abnormal expression of
NKX2-3 may alter gut migration of antigen-responsive lympho-
cytes and influence the intestinal inflammatory response. NKX2-
3-deficient mice develop splenic and gut-associated lymphoid
tissue abnormalities with disordered segregation of T and B cells
[28].
A highly attractive candidate gene for IBD, owing to its anti-
inflammatory function and involvement in type 1 diabetes
susceptibility and rheumatoid arthritis [29], is the PTPN2 (protein
tyrosine phosphatase, non-receptor type 2) located on chromo-
some 18p11, which encodes for a tyrosine phosphatase expressed
in T cells, a negative regulator of inflammation. A novel
association at rs2542151 of the PTPN2 gene and CD was
identified [6], replicated [26,30] and confirmed [3]. In contrast,
Franke et al [7] observed this association with UC, a finding
replicated in the recent GWA on UC [17]. In addition,
associations with CD pediatric was also demonstrated [16,31](Ta-
ble S1). In the present investigation the association was observed
only in adult CD (P = 0.015) confirming its role as an adult-
susceptibility gene.
The UC GWAS in European ancestry samples [9] indentified
loci on chromosomes 1p36 and 12q15 where are genes involved in
inflammation and immunity, such as PLA2GIIE (phospholipase
A2, group IIE), IFNG (interferon-c), IL22 (interleukin-22), and
IL26 (interleukin-26); previous associations were replicated in
GWA meta-analysis [18](Table S1). We were able to replicate the
association between the rs1558744 SNP on chromosome 12q15
and UC (P = 0.007), and interestingly after stratifying the cohort
with respect to age at diagnosis, the association was observed also
in the pediatric-onset patients (adult P = 0.03; pediatric P =
0.006).
Several independent genome-wide scans in inflammatory bowel
disease have shown evidence of linkage to the MHC region
[32,33], which is characterized by extensive LD blocks (up to
3 Mb) and several genes (250 genes), mainly involved in immune-
related functions. Recent genome-wide association studies in UC
confirmed the association with the HLA with the maximal
association signal at rs2395185, in a region spanning BTNL2 to
HLA-DQB1 genes [9,18]. Recently, SNP and the HLA data
convincingly show that the main signal is located in a narrow
genomic window containing the HLA-DRB1 gene and strongly
suggest that the more common HLA-DRB1*1101 allele plays a
primary role in both UC and CD susceptibility [34]. This
association to the DRB1 locus is consistent with other published
GWAs in UC [9,35,36], also in Japanese population [37]. The
same region was identified in the meta-analysis of CD genome-
wide association studies [3]. Recent genome-wide association
study in early-onset IBD [16] validated this known adult-onset
IBD locus in their CD, UC, and IBD dataset, further supporting
the importance of this region in IBD risk (Table S1). Allelic and
genotype association analysis in our cohort showed that the
polymorphism was significantly associated with overall suscepti-
bility to UC (P = 0.001), and in particular with adult subset (P =
0.001).
In the study of Libioulle et al [38] a region on chromosome
5p13.1 contributing to CD susceptibility was identified. The
disease-associated alleles were found to correlate with expression
levels of the prostaglandin receptor EP4, which binds prostaglan-
din E2 (PGE2) and is encoded by PTGER4. In the same region the
meta-analysis of three GWAS [3] identified rs4613763 as the most
strongly associated SNP. No highly significant association of the
PTGER4 region was documented in the studies of the NIDDK
[39] and the WTCCC [6]. Recently GWAS showed significant
evidence for association also with UC [18] (Table S1). Similarly,
our data indicated significant association with adult CD (P =
0.031), and early-onset UC (P = 0.002).
In the WTCCC GWA [6], a locus at chromosome 10q21
around rs10761659, a non-coding intergenic SNP mapping 14-kb
telomeric to a zinc finger gene known as ZNF365, was detected.
The locus was replicated both in pediatric CD and UC [16,31],
and adult-onset CD [3,40] (Table S1). We were able to confirm
this association with CD (P = 0.007), and UC (P = 461025), and
after stratifying the cohort with respect to age at diagnosis the
association was confirmed in adult CD (P = 0.002), and either
adult (P = 0.0002), and in pediatric UC patients (P = 0.005).
In conclusion, our study has confirmed recently described
associations, in particular between the PTGER4, HLA, TNFSF15,
NKX2-3, ZNF365, IFNG, PTPN2, and PSMG1 genes and IBD in
adults and in some cases for the first time also in children.
Furthermore, we were able to identify the influence of the
investigated genes on clinical expression and localization on CD
Table 6. Correlation between risk alleles and clinical characteristics in Crohn’s disease (CD) and ulcerative colitis (UC) patients at
Multivariate Analysis.
CD UC
Adult n=728Pediatric n=342Adult n=952 Pediatric n=261
HLA
rs2395185
steroid-responsive P=0.024
OR 2.07 CI 95% (1.10–3.89)
family history P,0.01
OR 3.68 CI 95% (1.93–7.04)
Adult P=0.003
OR 4.16 CI 95% (1.62–10.71)
Pediatric P=0.006
OR 3.81 CI 95%(1.45–9.97)
ZNF365
rs10761659
colon vs ileum P=0.024
OR 1.67 CI 95% (1.06–2.62)
TNFSF15
rs4263839
colon vs ileum P=0.021
OR 1.54 CI 95% (1.06–2.22)
NKX2-3
rs11190140
smoking P=0.018
OR 1.46 CI 95% (1.06–2.00)
surgery P=0.038
OR 0.64 CI 95% (0.42–0.97)
doi:10.1371/journal.pone.0022688.t006
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org6 July 2011 | Volume 6 | Issue 7 | e22688

Page 7

and UC patients, but the clinical significance of these associations
remains to be investigated and replicated. Further characteriza-
tion, fine mapping, and functional studies of these genetic regions
are needed to discover the pathogenetic role of these newly
identified genes/loci.
Supporting Information
Table S1
phisms (SNP) analyzed in the previously published studies and in
the current study.
(DOCX)
Allelic distributions of single nucleotide polymor-
Acknowledgments
The authors wish to thank all the patients and families that participated in
this study. The following physicians of the SIGENP* and adult
Gastrointestinal Units contributed to the study by providing DNA samples
and clinical information of their patients: *Ancona: Catassi, Nobile S;
*Bari: Rutigliano V, De Venuto D; *Foggia: Campanozzi A; *Messina:
Vieni G, Sferlazzas C; Milano: Bianchi Porro G, Vecchi M, Saibeni S;
Napoli: Riegler G, *Napoli: Berni Canani E, Staiano AM; Padova: D’inca `
R, Sturniolo GC; *Padova: Guariso G, Lodde V; *Palermo: Accomando S;
*Parma: de Angelis GL; *Pescara: Lombardi G; *Reggio Calabria:
Romano C; *Roma: Cucchiara S, Borrelli O, Bascietto C; S. Giovanni
Rotondo: *Firenze: Lionetti P; *Genova: Barabino A; *S. Giovanni
Rotondo: D’Altilia M.
Author Contributions
Conceived and designed the experiments: LA PO AV. Performed the
experiments: LT CG. Analyzed the data: VMR. Contributed reagents/
materials/analysis tools: BF MG BG SD PM MA DIR. Wrote the paper:
LA. Supervised manuscript preparation and patient recruitment: AA AV.
Supervised writing of the manuscript: AA.
References
1. Xavier RJ, Podolsky DK (2007) Unravelling the pathogenesis of inflammatory
bowel disease. Nature 448: 427–434.
2. Sartor RB (2008) Microbial influences in inflammatory bowel diseases.
Gastroenterology 134: 577–594.
3. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, et al. (2008) Genome-
wide association defines more than 30 distinct susceptibility loci for Crohn’s
disease. Nat Genet 40: 955–26.
4. Anderson CA, Boucher G, Lees CW, Franke A, D’Amato M, et al. (2011) Meta-
analysis identifies 29 additional ulcerative colitis risk loci, increasing the number
of confirmed associations to 47. Nat Genet 43: 246–52.
5. Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, et al. (2010)
Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s
disease susceptibility loci. Nat Genet 42: 1118–25.
6. Wellcome Trust Case Control Consortium (2007) Genome-wide association
study of 14,000 cases of seven common diseases and 3,000 shared controls.
Nature 47: 61–87.
7. Franke A, Balschun T, Karlsen TH, Hedderich J, May S, et al. (2008)
Replication of signals from recent studies of Crohn’s disease identifies previously
unknown disease loci for ulcerative colitis. Nat Genet 40: 713–5.
8. Thie ´baut R, Kotti S, Jung C, Merlin F, Colombel JF, et al. (2009) TNFSF15
polymorphisms are associated with susceptibility to inflammatory bowel disease
in a new European cohort. Am J Gastroenterol 104: 384–91.
9. Silverberg MS, Cho JH, Rioux JD, McGovern DP, Wu J, et al. (2009) Ulcerative
colitis-risk loci on chromosomes 1p36 and 12q15 found by genome-wide
association study. Nat Genet 41: 216–20.
10. Kugathasan S, Baldassano RN, Bradfield JP, Sleiman PM, Imielinski M, et al.
(2008) Loci on 20q13 and 21q22 are associated with pediatric-onset
inflammatory bowel disease. Nat Genet 40: 1211–1215.
11. Podolsky DK (2002) Inflammatory bowel disease. N Engl J Med 347: 417–29.
12. Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, et al. (2005)
Toward an integrated clinical, molecular and serological classification of
inflammatory bowel disease: Report of a Working Party of the 2005 Montreal
World Congress of Gastroenterology. Can J Gastroenterol 19(Suppl A): 5–36.
13. IBD Working Group of the European Society for Paediatric Gastroenterology,
Hepatology and Nutrition (2005) Inflammatory bowel disease in children and
adolescents: recommendations for diagnosis–the Porto criteria. J Pediatr
Gastroenterol Nutr 41: 1–7.
14. Munkholm P, Langholz E, Davidsen M, Binder V (1994) Frequency of
glucocorticoid resistance and dependency in Crohn’s disease. Gut 35: 360–2.
15. Faubion WA, Jr., Loftus EV, Jr., Harmsen WS, Zinsmeister AR, Sandborn WJ
(2001) The natural history of corticosteroid therapy for inflammatory bowel
disease: a population-based study. Gastroenterology 121: 255–60.
16. Imielinski M, Baldassano RN, Griffiths A, Russell RK, Annese V, et al. (2009)
Common variants at five new loci associated with early-onset inflammatory
bowel disease. Nat Genet 41: 1335–1340.
17. The UK IBD Genetics Consortium, the Wellcome Trust Case Control
Consortium 2 (2009) Genome-wide association study of ulcerative colitis
identifies three new susceptibility loci, including the HNF4A region. Nat Genet
12: 1330–1334.
18. McGovern DP, Gardet A, To ¨rkvist L, Goyette P, Essers J, et al. (2010) Genome-
wide association identifies multiple ulcerative colitis susceptibility loci. Nat Genet
42: 332–7.
19. Amre DK, Mack DR, Morgan K, Fujiwara M, Israel D, et al. (2009)
Investigation of Reported Associations Between the 20q13 and 21q22 Loci and
Pediatric-Onset Crohn’s Disease in Canadian Children. Am J Gastroenterol
104: 2824–8.
20. Yamazaki K, McGovern D, Ragoussis J, Paolucci M, Butler H, et al. (2005)
Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn’s
disease. Hum Mol Genet 14: 3499–3506.
21. Tremelling M, Berzuini C, Massey D, Bredin F, Price C, et al. (2008)
Contribution of TNFSF15 gene variants to Crohn’s disease susceptibility
confirmed in UK population. Inflamm Bowel Dis 14: 733–7.
22. Picornell Y, Mei L, Taylor K, Yang H, Targan SR, et al. (2007) TNFSF15 is an
ethnic-specific IBD gene. Inflamm Bowel Dis 13: 1333–1338.
23. Yang SK, Lim J, Chang HS, Lee I, Li Y, et al. (2008) Association of TNFSF15
with Crohn’s disease in Koreans. Am J Gastroenterol 103: 1437–42.
24. Migone TS, Zhang J, Luo X, Zhuang L, Chen C, et al. (2002) TL1A is a TNF-
like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator.
Immunity 16: 479–92.
25. Prehn JL, Mehdizadeh S, Landers CJ, Luo X, Cha SC, et al. (2004) Potential
role for TL1A, the new TNF-family member and potent costimulator of IFN-
gamma, in mucosal inflammation. Clin Immunol 112: 66–77.
26. Parkes M, Barrett JC, Prescott NJ, Tremelling M, Anderson CA, et al. (2007)
Sequence variants in the autophagy gene IRGM and multiple other replicating
loci contribute to Crohn’s disease susceptibility. Nat Genet 39: 830–2.
27. Fisher SA, Tremelling M, Anderson CA, Gwilliam R, Bumpstead S, et al. (2008)
Genetic determinants of ulcerative colitis include the ECM1 locus and five loci
implicated in Crohn’s disease. Nat Genet 40: 710–2.
28. Pabst O, Fo ¨rster R, Lipp M, Engel H, Arnold HH (2000) NKX2.3 is required
for MAdCAM-1 expression and homing of lymphocytes in spleen and mucosa-
associated lymphoid tissue. EMBO J 2: 2015–23.
29. Todd JA, Walker NM, Cooper JD, Smyth DJ, Downes K, et al. (2007) Robust
associations of four new chromosome regions from genome-wide analyses of type
1 diabetes. Nat Genet 7: 857–64.
30. Weersma RK, Stokkers PC, Cleynen I, Wolfkamp SC, Henckaerts L, et al.
(2009) Confirmation of multiple Crohn’s disease susceptibility loci in a large
Dutch-Belgian cohort. Am J Gastroenterol 104: 630–8.
31. Amre DK, Mack DR, Morgan K, Israel D, Deslandres C, et al. (2010)
Susceptibility loci reported in genome-wide association studies are associated with
Crohn’s disease in Canadian children. Aliment Pharmacol Ther 31: 1186–91.
32. Satsangi J, Welsh KI, Bunce M, Julier C, Farrant JM, et al. (1996) Contribution
of genes of the major histocompatibility complex to susceptibility and disease
phenotype in inflammatory bowel disease. Lancet 4: 1212–7.
33. Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, McLeod RS, et al. (2000)
Genomewide search in Canadian families with inflammatory bowel disease
reveals two novel susceptibility loci. Am J Hum Genet 66: 1863–70.
34. International MHC and Autoimmunity Genetics Network, Rioux JD, Goyette P,
Vyse TJ, Hammarstro ¨m L, et al. (2009) Mapping of multiple susceptibility
variants within the MHC region for 7 immune-mediated diseases. Proc Natl
Acad Sci U S A 106: 18680–5.
35. Fisher SA, Tremelling M, Anderson CA, Gwilliam R, Bumpstead S, et al. (2008)
Genetic determinants of ulcerative colitis include the ECM1 locus and five loci
implicated in Crohn’s disease. Nat Genet 40: 710–2.
36. Franke A, Balschun T, Karlsen TH, Sventoraityte J, Nikolaus S, et al. (2008)
Sequence variants in IL10, ARPC2 and multiple other loci contribute to
ulcerative colitis susceptibility. Nat Genet 40: 1319–23.
37. Asano K, Matsushita T, Umeno J, Hosono N, Takahashi A, et al. (2009) A
genome-wide association study identifies three new susceptibility loci for
ulcerative colitis in the Japanese population. Nat Genet 41: 1325–9.
38. Libioulle C, Louis E, Hansoul S, Sandor C, Farnir F, et al. (2007) Novel Crohn
disease locus identified by genome-wide association maps to a gene desert on
5p13.1 and modulates expression of PTGER4. PLoS Genet 3: e58.
39. Rioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, et al. (2007)
Genome-wide association study identifies new susceptibility loci for Crohn disease
and implicates autophagy in disease pathogenesis. Nat Genet 39: 596–604.
40. Essers JB, Lee JJ, Kugathasan S, Stevens CR, Grand RJ, et al. (2009) Established
genetic risk factors do not distinguish early and later onset Crohn’s disease.
Inflamm Bowel Dis 15: 1508–14.
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org7 July 2011 | Volume 6 | Issue 7 | e22688