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.
[show abstract] [hide abstract]
ABSTRACT: Recently, substantial advances in the understanding of the molecular pathogenesis of inflammatory bowel disease (IBD) have been made owing to three related lines of investigation. First, IBD has been found to be the most tractable of complex disorders for discovering susceptibility genes, and these have shown the importance of epithelial barrier function, and innate and adaptive immunity in disease pathogenesis. Second, efforts directed towards the identification of environmental factors implicate commensal bacteria (or their products), rather than conventional pathogens, as drivers of dysregulated immunity and IBD. Third, murine models, which exhibit many of the features of ulcerative colitis and seem to be bacteria-driven, have helped unravel the pathogenesis/mucosal immunopathology of IBD.Nature 08/2007; 448(7152):427-34. · 36.28 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The predominantly anaerobic microbiota of the distal ileum and colon contain an extraordinarily complex variety of metabolically active bacteria and fungi that intimately interact with the host's epithelial cells and mucosal immune system. Crohn's disease, ulcerative colitis, and pouchitis are the result of continuous microbial antigenic stimulation of pathogenic immune responses as a consequence of host genetic defects in mucosal barrier function, innate bacterial killing, or immunoregulation. Altered microbial composition and function in inflammatory bowel diseases result in increased immune stimulation, epithelial dysfunction, or enhanced mucosal permeability. Although traditional pathogens probably are not responsible for these disorders, increased virulence of commensal bacterial species, particularly Escherichia coli, enhance their mucosal attachment, invasion, and intracellular persistence, thereby stimulating pathogenic immune responses. Host genetic polymorphisms most likely interact with functional bacterial changes to stimulate aggressive immune responses that lead to chronic tissue injury. Identification of these host and microbial alterations in individual patients should lead to selective targeted interventions that correct underlying abnormalities and induce sustained and predictable therapeutic responses.Gastroenterology 03/2008; 134(2):577-94. · 11.68 Impact Factor
Article: TNFSF15 polymorphisms are associated with susceptibility to inflammatory bowel disease in a new European cohort.[show abstract] [hide abstract]
ABSTRACT: Inflammatory bowel disease (IBD), e.g., Crohn's disease (CD) and ulcerative colitis (UC), is a complex genetic disorder. Tumor necrosis factor (ligand) superfamily, member 15 (TNFSF15) has been previously identified as a susceptibility gene for CD in Japanese and UK cohorts. This replication study was designed in order to confirm and further validate the role of TNFSF15 in IBD. A total of 666 IBD families (corresponding to 2,982 relatives) with European ancestry were genotyped for the rs6478108 and rs7869487 polymorphisms, which define the main TNFSF15 haplotypes previously associated with CD. An association between the main haplotypes and CD, UC and IBD was tested using the Genehunter TDT and Unphased statistics. Caspase recruitment domain 15 (CARD15)/TNFSF15 interaction and genotype/phenotype correlations were also studied. The previously reported "high-risk" haplotype (A) was associated with IBD (P=0.001) (OR=1.25 (1.05-1.50)) and CD (P=0.02) (OR=1.31 (1.03-1.67)) whereas the "protective" (B) haplotype was significantly less transmitted to IBD and CD patients. No interaction between CARD15 and TNFSF15 was detected. We also failed to define a clinical subgroup of CD patients specifically associated with TNFSF15 haplotype A. This study confirms that TNFSF15 or a closely linked gene is involved in the genetic predisposition to CD.The American Journal of Gastroenterology 02/2009; 104(2):384-91. · 7.28 Impact Factor
Investigation of Multiple Susceptibility Loci for
Inflammatory Bowel Disease in an Italian Cohort of
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,
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
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 .
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 .
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
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
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
Extensive clinical characterization was available for all patients.
The diagnosis of CD or UC was established by conventional
clinical, radiological, endoscopic, and histological findings .
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 , two (rs10761659, rs2542151) by the
WTCCC  as showing the strongest association signals, two
(rs2395185, rs1558744) by the first UC GWA study , and one
(rs2836878) by a GWA analysis for early-onset IBD . 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 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
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
rs2395185 (P= 0.001), IFNG-IL22-IL26 rs1558744 (P
0.007), and PSMG1 rs2836878 (P = 0.047) were exclusively
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
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
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/
rs11190140 SNPs, but did not reach statistical significance.
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.
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
Table 1. Clinical and demographic characteristics of the
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 (%)
Penetrating 114 (11)
yes 188 (18)
No 651 (61) 908 (75)
yes419 (39) 305 (25)
No 959 (90)1107 (91)
yes 111 (10)106 (9)
Yes292 (32) 164 (15)
No 490 (53)622 (57)
Ex 140 (15) 311 (28)
No749 (70)1082 (89)
yes 321 (30) 131 (11)
Refractory83 (13) 101(13)
Responder569 (87) 673 (87)
IMS (AZT/6MP, Ciclo, MTX)
No 676 (42) 927(58)
Yes394 (58) 286 (42)
No 914 (85) 1168 (96)
Yes 156 (15) 45 (4)
CD: Crohn’s disease; UC: ulcerative colitis; EIM: extra-intestinal manifestations.
*For 148 CD and 116 UC patients the information is missing.
Table 1. Cont.
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org3 July 2011 | Volume 6 | Issue 7 | e22688
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
A summary of previously described allelic distributions of SNPs
of genes/loci analyzed is depicted in Supplementary Table S1.
Kugathasan et al  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). In keeping with previous finding is
the contributionof the PSMG1 locus to disease susceptibilityin adult
UC . McGovern et al  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 (Table S1).
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.
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.
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org4July 2011 | Volume 6 | Issue 7 | e22688
A significant association of geneticvariants of the TNFSF15 (TL1A)
Japanese patients, in several European cohorts [8,20,21], in US
Jewish patients , in the combined data from the NIDDK IBD
Consortium and the WTCCC , in Koreans patients , and in
UC GWA study . 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  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  supported this findings with an
independent set of CD cases and controls of European descent. In
addition, a large-scale meta-analysis  on CD cohort and
replication study on IBD samples  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 , in GWA
scan for UC , 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%)
rs426383935 (5.5%) 223 (35.2%) 376 (59.3%)22 (7.8%) 99 (35.4%) 159 (56.8%)
rs11190140 116 (19.4%)301 (50.4%)180 (30.2%) 47 (17.2%) 136 (49.8%) 90 (33.0%)
rs10761659 187 (30.4%)301 (48.8%) 128 (20.8%)53 (29.8%)78 (43.8%) 47 (26.4%)
rs2542151 13 (2.1%)169 (27.8%) 426 (70.1%)8 (3.0%) 60 (22.8%)196 (74.2%)Adult
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%)
rs2395185 19 (3.1%) 176 (28.4%)425 (68.5%)10 (6.0%) 45 (26.9%) 112 (67.1%)
rs4263839 50 (7.5%) 289 (43.4%) 327 (49.1%)16 (6.8%)86(36.6%) 133 (56.6%)
rs11190140132 (19.8%)313 (46.9%) 222 (33.3%)30 (12.6%) 125 (52.3%)84 (35.1%)
rs10761659 121 (27.7%)235 (53.8%)81 (18.5%)47 (32.2%) 74 (50.7%)25 (17.1%)
rs1558744127 (18.7%)329 (48.5%) 223 (32.8%)40 (26.1%)72 (47.1%) 41 (26.8%)
rs25421518 (1.2%) 169 (24.9%)500 (73.9%)2 (0.8%) 63 (26.6%) 172 (72.6%)
rs2836878 30 (4.2%)252 (35.6%) 426 (60.2%)2 (1.2%) 50 (30.1%)114 (68.7%)
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org5 July 2011 | Volume 6 | Issue 7 | e22688
replication studies (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
A highly attractive candidate gene for IBD, owing to its anti-
inflammatory function and involvement in type 1 diabetes
susceptibility and rheumatoid arthritis , 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 , replicated [26,30] and confirmed . In contrast,
Franke et al  observed this association with UC, a finding
replicated in the recent GWA on UC . 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-
The UC GWAS in European ancestry samples  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 (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 =
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 . This
association to the DRB1 locus is consistent with other published
GWAs in UC [9,35,36], also in Japanese population . The
same region was identified in the meta-analysis of CD genome-
wide association studies . Recent genome-wide association
study in early-onset IBD  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 =
In the study of Libioulle et al  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  identified rs4613763 as the most
strongly associated SNP. No highly significant association of the
PTGER4 region was documented in the studies of the NIDDK
 and the WTCCC . Recently GWAS showed significant
evidence for association also with UC  (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 , 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
Adult n=728Pediatric n=342Adult n=952 Pediatric n=261
OR 2.07 CI 95% (1.10–3.89)
family history P,0.01
OR 3.68 CI 95% (1.93–7.04)
OR 4.16 CI 95% (1.62–10.71)
OR 3.81 CI 95%(1.45–9.97)
colon vs ileum P=0.024
OR 1.67 CI 95% (1.06–2.62)
colon vs ileum P=0.021
OR 1.54 CI 95% (1.06–2.22)
OR 1.46 CI 95% (1.06–2.00)
OR 0.64 CI 95% (0.42–0.97)
SNPs Analysis in Italian IBD
PLoS ONE | www.plosone.org6 July 2011 | Volume 6 | Issue 7 | e22688
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
phisms (SNP) analyzed in the previously published studies and in
the current study.
Allelic distributions of single nucleotide polymor-
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.
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.
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PLoS ONE | www.plosone.org7 July 2011 | Volume 6 | Issue 7 | e22688