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Relationships between genetic polymorphisms of triggering receptor expressed on myeloid cells-1 and inflammatory bowel diseases in the Korean population
Life Sciences 89 (2011) 289–294
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Relationships between genetic polymorphisms of triggering receptor expressed on myeloid cells-1 and inflammatory bowel diseases in the Korean population Eun Suk Jung a, b, 1, Seung Won Kim b, 1, Chang Mo Moon a, Dong-Jik Shin c, Nak-Hoon Son c, Eun Soo Kim a, Hyun Jung Lee a, Sung Pil Hong a, Tae Il Kim a, Won Ho Kim a, b, Jae Hee Cheon a, b,⁎ a b c
Department of Internal Medicine and Institute of Gastroentorology, Yonsei University College of Medicine, Seoul, Republic of Korea Department of Internal Medicine and Institute of Gastroenterology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea Cardiovascular Genome Center, Yonsei University College of Medicine & Yonsei University Research Institute of Science for Aging, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 13 January 2011 Accepted 7 June 2011 Keywords: Crohn's disease Genetic polymorphism Inflammatory bowel diseases Intestinal Behcet disease TREM1 Ulcerative colitis
a b s t r a c t Aims: Triggering receptor expressed on myeloid cells-1 (TREM-1) has been shown to play a crucial role in the propagation of inflammatory responses. Recent studies have reported that TREM-1 expression is up-regulated in patients with inflammatory bowel disease (IBD). Therefore, we investigated the associations between TREM-1 genetic polymorphisms and IBD development and its phenotypes in the Korean population. Main methods: Three TREM-1 single nucleotide polymorphisms (SNPs, rs2234237, rs3789205, and rs9471535) were genotyped by Taqman technology on 202 Crohn's disease (CD), 265 ulcerative colitis (UC), 138 with intestinal Behcet's disease (BD), and 234 healthy controls and the relationships between these SNPs and IBD development and phenotypes were evaluated. Key findings: We found that TREM-1 SNPs are significantly associated with the development of intestinal Behcet's disease (rs9471535: odds ratio [OR] = 1.637, P = 0.025; rs3789205: OR = 1.668, P = 0.019; rs2234237: OR = 1.691, P = 0.016), and in particular with skin involvement (rs9471535: OR = 2.723, P = 0.009; rs3789205: OR = 2.477, P = 0.017; rs2234237: OR = 2.278, P = 0.030) and the risk of azathioprine use (rs9471535: OR = 2.722, P = 0.021; rs3789205: OR = 2.493, P = 0.032; rs2234237: OR = 2.638, P = 0.026). However, TREM-1 SNPs were not significantly associated with the development of Crohn's disease or ulcerative colitis. Significance: The results of our study suggest that TREM-1 SNPs may play a significant role in the development of intestinal Behcet's disease and may have modest effects on disease severity. © 2011 Elsevier Inc. All rights reserved.
Introduction Inflammatory bowel disease (IBD) is a chronic intestinal disease with repeated episodes of relapse and remission due to unknown causes (Hendriksen et al., 1985; Moum et al., 1997; Munkholm et al., 1995; Stonnington et al., 1987). The prevalence and incidence of IBD differ among regions. IBD is most common in people of European descent who live in North America and Northern Europe. Recently, the number of IBD patients has rapidly increased in Korea due to the westernization of the Korean diet, improvement of hygiene status, and reduction of infectious diseases (Yang et al., 2001; Kim and Kim, 2010). In contrast, another form of multisystemic, chronic, relapsing intestinal inflammatory disorder, intestinal Behcet's disease (BD), is
⁎ Corresponding author at: Department of Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Republic of Korea. Tel.: +82 2 2228 1990; fax: +82 2 393 6884. E-mail address: [email protected] (J.H. Cheon). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.06.018
most prevalent in Eastern Mediterranean countries and East Asian populations including Koreans (Kaklamani et al., 1998). Although the pathogenesis of IBD, including intestinal BD, is still unclear, genetic and environmental factors are thought to be involved (Bayraktar et al., 2000; Duchmann et al., 1997; Kasahara et al., 1981; MacDermott, 1996; Reif et al., 1997). Additional evidence of genetic influences on IBD development include a higher frequency in identical twins compared to fraternal twins, people with a family history of IBD, and differences in the prevalence of IBD between races (Orholm et al., 1991; Probert et al., 1993). IBD is considered an autoimmune disease caused by an abnormal immune reaction to intestinal bacteria that occurs in patients with genetic susceptibilities (Sartor, 2006; Xavier and Podolsky, 2007). Triggering receptor expressed on myeloid cell-1 (TREM-1), a 30kDA immunoglobulin superfamily expressed on blood neutrophils and a subset of monocytes, has been shown to play a crucial role in the propagation of local or systemic inflammatory responses (Colonna, 2003). Elevated levels of soluble form of TREM-1 (sTREM-1) have been observed in several infectious diseases (Cohen, 2001; Gibot and Cravoisy, 2004). A sTREM-1 was identified at significant levels in the
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sera of patients with sepsis and appeared to be the most helpful parameter in differentiating patients with sepsis from those with systemic inflammatory response syndrome (Gibot et al., 2004b). In addition, an elevated level of sTREM-1 in bronchoavelolar lavage fluid was found to be a strong predictor of pneumonia (Gibot et al., 2004a). As for gastroenterological diseases, pancreatic tissue TREM-1 mRNA expression was correlated with the severity of pancreatitis (Wang et al., 2004), and the sTREM-1 level in gastric juice was also correlated with a degree of gastric inflammation scores (Koussoulas et al., 2006). Recently, it was reported that TREM-1 is normally underexpressed in the human intestine, which causes immune tolerance to intestinal bacteria and antigens (Schenk et al., 2005). However, TREM-1 is up-regulated in the intestines of patients with IBD (Schenk et al., 2007). We previously reported that sTREM-1 levels are upregulated in the sera of IBD patients and correlate with disease activity in patients with ulcerative colitis (Park et al., 2009). Nevertheless, it remains unknown whether genetic polymorphisms of TREM-1 influence genotypes and phenotypes in IBD patients. We hypothesized that genetic polymorphisms of TREM-1 might be associated with the development of IBD and correlated with disease phenotypes in IBD. Accordingly, we analyzed TREM-1 single nucleotide polymorphisms (SNPs) and evaluated the relationships between TREM-1 polymorphisms and the development and phenotypes of IBD. Materials and methods Study sample We surveyed 839 unrelated Korean subjects including 202 patients with Crohn's disease (CD), 265 with ulcerative colitis (UC), 138 with intestinal Behcet's disease (BD), and 234 healthy controls between July 2006 and October 2007. All patients were diagnosed and managed at the gastroenterology clinics of Yonsei University Health System, Severance Hospital, Seoul, Republic of Korea. The diagnoses of CD, UC, or intestinal BD were based on established clinical, radiographic, endoscopic, and histopathologic criteria as previously described. CD and UC were diagnosed according to the Lennard–Jones criteria (Lennard–Jones, 1989). Intestinal BD was diagnosed according to the criteria suggested by our group and the Behcet's Disease Research Committee of Japan in 1987 (Cheon et al., 2009; Mizushima et al., 1988). Exclusion criteria for this study were indeterminate colitis, other autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.), chronic hepatitis C, asthma, infection, or coexistence of serious medical or surgical conditions. Healthy controls were recruited among health care center customers who had no gastrointestinal symptoms, took no regular medications and had a normal complete blood count and a normal biochemical profile, including ESR and CRP. All control subjects underwent colonoscopy for routine check-up which revealed grossly normal findings. Demographic and clinical characteristics were obtained by reviewing the medical records of the subjects and through detailed questionnaires. The CD patients were subdivided into groups according to age at diagnosis, location, and behavior of disease using the Montreal Classification (Silverberg et al., 2005). In patients with UC, anatomic location was also sub-grouped using the Montreal Classification as being ulcerative proctitis (E1), left-sided UC (E2), and extensive UC (E3). Other characteristics including age at diagnosis, gender, extraintestinal manifestations (EIMs), total follow-up period, the use of immunosuppressive drugs, and the need for surgical intervention were also assessed. The intestinal BD patients were classified into 3 groups using colonoscopic findings and extraintestinal systemic manifestations: (i) complete group, (ii) probable group, or (iii) suspected group (Cheon et al., 2009). This study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki and was approved by the
Institutional Review Board of Yonsei University College of Medicine. Informed written consent was obtained from all subjects.
TREM-1 single nucleotide polymorphism selection and genotyping We genotyped 3 TREM-1 SNPs (rs9471535, rs2234237, and rs3789205). These SNPs were chosen for this study based on a previous Chinese cohort study (Chen et al., 2008) and International HapMap data set (http://www.hapmap.org) regarding the TREM-1 gene (GenBank access no. DQ217941) in patients and healthy controls, of which SNP rs9471535 was located in promoter region of the TREM-1 gene, rs3789205 in intron 1 and rs2234237 in exon 2. Genomic DNA was isolated from whole blood using commercially available kits (Qiagen, Chatsworth, CA, USA). SNPs were genotyped using the TaqMan® fluorogenic 5′ nuclease assay (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. The final volume of each polymerase chain reaction (PCR) was 5 μL, including 20 ng of genomic DNA, 2.5 μL TaqMan® Universal PCR master mix (Applied Biosystems), and 0.22 μL of TaqMan® SNP Genotyping Assay Mix (40×). Amplification conditions were 50 °C for 2 min to activate the uracil N-glycosylase and to prevent carryover contamination, then 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The reaction was performed in a 384-well format using a Dual 384-Well GeneAmp® PCR System 9700 (Applied Biosystems). The TaqMan® assay plate was transferred to ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems), where the endpoint fluorescence intensity of each well was read. The calculation of the fluorescence data from each plate was performed using SDS software version 2.3 (Applied Biosystems). Statistical analysis All SNPs investigated in this study were tested for Hardy– Weinberg equilibrium (HWE) in controls using the χ 2-test. Associations between SNPs and disease susceptibility were determined by comparing allele, and genotype frequencies between cases and controls using the χ 2-test or Fisher's exact test. Relationships between TREM-1 polymorphisms and phenotypes were assessed using logistic regression analysis under a dominant (risk allele homozygotes plus heterozygotes versus wild type allele homozygotes), a recessive (risk allele homozygotes versus heterozygotes plus wild type allele homozygotes), and a codominant (wild type allele homozygotes versus heterozygotes versus risk allele homozygotes) genetic model. Odds ratios (OR) with 95% confidence intervals (CIs) were determined. To identify significant associations in disease phenotypes and genotypes, we performed comparative analyses within subgroups encompassing UC, CD, and intestinal BD patients by clinical characteristics such as gender, mean age at diagnosis, disease location and behavior, EIMs, history of immunosuppressive drug use, and surgical intervention using logistic regression analysis. P-values of less than 0.05 were considered statistically significant. The data were analyzed using PASW v.18.0 for Windows (SPSS Inc., Chicago, IL, USA). Results Demographic and clinical characteristics of study sample Demographic and clinical characteristics of the patients with CD, UC, intestinal BD, and healthy controls who were included in this study are summarized in Table 1. The mean age of the patients at the diagnosis of CD was 26.0 ± 11.2 years, with a gender ratio of 1.58:1 (male:female), that of UC was 36.4 ± 12.4 years, with a gender ratio of 1.01:1, and that of intestinal BD was 38.1 ± 11.1 years, with a gender ratio of 0.92:1. We had only one patient with UC accompanied with primary sclerosing cholangitis.
E.S. Jung et al. / Life Sciences 89 (2011) 289–294 Table 1 Demographic and clinical characteristics of patients with inflammatory bowel diseases and healthy controls. Characteristics
CD (n = 202)
UC (n = 265)
Intestinal BD Control (n=138) (n = 234)
Mean age at diagnosis (years)a ≤16 years 17–40 years N40 years Gender (male/female) Follow-up (months) a Disease localization (%)b Ileal (L1) ± L4 Colonic (L2) ± L4 Ileocolonic (L3) ± L4 Proctitis (E1) Left-sided (E2) Pancolitis (E3) Ileocecal region Other colonic region Disease behavior (%)b Inflammatory (B1) ± p Stricturing (B2) ± p Penetrating (B3) ± p Clinical subtype of BD (%) Complete Probable Suspected Extraintestinal manifestations (%) Joint Skin Eye Oral Genital Liver Use of immunosuppressive drugs (%) Use of infliximab (%) History of surgical interventions (%)
26.0±11.2 26 (12.9%) 156 (77.2%) 20 (9.9%) 124/78 89.3±51.7
36.4±12.4 8 (3.0%) 167 (63.0%) 90 (34.0%) 133/132 103.6±61.3
38.1±11.1 4 (2.9%) 83 (60.1%) 51 (37.0%) 66/72 89.9±54.7
44.0±10.6
133/101
60 (29.7) 24 (11.9) 115 (56.9) 64 (24.2) 91 (34.3) 110 (41.5) 116(84.1) 22 (15.9) 81 (40.1) 36 (17.8) 78 (38.6) 107 (77.5) 7 (5.1) 24 (17.4)
19 (9.4) 10 (5.0) 11 (5.4)
41 (15.5) 25 (9.4) 37 (14.0)
48 (34.8) 60 (43.5) 24 (17.4) 111 (80.4) 42 (30.4)
110 (54.5)
1 (0.4) 69 (26.0)
43 (31.1)
36 (17.8) 80 (39.6)
10 (3.8)
41 (29.7)
Abbreviations: CD, Crohn's disease; UC, ulcerative colitis; BD, Behcet's disease. a Mean ± standard deviation. b Disease localization and behavior according to Montreal Classification (L4, upper gastrointestinal involvement; p, perianal disease).
Associations of TREM-1 genetic polymorphisms with disease susceptibility to inflammatory bowel disease Three candidate SNPs in TREM-1 were genotyped to determine their relationships with disease susceptibility to IBD. No significant deviations from HWE were observed in controls. We evaluated the relationships of TREM-1 genetic polymorphisms with disease susceptibility to CD, UC, and intestinal BD using a multiple logistic regression model (Table 2). We found that TREM-1 genetic polymorphisms were significantly correlated only with intestinal BD in terms of disease development. TREM-1 rs9471535 C allele in promoter region was significantly associated with the occurrence of intestinal BD under dominant model (TT vs CT/CC, OR = 1.637, 95% CI, 1.063 to 2.518, P = 0.025). In rs3789205 at intron 1 and rs2234237 at the exon 2 of the TREM-1 gene, we observed similarly significant results (rs3789205: AA vs AG/GG, OR = 1.668, 95% CI, 1.086 to 2.562, P = 0.019 in dominant model; rs2234237: TT vs AT/AA, OR = 1.691, 95% CI, 1.100 to 2.602, P = 0.016 in dominant model). In CD and UC patients, however, we observed no significant correlations between genotype distributions or combined genotype frequencies of any 3 SNPs in TREM-1. The linkage disequilibrium (LD) pattern of the 3 SNPs of TREM-1 gene is shown in Fig. 1. All of the 3 SNPs were located in the same haplotype block and were in strong LD (D′ N 0.99, r 2 N 0.96).
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Associations of TREM-1 genetic polymorphisms with intestinal BD phenotypes We also evaluated the relationships between the TREM-1 SNPs and phenotypes of intestinal BD (Table 3). We included all other covariates (gender, age at diagnosis, and use of immunosuppressive drugs) in a multiple logistic regression analysis in order to adjust for the different clinical manifestations. We found that dominant models of all 3 SNPs were associated with skin involvement (rs9471535: OR = 2.723, 95% CI, 1.285 to 5.770, P = 0.009; rs3789205: OR= 2.477, 95% CI, 1.179 to 5.203 P = 0.017; rs2234237: OR = 2.278, 95% CI, 1.085 to 4.785 P = 0.030) and a higher risk of azathioprine use (rs9471535: OR = 2.722, 95% CI, 1.167 to 6.352, P = 0.021; rs3789205: OR= 2.493, 95% CI, 1.079 to 5.759 P = 0.032; rs2234237: OR = 2.638, 95% CI, 1.120 to 6.211 P = 0.026) in intestinal BD. Discussion In this study, we examined the relationships between TREM-1 genetic polymorphisms and susceptibility to IBD and disease phenotypes. It was previously reported that TREM-1 is under-expressed in the human intestine which results in immune tolerance to intestinal bacteria and antigens (Schenk et al., 2005), while TREM-1 is upregulated in the intestine of patients with IBD (Schenk et al., 2007). In an experimental mouse model with colitis, TREM-1-expressing intestinal macrophages contributed to amplifying chronic inflammation (Schenk et al., 2007). Our group also reported that sTREM-1 levels are upregulated in the sera of IBD patients and correlate with disease activity in patients with UC (Park et al., 2009) and intestinal BD (Jung et al., in press). Furthermore, a study investigating an association between TREM-1 gene polymorphisms and severe sepsis was conducted in a Chinese cohort (Chen et al., 2008). Based on these observations, development and phenotypes of IBD could be different among TREM-1 SNPs. Therefore, we hypothesized that TREM-1 genetic polymorphisms might be associated with the development of CD and UC. To the authors' knowledge, this is the first attempt to investigate the correlations of TREM-1 SNPs with IBD genotypes and phenotypes. However, TREM-1 genetic polymorphisms did not show a significant association with the development of CD and UC. These findings suggest that TREM-1 genetic polymorphisms do not have a strong impact on CD or UC susceptibility. However, further studies are needed to exclude the possibility that this finding may have been due to the small sample size of this study, or to exclude other unknown SNPs from consideration. We also examined the relationships between TREM-1 genetic polymorphisms and susceptibility to intestinal BD. Three common markers showed a similarly significant association. The SNP (rs9471535) located within the 5′ flanking region of the reference TREM-1 gene may impact the transcriptional activity, and other nonsynonymous variation Ser25Thr (rs2234237) in the exon 2 of the TREM-1 gene may influence the biologic function of TREM-1. All of the 3 SNPs were located in the same haplotype block and were in tight LD, which suggests that the TREM-1 gene may be strongly involved in intestinal BD susceptibility. Intestinal BD is similar to CD in terms of its clinical features and therapeutic implications (Kim et al., 1999). Both diseases develop at a young age, have similar gastrointestinal and extraintestinal manifestations, and have chronic relapsing and remission courses. Because of these clinical similarities, it is occasionally difficult to distinguish between intestinal BD and CD. Despite similar manifestations in clinical practice, intestinal BD is generally agreed to be a distinct entity (Lee et al., 2001; Smith et al., 1973). Previous genetic studies of BD have shown that HLAB⁎51 carriers were more likely to develop BD, and that the frequency of HLA-B⁎51 was much higher in male patients (de Menthon et al., 2009; Ohno et al., 1982; Ono et al., 1973). Moreover, tumor necrosis factor-α1031 T/C polymorphisms were reported be associated with BD susceptibility (Akman et al., 2006). The distribution of IL-8 gene haplotype was also associated with BD (Lee et al., 2007). Recent studies
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Table 2 Associations of triggering receptor expressed on myeloid cells-1 (TREM-1) single nucleotide polymorphism genotypes between patients with inflammatory bowel diseases and healthy controls. rs number
Genotype
Intestinal BD
P-value
OR (95% CI)
rs9471535 Dominant
TT
(n = 134) 53
0.025
1.637 (1.063–2.518)
CT/CC TT/CT
81 125
0.372
0.694 (0.310–1.554)
CC TT CT
9 53 72
0.001
CC
9
0.805
AA
(n = 137) 53
0.019
1.668 (1.086–2.562)
AG/GG AA/AG
84 127
0.486
0.759 (0.348–1.654)
GG AA AG
10 53 74
0.008
GG
10
0.984
TT
(n = 138) 51
0.016
1.691 (1.100–2.602)
AT/AA TT/AT
87 127
0.630
0.831 (0.390–1.770)
TT TT AT
11 51 76
0.009
AA
11
0.824
Recessive
Codominant
rs3789205 Dominant
Recessive
Codominant
rs2234237 Dominant
Recessive
Codominant
1.813 (1.166–2.820) 0.900 (0.389–2.082)
1.802 (1.160–2.800) 0.991 (0.440–2.236)
1.797 (1.155–2.796) 1.094 (0.494–2.419)
CD
P-value
OR (95% CI)
(n = 196) 96
0.573
1.115 (0.763–1.631)
100 183
0.296
0.685 (0.335–1.397)
13 96 87
0.407
13
0.401
(n = 196) 95
0.561
1.119 (0.766–1.636)
101 184
0.209
0.628 (0.303–1.305)
1.182 (0.796–1.756) 0.729 (0.349–1.525)
12 95 89
0.297
112
0.453
(n = 197) 95
0.747
1.065 (0.728–1.556)
102 185
0.200
0.622 (0.300–1.292)
12 95 90
0.644
12
0.884
1.234 (0.830–1.837) 0.754 (0.361–1.576)
0.915 (0.630–1.330) 1.046 (0.566–1.936)
UC
P-value
OR (95% CI)
(n = 256) 137
0.689
0.930 (0.652–1.327)
119 231
0.891
1.043 (0.571–1.905)
25 137 94
0.657
25
0.973
(n = 261) 137
0.788
0.953 (0.669–1.356)
124 234
0.726
1.112 (0.615–2.011)
0.918 (0.630–1.338) 0.989 (0.531–1.844)
27 137 97
0.297
27
0.453
(n = 260) 134
0.698
0.932 (0.655–1.328)
126 233
0.727
1.111 (0.614–2.011)
27 134 99
0.407
27
0.401
1.234 (0.830–1.837) 0.754 (0.361–1.576)
1.182 (0.795–1.756) 0.729 (0.348–1.525)
Control (n = 234) 121 113 212 22 121 91 22 (n = 234) 120 114 212 22 120 92 22 (n = 233) 116 117 211 22 116 95 22
Abbreviations: BD, Behcet's disease; CD, Crohn's disease; UC, ulcerative colitis; OR, odds ratio; CI, confidence interval.
Fig. 1. Linkage disequilibrium structure of the 3 triggering receptor expressed on myeloid cell-1 (TREM-1) single nucleotide polymorphisms (SNPs) in Koreans. The linkage disequilibrium (LD) patterns of the 3 SNPs in TREM-1 gene with D′ values are shown (D′ N 0.99, r2 N 0.96).
showed that SNPs of TLR-4 (Horie et al., 2009) and IL-17 F may influence the susceptibility of BD (Jang et al., 2008). Baranathan et al. (2007) reported that protein tyrosine phosphatase type 22 (PTPN22 620W) was inversely associated with BD susceptibility. Gunesacar et al. (2007) reported that SNP of the CD 28 gene was increased in BD patients, whereas the CTLA-4 gene was decreased. With regard to intestinal BD, however, there have been no reports on genetic polymorphisms. Therefore, a genetic polymorphism study is necessary with respect to intestinal BD. Accordingly, we hypothesized that TREM-1 genetic polymorphisms might be associated with intestinal BD susceptibility. To the best of our knowledge, this is the first trial to explore the relationships between genetic polymorphisms and intestinal BD. Our data indicate that all 3 TREM-1 SNPs were significantly associated with susceptibility to intestinal BD. According to these results, intestinal BD may differ genetically from other IBDs such as CD or UC. Some groups have claimed that intestinal BD and CD are the same disease with different spectra (Kim et al., 2007; Yim and White, 1985). However, based on our observations, these two diseases should be considered different disease entities. One of the main histological findings of intestinal BD is neutrophil infiltrations around the ulcers. Hayasaki et al. (2004) showed that neutrophilic phlebitis may be involved in the pathogenesis of intestinal BD. Because TREM-1 is mainly expressed in neutrophils and monocytes, TREM-1 might contribute to the pathogenesis of intestinal BD. Moreover, we found that TREM-1 SNPs are associated with skin involvement as well as the use of azathioprine as a treatment for intestinal BD. The association of TREM-1 SNPs and use of
E.S. Jung et al. / Life Sciences 89 (2011) 289–294
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Table 3 Associations of triggering receptor expressed on myeloid cells-1 (TREM-1) single nucleotide polymorphism genotypes with intestinal Behcet's disease phenotypes. Disease phenotype
rs number
Genotype
Patients without phenotype (%)
Patients with phenotype (%)
Unadjusted OR (95% CI)
Adjusted OR (95% CI)
Skin
rs9471535 Dominant
TT
37 (49.3) 38 (50.7)
16 (27.1) 43 (72.9)
2.617⁎⁎ (1.260–5.435)
2.723⁎⁎ (1.285–5.770)
37 (47.4) 41 (52.6)
16 (27.1) 43 (72.9)
2.425⁎ (1.174–5.012)
2.477⁎ (1.179–5.203)
35 (44.9) 43 (55.1)
16 (26.7) 44 (73.3)
2.238⁎ (1.083–4.624)
2.278⁎ (1.085–4.785)
42 (45.7) 50 (54.3)
11 (26.2) 31 (73.8)
2.367⁎ (1.063–5.273)
2.722⁎ (1.167–6.352)
42 (44.2) 53 (55.8)
11 (26.2) 31 (73.8)
2.233⁎ (1.005–4.961)
2.493⁎ (1.079–5.759)
41 (42.7) 55 (57.3)
10 (23.8) 32 (76.2)
2.385⁎ (1.054–5.401)
2.638⁎ (1.120–6.211)
CT/CC rs3789205 Dominant
AA AG/GG
rs2234237 Dominant
TT AT/AA
Azathioprine use
rs9471535 Dominant
TT CT/CC
rs3789205 Dominant
AA AG/GG
rs2234237 Dominant
TT AT/AA
a
Abbreviations: OR, odds ratio; CI, confidence interval. a After adjustment for all other covariates (gender, age at diagnosis, and use of immunosuppressive drugs). ⁎ P b 0.05. ⁎⁎ P b 0.01.
azathioprine suggests that the presence of TREM-1 SNPs are associated with higher disease activity of intestinal BD. TREM-1 SNPs, however, are not associated with increased necessity of operation. Moreover, we found that these 3 SNPs are not correlated with sudden serious complications such as perforation. Therefore, we hypothesize that TREM-1 SNPs have a modest effect on intestinal BD manifestations. However, there were no significant associations between TREM-1 SNPs and oral, genital tract, joint, or eye involvement. Further study is warranted to determine the relationships of TREM-1 SNPs with not only intestinal BD but also systemic BD. Our study has several limitations. First, our study is limited by its small sample size. Because of this small sample size, we were unable to demonstrate strong associations between TREM-1 genetic polymorphisms and disease phenotypes in recessive disease models. A larger Korean cohort study is necessary to validate our data. Second, our study investigated only 3 SNPs of TREM-1. Supplementary research is necessary in order to uncover new SNPs that might be further related to IBD susceptibility through the full sequencing of TREM-1 gene and haplotype-based association study. In addition, it would be necessary to search new genes that have similar action to TREM-1, and then analyze the gene-to-gene interaction and functional relevance of these genes. Finally, our results were obtained in a Korean sample and should be validated in other ethnic groups.
Conclusion In conclusion, TREM-1 SNPs have a significant association with the development of intestinal BD; in particular, its skin involvement and use of azathioprine for treatment. However, TREM-1 SNPs do not have a significant influence on the development of CD and UC. The results of our study suggest that TREM-1 SNPs could play a significant role in
the development of intestinal BD and may have modest effects on its disease severity. Conflict of interest statement The authors declare no conflict of interest.
Acknowledgments This study was supported by the Yonsei University College of Medicine, Internal Medicine Research Grant 2010, and the BumSuk Academic Research Fund of 2009. This paper was presented during the United European Gastroenterology Week 2010, in Barcelona, Spain. References Akman A, Sallakci N, Coskun M, Bacanli A, Yavuzer U, Alpsoy E, et al. TNF-alpha gene 1031 T/C polymorphism in Turkish patients with Behcet's disease. Br J Dermatol 2006;155(2):350–6. Baranathan V, Stanford MR, Vaughan RW, Kondeatis E, Graham E, Fortune F, et al. The association of the PTPN22 620W polymorphism with Behcet's disease. Ann Rheum Dis 2007;66(11):1531–3. Bayraktar Y, Ozaslan E, Van Thiel DH. Gastrointestinal manifestations of Behcet's disease. J Clin Gastroenterol 2000;30(2):144–54. Chen Q, Zhou H, Wu S, Wang H, Lv C, Cheng B, et al. Lack of association between TREM-1 gene polymorphisms and severe sepsis in a Chinese Han population. Hum Immunol 2008;69(3):220–6. Cheon JH, Kim ES, Shin SJ, Kim TI, Lee KM, Kim SW, et al. Development and validation of novel diagnostic criteria for intestinal Behcet's disease in Korean patients with ileocolonic ulcers. Am J Gastroenterol 2009;104(10):2492–9. Cohen J. TREM-1 in sepsis. Lancet 2001;358(9284):776–8. Colonna M. TREMs in the immune system and beyond. Nat Rev Immunol 2003;3(6): 445–53. de Menthon M, Lavalley MP, Maldini C, Guillevin L, Mahr A. HLA-B51/B5 and the risk of Behcet's disease: a systematic review and meta-analysis of case-control genetic association studies. Arthritis Rheum 2009;61(10):1287–96.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Relationships between genetic polymorphisms of triggering receptor expressed on myeloid cells-1 and inflammatory bowel diseases in the Korean population Eun Suk Jung a, b, 1, Seung Won Kim b, 1, Chang Mo Moon a, Dong-Jik Shin c, Nak-Hoon Son c, Eun Soo Kim a, Hyun Jung Lee a, Sung Pil Hong a, Tae Il Kim a, Won Ho Kim a, b, Jae Hee Cheon a, b,⁎ a b c
Department of Internal Medicine and Institute of Gastroentorology, Yonsei University College of Medicine, Seoul, Republic of Korea Department of Internal Medicine and Institute of Gastroenterology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea Cardiovascular Genome Center, Yonsei University College of Medicine & Yonsei University Research Institute of Science for Aging, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 13 January 2011 Accepted 7 June 2011 Keywords: Crohn's disease Genetic polymorphism Inflammatory bowel diseases Intestinal Behcet disease TREM1 Ulcerative colitis
a b s t r a c t Aims: Triggering receptor expressed on myeloid cells-1 (TREM-1) has been shown to play a crucial role in the propagation of inflammatory responses. Recent studies have reported that TREM-1 expression is up-regulated in patients with inflammatory bowel disease (IBD). Therefore, we investigated the associations between TREM-1 genetic polymorphisms and IBD development and its phenotypes in the Korean population. Main methods: Three TREM-1 single nucleotide polymorphisms (SNPs, rs2234237, rs3789205, and rs9471535) were genotyped by Taqman technology on 202 Crohn's disease (CD), 265 ulcerative colitis (UC), 138 with intestinal Behcet's disease (BD), and 234 healthy controls and the relationships between these SNPs and IBD development and phenotypes were evaluated. Key findings: We found that TREM-1 SNPs are significantly associated with the development of intestinal Behcet's disease (rs9471535: odds ratio [OR] = 1.637, P = 0.025; rs3789205: OR = 1.668, P = 0.019; rs2234237: OR = 1.691, P = 0.016), and in particular with skin involvement (rs9471535: OR = 2.723, P = 0.009; rs3789205: OR = 2.477, P = 0.017; rs2234237: OR = 2.278, P = 0.030) and the risk of azathioprine use (rs9471535: OR = 2.722, P = 0.021; rs3789205: OR = 2.493, P = 0.032; rs2234237: OR = 2.638, P = 0.026). However, TREM-1 SNPs were not significantly associated with the development of Crohn's disease or ulcerative colitis. Significance: The results of our study suggest that TREM-1 SNPs may play a significant role in the development of intestinal Behcet's disease and may have modest effects on disease severity. © 2011 Elsevier Inc. All rights reserved.
Introduction Inflammatory bowel disease (IBD) is a chronic intestinal disease with repeated episodes of relapse and remission due to unknown causes (Hendriksen et al., 1985; Moum et al., 1997; Munkholm et al., 1995; Stonnington et al., 1987). The prevalence and incidence of IBD differ among regions. IBD is most common in people of European descent who live in North America and Northern Europe. Recently, the number of IBD patients has rapidly increased in Korea due to the westernization of the Korean diet, improvement of hygiene status, and reduction of infectious diseases (Yang et al., 2001; Kim and Kim, 2010). In contrast, another form of multisystemic, chronic, relapsing intestinal inflammatory disorder, intestinal Behcet's disease (BD), is
⁎ Corresponding author at: Department of Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Republic of Korea. Tel.: +82 2 2228 1990; fax: +82 2 393 6884. E-mail address: [email protected] (J.H. Cheon). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.06.018
most prevalent in Eastern Mediterranean countries and East Asian populations including Koreans (Kaklamani et al., 1998). Although the pathogenesis of IBD, including intestinal BD, is still unclear, genetic and environmental factors are thought to be involved (Bayraktar et al., 2000; Duchmann et al., 1997; Kasahara et al., 1981; MacDermott, 1996; Reif et al., 1997). Additional evidence of genetic influences on IBD development include a higher frequency in identical twins compared to fraternal twins, people with a family history of IBD, and differences in the prevalence of IBD between races (Orholm et al., 1991; Probert et al., 1993). IBD is considered an autoimmune disease caused by an abnormal immune reaction to intestinal bacteria that occurs in patients with genetic susceptibilities (Sartor, 2006; Xavier and Podolsky, 2007). Triggering receptor expressed on myeloid cell-1 (TREM-1), a 30kDA immunoglobulin superfamily expressed on blood neutrophils and a subset of monocytes, has been shown to play a crucial role in the propagation of local or systemic inflammatory responses (Colonna, 2003). Elevated levels of soluble form of TREM-1 (sTREM-1) have been observed in several infectious diseases (Cohen, 2001; Gibot and Cravoisy, 2004). A sTREM-1 was identified at significant levels in the
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sera of patients with sepsis and appeared to be the most helpful parameter in differentiating patients with sepsis from those with systemic inflammatory response syndrome (Gibot et al., 2004b). In addition, an elevated level of sTREM-1 in bronchoavelolar lavage fluid was found to be a strong predictor of pneumonia (Gibot et al., 2004a). As for gastroenterological diseases, pancreatic tissue TREM-1 mRNA expression was correlated with the severity of pancreatitis (Wang et al., 2004), and the sTREM-1 level in gastric juice was also correlated with a degree of gastric inflammation scores (Koussoulas et al., 2006). Recently, it was reported that TREM-1 is normally underexpressed in the human intestine, which causes immune tolerance to intestinal bacteria and antigens (Schenk et al., 2005). However, TREM-1 is up-regulated in the intestines of patients with IBD (Schenk et al., 2007). We previously reported that sTREM-1 levels are upregulated in the sera of IBD patients and correlate with disease activity in patients with ulcerative colitis (Park et al., 2009). Nevertheless, it remains unknown whether genetic polymorphisms of TREM-1 influence genotypes and phenotypes in IBD patients. We hypothesized that genetic polymorphisms of TREM-1 might be associated with the development of IBD and correlated with disease phenotypes in IBD. Accordingly, we analyzed TREM-1 single nucleotide polymorphisms (SNPs) and evaluated the relationships between TREM-1 polymorphisms and the development and phenotypes of IBD. Materials and methods Study sample We surveyed 839 unrelated Korean subjects including 202 patients with Crohn's disease (CD), 265 with ulcerative colitis (UC), 138 with intestinal Behcet's disease (BD), and 234 healthy controls between July 2006 and October 2007. All patients were diagnosed and managed at the gastroenterology clinics of Yonsei University Health System, Severance Hospital, Seoul, Republic of Korea. The diagnoses of CD, UC, or intestinal BD were based on established clinical, radiographic, endoscopic, and histopathologic criteria as previously described. CD and UC were diagnosed according to the Lennard–Jones criteria (Lennard–Jones, 1989). Intestinal BD was diagnosed according to the criteria suggested by our group and the Behcet's Disease Research Committee of Japan in 1987 (Cheon et al., 2009; Mizushima et al., 1988). Exclusion criteria for this study were indeterminate colitis, other autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.), chronic hepatitis C, asthma, infection, or coexistence of serious medical or surgical conditions. Healthy controls were recruited among health care center customers who had no gastrointestinal symptoms, took no regular medications and had a normal complete blood count and a normal biochemical profile, including ESR and CRP. All control subjects underwent colonoscopy for routine check-up which revealed grossly normal findings. Demographic and clinical characteristics were obtained by reviewing the medical records of the subjects and through detailed questionnaires. The CD patients were subdivided into groups according to age at diagnosis, location, and behavior of disease using the Montreal Classification (Silverberg et al., 2005). In patients with UC, anatomic location was also sub-grouped using the Montreal Classification as being ulcerative proctitis (E1), left-sided UC (E2), and extensive UC (E3). Other characteristics including age at diagnosis, gender, extraintestinal manifestations (EIMs), total follow-up period, the use of immunosuppressive drugs, and the need for surgical intervention were also assessed. The intestinal BD patients were classified into 3 groups using colonoscopic findings and extraintestinal systemic manifestations: (i) complete group, (ii) probable group, or (iii) suspected group (Cheon et al., 2009). This study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki and was approved by the
Institutional Review Board of Yonsei University College of Medicine. Informed written consent was obtained from all subjects.
TREM-1 single nucleotide polymorphism selection and genotyping We genotyped 3 TREM-1 SNPs (rs9471535, rs2234237, and rs3789205). These SNPs were chosen for this study based on a previous Chinese cohort study (Chen et al., 2008) and International HapMap data set (http://www.hapmap.org) regarding the TREM-1 gene (GenBank access no. DQ217941) in patients and healthy controls, of which SNP rs9471535 was located in promoter region of the TREM-1 gene, rs3789205 in intron 1 and rs2234237 in exon 2. Genomic DNA was isolated from whole blood using commercially available kits (Qiagen, Chatsworth, CA, USA). SNPs were genotyped using the TaqMan® fluorogenic 5′ nuclease assay (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. The final volume of each polymerase chain reaction (PCR) was 5 μL, including 20 ng of genomic DNA, 2.5 μL TaqMan® Universal PCR master mix (Applied Biosystems), and 0.22 μL of TaqMan® SNP Genotyping Assay Mix (40×). Amplification conditions were 50 °C for 2 min to activate the uracil N-glycosylase and to prevent carryover contamination, then 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The reaction was performed in a 384-well format using a Dual 384-Well GeneAmp® PCR System 9700 (Applied Biosystems). The TaqMan® assay plate was transferred to ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems), where the endpoint fluorescence intensity of each well was read. The calculation of the fluorescence data from each plate was performed using SDS software version 2.3 (Applied Biosystems). Statistical analysis All SNPs investigated in this study were tested for Hardy– Weinberg equilibrium (HWE) in controls using the χ 2-test. Associations between SNPs and disease susceptibility were determined by comparing allele, and genotype frequencies between cases and controls using the χ 2-test or Fisher's exact test. Relationships between TREM-1 polymorphisms and phenotypes were assessed using logistic regression analysis under a dominant (risk allele homozygotes plus heterozygotes versus wild type allele homozygotes), a recessive (risk allele homozygotes versus heterozygotes plus wild type allele homozygotes), and a codominant (wild type allele homozygotes versus heterozygotes versus risk allele homozygotes) genetic model. Odds ratios (OR) with 95% confidence intervals (CIs) were determined. To identify significant associations in disease phenotypes and genotypes, we performed comparative analyses within subgroups encompassing UC, CD, and intestinal BD patients by clinical characteristics such as gender, mean age at diagnosis, disease location and behavior, EIMs, history of immunosuppressive drug use, and surgical intervention using logistic regression analysis. P-values of less than 0.05 were considered statistically significant. The data were analyzed using PASW v.18.0 for Windows (SPSS Inc., Chicago, IL, USA). Results Demographic and clinical characteristics of study sample Demographic and clinical characteristics of the patients with CD, UC, intestinal BD, and healthy controls who were included in this study are summarized in Table 1. The mean age of the patients at the diagnosis of CD was 26.0 ± 11.2 years, with a gender ratio of 1.58:1 (male:female), that of UC was 36.4 ± 12.4 years, with a gender ratio of 1.01:1, and that of intestinal BD was 38.1 ± 11.1 years, with a gender ratio of 0.92:1. We had only one patient with UC accompanied with primary sclerosing cholangitis.
E.S. Jung et al. / Life Sciences 89 (2011) 289–294 Table 1 Demographic and clinical characteristics of patients with inflammatory bowel diseases and healthy controls. Characteristics
CD (n = 202)
UC (n = 265)
Intestinal BD Control (n=138) (n = 234)
Mean age at diagnosis (years)a ≤16 years 17–40 years N40 years Gender (male/female) Follow-up (months) a Disease localization (%)b Ileal (L1) ± L4 Colonic (L2) ± L4 Ileocolonic (L3) ± L4 Proctitis (E1) Left-sided (E2) Pancolitis (E3) Ileocecal region Other colonic region Disease behavior (%)b Inflammatory (B1) ± p Stricturing (B2) ± p Penetrating (B3) ± p Clinical subtype of BD (%) Complete Probable Suspected Extraintestinal manifestations (%) Joint Skin Eye Oral Genital Liver Use of immunosuppressive drugs (%) Use of infliximab (%) History of surgical interventions (%)
26.0±11.2 26 (12.9%) 156 (77.2%) 20 (9.9%) 124/78 89.3±51.7
36.4±12.4 8 (3.0%) 167 (63.0%) 90 (34.0%) 133/132 103.6±61.3
38.1±11.1 4 (2.9%) 83 (60.1%) 51 (37.0%) 66/72 89.9±54.7
44.0±10.6
133/101
60 (29.7) 24 (11.9) 115 (56.9) 64 (24.2) 91 (34.3) 110 (41.5) 116(84.1) 22 (15.9) 81 (40.1) 36 (17.8) 78 (38.6) 107 (77.5) 7 (5.1) 24 (17.4)
19 (9.4) 10 (5.0) 11 (5.4)
41 (15.5) 25 (9.4) 37 (14.0)
48 (34.8) 60 (43.5) 24 (17.4) 111 (80.4) 42 (30.4)
110 (54.5)
1 (0.4) 69 (26.0)
43 (31.1)
36 (17.8) 80 (39.6)
10 (3.8)
41 (29.7)
Abbreviations: CD, Crohn's disease; UC, ulcerative colitis; BD, Behcet's disease. a Mean ± standard deviation. b Disease localization and behavior according to Montreal Classification (L4, upper gastrointestinal involvement; p, perianal disease).
Associations of TREM-1 genetic polymorphisms with disease susceptibility to inflammatory bowel disease Three candidate SNPs in TREM-1 were genotyped to determine their relationships with disease susceptibility to IBD. No significant deviations from HWE were observed in controls. We evaluated the relationships of TREM-1 genetic polymorphisms with disease susceptibility to CD, UC, and intestinal BD using a multiple logistic regression model (Table 2). We found that TREM-1 genetic polymorphisms were significantly correlated only with intestinal BD in terms of disease development. TREM-1 rs9471535 C allele in promoter region was significantly associated with the occurrence of intestinal BD under dominant model (TT vs CT/CC, OR = 1.637, 95% CI, 1.063 to 2.518, P = 0.025). In rs3789205 at intron 1 and rs2234237 at the exon 2 of the TREM-1 gene, we observed similarly significant results (rs3789205: AA vs AG/GG, OR = 1.668, 95% CI, 1.086 to 2.562, P = 0.019 in dominant model; rs2234237: TT vs AT/AA, OR = 1.691, 95% CI, 1.100 to 2.602, P = 0.016 in dominant model). In CD and UC patients, however, we observed no significant correlations between genotype distributions or combined genotype frequencies of any 3 SNPs in TREM-1. The linkage disequilibrium (LD) pattern of the 3 SNPs of TREM-1 gene is shown in Fig. 1. All of the 3 SNPs were located in the same haplotype block and were in strong LD (D′ N 0.99, r 2 N 0.96).
291
Associations of TREM-1 genetic polymorphisms with intestinal BD phenotypes We also evaluated the relationships between the TREM-1 SNPs and phenotypes of intestinal BD (Table 3). We included all other covariates (gender, age at diagnosis, and use of immunosuppressive drugs) in a multiple logistic regression analysis in order to adjust for the different clinical manifestations. We found that dominant models of all 3 SNPs were associated with skin involvement (rs9471535: OR = 2.723, 95% CI, 1.285 to 5.770, P = 0.009; rs3789205: OR= 2.477, 95% CI, 1.179 to 5.203 P = 0.017; rs2234237: OR = 2.278, 95% CI, 1.085 to 4.785 P = 0.030) and a higher risk of azathioprine use (rs9471535: OR = 2.722, 95% CI, 1.167 to 6.352, P = 0.021; rs3789205: OR= 2.493, 95% CI, 1.079 to 5.759 P = 0.032; rs2234237: OR = 2.638, 95% CI, 1.120 to 6.211 P = 0.026) in intestinal BD. Discussion In this study, we examined the relationships between TREM-1 genetic polymorphisms and susceptibility to IBD and disease phenotypes. It was previously reported that TREM-1 is under-expressed in the human intestine which results in immune tolerance to intestinal bacteria and antigens (Schenk et al., 2005), while TREM-1 is upregulated in the intestine of patients with IBD (Schenk et al., 2007). In an experimental mouse model with colitis, TREM-1-expressing intestinal macrophages contributed to amplifying chronic inflammation (Schenk et al., 2007). Our group also reported that sTREM-1 levels are upregulated in the sera of IBD patients and correlate with disease activity in patients with UC (Park et al., 2009) and intestinal BD (Jung et al., in press). Furthermore, a study investigating an association between TREM-1 gene polymorphisms and severe sepsis was conducted in a Chinese cohort (Chen et al., 2008). Based on these observations, development and phenotypes of IBD could be different among TREM-1 SNPs. Therefore, we hypothesized that TREM-1 genetic polymorphisms might be associated with the development of CD and UC. To the authors' knowledge, this is the first attempt to investigate the correlations of TREM-1 SNPs with IBD genotypes and phenotypes. However, TREM-1 genetic polymorphisms did not show a significant association with the development of CD and UC. These findings suggest that TREM-1 genetic polymorphisms do not have a strong impact on CD or UC susceptibility. However, further studies are needed to exclude the possibility that this finding may have been due to the small sample size of this study, or to exclude other unknown SNPs from consideration. We also examined the relationships between TREM-1 genetic polymorphisms and susceptibility to intestinal BD. Three common markers showed a similarly significant association. The SNP (rs9471535) located within the 5′ flanking region of the reference TREM-1 gene may impact the transcriptional activity, and other nonsynonymous variation Ser25Thr (rs2234237) in the exon 2 of the TREM-1 gene may influence the biologic function of TREM-1. All of the 3 SNPs were located in the same haplotype block and were in tight LD, which suggests that the TREM-1 gene may be strongly involved in intestinal BD susceptibility. Intestinal BD is similar to CD in terms of its clinical features and therapeutic implications (Kim et al., 1999). Both diseases develop at a young age, have similar gastrointestinal and extraintestinal manifestations, and have chronic relapsing and remission courses. Because of these clinical similarities, it is occasionally difficult to distinguish between intestinal BD and CD. Despite similar manifestations in clinical practice, intestinal BD is generally agreed to be a distinct entity (Lee et al., 2001; Smith et al., 1973). Previous genetic studies of BD have shown that HLAB⁎51 carriers were more likely to develop BD, and that the frequency of HLA-B⁎51 was much higher in male patients (de Menthon et al., 2009; Ohno et al., 1982; Ono et al., 1973). Moreover, tumor necrosis factor-α1031 T/C polymorphisms were reported be associated with BD susceptibility (Akman et al., 2006). The distribution of IL-8 gene haplotype was also associated with BD (Lee et al., 2007). Recent studies
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Table 2 Associations of triggering receptor expressed on myeloid cells-1 (TREM-1) single nucleotide polymorphism genotypes between patients with inflammatory bowel diseases and healthy controls. rs number
Genotype
Intestinal BD
P-value
OR (95% CI)
rs9471535 Dominant
TT
(n = 134) 53
0.025
1.637 (1.063–2.518)
CT/CC TT/CT
81 125
0.372
0.694 (0.310–1.554)
CC TT CT
9 53 72
0.001
CC
9
0.805
AA
(n = 137) 53
0.019
1.668 (1.086–2.562)
AG/GG AA/AG
84 127
0.486
0.759 (0.348–1.654)
GG AA AG
10 53 74
0.008
GG
10
0.984
TT
(n = 138) 51
0.016
1.691 (1.100–2.602)
AT/AA TT/AT
87 127
0.630
0.831 (0.390–1.770)
TT TT AT
11 51 76
0.009
AA
11
0.824
Recessive
Codominant
rs3789205 Dominant
Recessive
Codominant
rs2234237 Dominant
Recessive
Codominant
1.813 (1.166–2.820) 0.900 (0.389–2.082)
1.802 (1.160–2.800) 0.991 (0.440–2.236)
1.797 (1.155–2.796) 1.094 (0.494–2.419)
CD
P-value
OR (95% CI)
(n = 196) 96
0.573
1.115 (0.763–1.631)
100 183
0.296
0.685 (0.335–1.397)
13 96 87
0.407
13
0.401
(n = 196) 95
0.561
1.119 (0.766–1.636)
101 184
0.209
0.628 (0.303–1.305)
1.182 (0.796–1.756) 0.729 (0.349–1.525)
12 95 89
0.297
112
0.453
(n = 197) 95
0.747
1.065 (0.728–1.556)
102 185
0.200
0.622 (0.300–1.292)
12 95 90
0.644
12
0.884
1.234 (0.830–1.837) 0.754 (0.361–1.576)
0.915 (0.630–1.330) 1.046 (0.566–1.936)
UC
P-value
OR (95% CI)
(n = 256) 137
0.689
0.930 (0.652–1.327)
119 231
0.891
1.043 (0.571–1.905)
25 137 94
0.657
25
0.973
(n = 261) 137
0.788
0.953 (0.669–1.356)
124 234
0.726
1.112 (0.615–2.011)
0.918 (0.630–1.338) 0.989 (0.531–1.844)
27 137 97
0.297
27
0.453
(n = 260) 134
0.698
0.932 (0.655–1.328)
126 233
0.727
1.111 (0.614–2.011)
27 134 99
0.407
27
0.401
1.234 (0.830–1.837) 0.754 (0.361–1.576)
1.182 (0.795–1.756) 0.729 (0.348–1.525)
Control (n = 234) 121 113 212 22 121 91 22 (n = 234) 120 114 212 22 120 92 22 (n = 233) 116 117 211 22 116 95 22
Abbreviations: BD, Behcet's disease; CD, Crohn's disease; UC, ulcerative colitis; OR, odds ratio; CI, confidence interval.
Fig. 1. Linkage disequilibrium structure of the 3 triggering receptor expressed on myeloid cell-1 (TREM-1) single nucleotide polymorphisms (SNPs) in Koreans. The linkage disequilibrium (LD) patterns of the 3 SNPs in TREM-1 gene with D′ values are shown (D′ N 0.99, r2 N 0.96).
showed that SNPs of TLR-4 (Horie et al., 2009) and IL-17 F may influence the susceptibility of BD (Jang et al., 2008). Baranathan et al. (2007) reported that protein tyrosine phosphatase type 22 (PTPN22 620W) was inversely associated with BD susceptibility. Gunesacar et al. (2007) reported that SNP of the CD 28 gene was increased in BD patients, whereas the CTLA-4 gene was decreased. With regard to intestinal BD, however, there have been no reports on genetic polymorphisms. Therefore, a genetic polymorphism study is necessary with respect to intestinal BD. Accordingly, we hypothesized that TREM-1 genetic polymorphisms might be associated with intestinal BD susceptibility. To the best of our knowledge, this is the first trial to explore the relationships between genetic polymorphisms and intestinal BD. Our data indicate that all 3 TREM-1 SNPs were significantly associated with susceptibility to intestinal BD. According to these results, intestinal BD may differ genetically from other IBDs such as CD or UC. Some groups have claimed that intestinal BD and CD are the same disease with different spectra (Kim et al., 2007; Yim and White, 1985). However, based on our observations, these two diseases should be considered different disease entities. One of the main histological findings of intestinal BD is neutrophil infiltrations around the ulcers. Hayasaki et al. (2004) showed that neutrophilic phlebitis may be involved in the pathogenesis of intestinal BD. Because TREM-1 is mainly expressed in neutrophils and monocytes, TREM-1 might contribute to the pathogenesis of intestinal BD. Moreover, we found that TREM-1 SNPs are associated with skin involvement as well as the use of azathioprine as a treatment for intestinal BD. The association of TREM-1 SNPs and use of
E.S. Jung et al. / Life Sciences 89 (2011) 289–294
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Table 3 Associations of triggering receptor expressed on myeloid cells-1 (TREM-1) single nucleotide polymorphism genotypes with intestinal Behcet's disease phenotypes. Disease phenotype
rs number
Genotype
Patients without phenotype (%)
Patients with phenotype (%)
Unadjusted OR (95% CI)
Adjusted OR (95% CI)
Skin
rs9471535 Dominant
TT
37 (49.3) 38 (50.7)
16 (27.1) 43 (72.9)
2.617⁎⁎ (1.260–5.435)
2.723⁎⁎ (1.285–5.770)
37 (47.4) 41 (52.6)
16 (27.1) 43 (72.9)
2.425⁎ (1.174–5.012)
2.477⁎ (1.179–5.203)
35 (44.9) 43 (55.1)
16 (26.7) 44 (73.3)
2.238⁎ (1.083–4.624)
2.278⁎ (1.085–4.785)
42 (45.7) 50 (54.3)
11 (26.2) 31 (73.8)
2.367⁎ (1.063–5.273)
2.722⁎ (1.167–6.352)
42 (44.2) 53 (55.8)
11 (26.2) 31 (73.8)
2.233⁎ (1.005–4.961)
2.493⁎ (1.079–5.759)
41 (42.7) 55 (57.3)
10 (23.8) 32 (76.2)
2.385⁎ (1.054–5.401)
2.638⁎ (1.120–6.211)
CT/CC rs3789205 Dominant
AA AG/GG
rs2234237 Dominant
TT AT/AA
Azathioprine use
rs9471535 Dominant
TT CT/CC
rs3789205 Dominant
AA AG/GG
rs2234237 Dominant
TT AT/AA
a
Abbreviations: OR, odds ratio; CI, confidence interval. a After adjustment for all other covariates (gender, age at diagnosis, and use of immunosuppressive drugs). ⁎ P b 0.05. ⁎⁎ P b 0.01.
azathioprine suggests that the presence of TREM-1 SNPs are associated with higher disease activity of intestinal BD. TREM-1 SNPs, however, are not associated with increased necessity of operation. Moreover, we found that these 3 SNPs are not correlated with sudden serious complications such as perforation. Therefore, we hypothesize that TREM-1 SNPs have a modest effect on intestinal BD manifestations. However, there were no significant associations between TREM-1 SNPs and oral, genital tract, joint, or eye involvement. Further study is warranted to determine the relationships of TREM-1 SNPs with not only intestinal BD but also systemic BD. Our study has several limitations. First, our study is limited by its small sample size. Because of this small sample size, we were unable to demonstrate strong associations between TREM-1 genetic polymorphisms and disease phenotypes in recessive disease models. A larger Korean cohort study is necessary to validate our data. Second, our study investigated only 3 SNPs of TREM-1. Supplementary research is necessary in order to uncover new SNPs that might be further related to IBD susceptibility through the full sequencing of TREM-1 gene and haplotype-based association study. In addition, it would be necessary to search new genes that have similar action to TREM-1, and then analyze the gene-to-gene interaction and functional relevance of these genes. Finally, our results were obtained in a Korean sample and should be validated in other ethnic groups.
Conclusion In conclusion, TREM-1 SNPs have a significant association with the development of intestinal BD; in particular, its skin involvement and use of azathioprine for treatment. However, TREM-1 SNPs do not have a significant influence on the development of CD and UC. The results of our study suggest that TREM-1 SNPs could play a significant role in
the development of intestinal BD and may have modest effects on its disease severity. Conflict of interest statement The authors declare no conflict of interest.
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