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Genetic polymorphisms of interleukin 20 (IL-20) in patients with ulcerative colitis
Immunology Letters 149 (2013) 50–53
Contents lists available at SciVerse ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Genetic polymorphisms of interleukin 20 (IL-20) in patients with ulcerative colitis Jesús K. Yamamoto-Furusho a , Jorge L. De-León-Rendón a , Monica García de la Torre b , Edith Alvarez-León b , Gilberto Vargas-Alarcón b,∗ a b
Inflammatory Bowel Disease Clinic, Department of Gastroenterology. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Department of Molecular Biology. Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
a r t i c l e
i n f o
Article history: Received 20 July 2012 Received in revised form 8 November 2012 Accepted 14 November 2012 Available online 23 November 2012 Keywords: Genetic susceptibility IL-20 polymorphisms Mexican Ulcerative colitis
a b s t r a c t Interleukin (IL)-20 belongs to the IL-10 family and is a potent immunomodulatory cytokine with implications for pathogenesis in the inflammatory bowel disease (IBD). The interleukin 20 gene is located within a 200 kb region of q31-32 locus of chromosome 1. No previous studies have reported this novel association between ulcerative colitis (UC) and IL-20 polymorphisms. In the present work, we evaluated the role of IL-20 gene polymorphisms as susceptibility markers for UC. Three polymorphisms of IL-20 gene (rs2981573, rs2232360, rs1518108) were genotyped by 5 exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real-Time PCR system in a group of 198 Mexican Mestizo patients with UC and 698 ethnically matched healthy unrelated individuals with no family history of UC. We found significant decreased frequencies of two IL-20 genotypes: GG (rs2981573) [10.6% vs. 17.6%, p = 0.017, OR = 0.55, 95% CI: 0.33–0.93] and GG (rs2232360) [10.6% vs. 17.6%, p = 0.017, OR = 0.55, 95% CI: 0.33–0.93] in UC patients as compared to healthy controls. No significant differences of gene frequencies were found between UC patients and healthy controls in the rs1518108 polymorphism. In the subgroup analysis, no differences were found between the IL-20 genotypes and the clinical characteristics of UC. The results suggest that the GG genotypes of the IL-20 polymorphisms (rs2981573 and rs2232360) might have an important role in the development of UC in the Mexican population. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ulcerative colitis (UC) is a multi-factorial disease; it means that several genes, together with environmental factors, contribute to the development of this disease. This disease is heterogeneous [1] and characterized by a chronic relapsing inflammation that is thought to result from an aberrant immune response to the intestinal flora in the context of genetic predisposition. Many common responses in the inflammatory bowel disease (IBD) are mediated by cytokines such as regulation of inflammatory mediators production, reactive oxygen metabolites, nitric oxide, leukotriens, platelet-activating factor, and prostaglandins, activation of the nuclear factor B (NF-B) and inhibition of apoptosis; how cytokines determine the nature of the immune response in IBD may be quite different among IBD forms [2].
∗ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez. Juan Badiano 1, Sección XVI, Tlalpan, Mexico City, Mexico. Tel.: +52 55 5573 2911x1460/1347; fax: +52 55 55135811. E-mail address: [email protected] (G. Vargas-Alarcón). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2012.11.008
Recently, new cytokines like IL-21, IL-22, and IL-24 have been implicated in the pathophysiology of UC [3–5]. Interleukin (IL)-20 belongs to the IL-10 family. IL-10 is an antiinflammatory cytokine that inhibits both antigen presentation and subsequent release of pro-inflammatory cytokines, thereby attenuating mucosal inflammation. Inflamed tissues and granulomas of Crohn disease (CD) show low IL-10 [6]. Melgar et al. [7] reported a highly significant increase in IL-10 mRNA levels in T lymphocytes and in IL-10-positive cells from colonic tissue of UC patients. A recent study found low expression of IL-10 gene in the colonic mucosa from patients with active UC compared to UC remission and healthy control groups [8]. IL-20 gene is located within a 200 kb region of chromosome 1 in q31–32 locus [6]. Linkage to several common autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis has been detected in this locus [9,10]. In a previous study, IL-20 polymorphisms (rs2981572, rs2981573, rs2232360 and rs1518108) were associated with the development of different types of psoriasis, such as familial, sporadic, early and late onset disease [11]. No previous studies have explored the role of IL-20 polymorphisms in patients with ulcerative colitis. Thus, the aim of the
J.K. Yamamoto-Furusho et al. / Immunology Letters 149 (2013) 50–53
present study was to establish the role of the IL-20 gene polymorphisms in the risk of developing UC in a well-characterized clinical cohort of Mexican Mestizo patients.
Table 1 Demographic and clinical characteristics of Mexican Mestizo patients with ulcerative colitis (UC). Gender (male/female)
2. Material and methods 2.1. Patients One hundred ninety eight Mexican Mestizo patients with diagnosis of ulcerative colitis confirmed by histology were studied, patients were from the Inflammatory Bowel Disease Clinic at the National Institute of Medical Sciences and Nutrition. Details of demographic and clinical characteristics of UC were obtained by a questionnaire, review of records, and personal interview. No patient had a family history for UC. Disease extension was defined by colonoscopy. Thus, disease was classified either as extensive colitis (inflammation proximal to the splenic flexure) and distal colitis. The clinical course of disease was defined as: Active then inactive (first episode with activity and then long-term remission for more than 5 years); intermittent activity (≥2 relapses in a year); chronic continual activity (persistent activity despite of medical conventional therapy) as previously described [12]. Their past and current therapy was assessed, as well as the clinical response to conventional medical therapy including 5-ASA agents, steroids and immune-modulator agents. Medical therapy failure was defined as lack of clinical response to maximal dose of 5-ASA (4.5 g/d), steroid resistance (persistent activity with prednisone above 40 mg/d), immune modulator resistance (persistent activity with azathioprine above 2.5 mg/kg/d at least for 6 months of therapy), and steroid dependence (relapse when tapering prednisone to less than 15 mg/d). The study complies with the Declaration of Helsinki. The Bioethics and Research Committee approved the present study and all study subjects signed an informed consent letter. 2.2. Controls Blood samples were obtained from 698 Mexican Mestizo healthy unrelated individuals with no family history of UC. All subjects included in the study (patients and controls) were ethnically matched, and we considered as Mexican Mestizos only those individuals who had been born in Mexico for three generations, including their own. A Mexican Mestizo is defined as someone born in Mexico, who is a descendant of the original autochthonous inhabitants of the region and of individuals, mainly Spaniards, of Caucasian and/or African origin, who came to America during the sixteenth century. 2.3. DNA extraction Genomic DNA from whole blood containing EDTA was isolated by standard technique [13]. 2.4. Determination of the IL-20 genotypes Polymorphisms (rs2981573, rs2232360, rs1518108) were genotyped using 5 exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real-Time PCR system according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). 2.5. Statistical analysis Gene frequencies of IL-20 polymorphisms in patients and controls were obtained by direct counting. The Hardy-Weinberg equilibrium was evaluated by chi-square test. Gene frequencies in patients were compared to those present in controls using a
51
Age at presentation (years) Duration of disease (years) Extent of UC (%) Pancolitis Distal Extraintestinal manifestations (%) Present No Clinical course of disease Active then inactive Intermitent activity Persistent activity Response to treatment Favorable Steroid dependant Steroid resistant Immumodulator resistant Colectomy (%) Yes No
91/107 31.5 ± 14 12.34 134 (67.6%) 64 (32.3%) 64 (32.3%) 134 (67.6%) 42% 30% 28% 68% 20.5% 7.5% 4% 29 (14.6%) 169 (85.3%)
2 × 2 contingency table and Chi-square. Odd ratios (OR) have been calculated for the disease in carriers of specific alleles. Comparisons of allele frequencies between subgroups were performed using the EPI-INFO statistical package (Version 5.0; USD incorporated 1990, Stone Mountain, Georgia). Statistical significance was considered as p < 0.05. Pair wise linkage disequilibrium (LD, D ) estimations between polymorphisms and haplotype reconstruction were performed with Haploview version 3:32 (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA). 3. Results 3.1. Clinical features Of the 198 patients, 107 (54%) were women and 91 (46%) men, with ages ranging from 20 to 78 (mean 31.5 years). The evolution of the disease was 12.34 years. Pancolitis was present in 134 patients (67.6%) and distal colitis in 64 (32.3%). Sixty-four patients presented extraintestinal manifestations (32.3%) that included arthropathy (18%), primary sclerosing cholangitis (8%), erythema nodosum (2%), sacroiliitis (1.5%), ankylosing spondilytis (1.5%), and aphthous ulceration (1.3%). According to the clinical course of the disease, 42% had a pattern of activity then inactivity, 30% intermittent activity, and 28% showed chronic continual activity. Regarding medical treatment, 68% had a favorable response; 20.5% were steroid-dependent, 7.5% steroid-resistant, and 4% were azathioprine resistant. None of the patients received treatment with cyclosporine or infliximab. In regard to surgical treatment, twenty-nine patients (14.6%) underwent radical colectomy due to refractory medical therapy, toxic megalon, perforation and uncontrolled bleeding (Table 1). 3.2. IL-20 gene polymorphisms All IL-20 gene polymorphisms were consistent with HWE (p > 0.05). No indication of genotype error was observed and results from duplicate samples (10%) were 100% concordant. We found significant decreased frequencies of two IL-20 genotypes: GG (rs2981573) [10.6% vs. 17.6%, p = 0.017, OR = 0.55 95% CI: 0.33–0.93] and GG (rs2232360) [10.6% vs. 17.6%, p = 0.017, OR = 0.55, 95% CI: 0.33–0.93] in UC patients as compared to healthy controls. Similar distribution of the rs1518108 polymorphism was observed in
52
J.K. Yamamoto-Furusho et al. / Immunology Letters 149 (2013) 50–53
Table 2 Allele (af) and genotype (gf) frequency (%) of IL-20 gene polymorphisms (rs2981573, rs2232360, and rs1518108) in UC patients and healthy controls.
rs2981573 Allele A G Genotypes AA AG GG rs2232360 Allele A G Genotypes AA AG GG rs1518108 Allele T C Genotype TT TC CC
UC
Controls
(n = 198) n 253 143 n 76 101 21 (n = 198) n 253 143 n 76 101 21 (n = 180) n 178 182
gf 0.494 0.505
(n = 698) n 827 569 0.407 n 252 323 123 (n = 698) n 828 568 n 253 322 123 (n = 698) n 658 738
0.238 0.511 0.25
150 358 190
43 92 45
af 0.638 0.361 gf 0.383 0.51 0.106 af 0.638 0.362 gf 0.383 0.51 0.106
p
OR
95%CI
gf 0.361 0.462 0.176
0.017
0.55
0.33–0.93
af 0.593 0.406 gf 0.362 0.461 0.176
0.017
0.55
0.33–0.93
af 0.592
gf 0.471 0.528 0.214 0.521 0.272
All populations were in Hardy-Weinberg equilibrium. Abbreviations: UC = ulcerative colitis; OR = odds ratio; CI = confidence intervals. The rs1518108 polymorphism was determined in 180 patients.
UC patients and healthy controls (Table 2). In the subgroup analysis, no differences were found between IL-20 genotypes and the clinical characteristics of UC (data not shown). 3.3. IL-20 haplotype analysis The analysis of linkage disequilibrium of the IL-20 gene polymorphisms was done using the Haploview program. In this analysis only two polymorphisms (rs2981573 and rs2232360) were in high linkage disequilibrium. The distribution of the constructed haplotypes was similar in UC patients and healthy controls (data not shown). 4. Discussion Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis are chronic inflammatory disorders of the gastrointestinal tract. Several genetic factors contribute to genetic susceptibility to these diseases. Some genome wide association studies (GWAS) have identified several risk conferring loci, some of them have been confirmed in different populations [14–17]. Polymorphisms of the IL-10 gene have been associated with UC in these studies [14–16]. In a previous study, four polymorphisms were associated with UC in European individuals; these results were replicated in three independent case-control panels [18]. The rs3024505 SNP immediately flanking the IL-10 gene showed the most significant association in the combined samples. The association of this SNP was corroborated in a sample of Indian patients [14]. Although several studies have linked the IL-10 gene with UC, few studies in other genes related with IL-10 (IL-19, IL-20, IL-22 and IL-24) have been reported. Recently, our group reported that the IL-19 gene polymorphisms (rs224188 and rs2243193) might have an important role in the development of UC in Mexican individuals [19]. Considering the important role of IL-10 cluster genes in UC, in the present work, two polymorphisms (rs2981573 and rs2232360) in the IL-20 gene were studied in UC patients. The frequencies of the GG genotypes in both polymorphisms were decreased in the group of patients. The role of these polymorphisms in the development of
UC confirms in part its similar effect in patients with psoriasis [11]. In psoriasis, the IL-20 polymorphisms were associated with different forms of the disease such as early-onset, late-onset, familial and sporadic disease [11]. A combined haplotype analysis of the IL-19 and IL-20 gene polymorphisms in psoriasis demonstrates the susceptibility effect of the IL-20 gene [20]. Kingo et al. studied the IL-20 rs2981573 polymorphism in patients with palmoplantar pustulosis and reported a decreased frequency of the G allele in these patients [21]. This is the same allele (in genotype form) that was decreased in our group of UC patients. At the present time there is no information about the functional effect of this polymorphism. Another study by Truelove et al. [22] showed association of the IL-20 rs1400986, rs3024517 and rs1518108 polymorphisms with chronic hepatitis B infection outcome. Only the rs1518108 polymorphism was analyzed in our study and this polymorphism was not associated with UC. The distribution of the polymorphisms in our population was compared to those reported in other ethnic groups [21,22] and in the HapMap project. Similar distribution of the rs1518108 polymorphism was observed in African and European populations when compared to Mexicans [21,22]. According to the HapMap information, the distribution of the rs2981573 and rs2232360 polymorphisms was similar in Mexicans when compared to Africans and Europeans but different when compared to Asian populations. In this case, the “A” allele of both polymorphisms was more frequent in Mexicans, Europeans and Africans (59% to 87%) when compared to Asians (25% to 33%). The pleiotropic inflammatory cytokine IL-20 is expressed in monocytes, epithelial cells, and endothelial cells and exerts its biological functions on multiple cell types by activating IL-20R1/IL20R2 or IL-22R1/IL- 20R2 complexes [23]. IL-20 is involved in various inflammatory diseases [24], such as psoriasis [25], rheumatoid arthritis [26], atherosclerosis [27], and renal failure [28]. In our study, no association of IL-20 polymorphisms with clinical features was observed. In our previous study of IL-19 polymorphisms, lack of association with clinical characteristics was also observed [19]. In both studies, this lack of associations between interleukin polymorphisms and clinical features could be caused
J.K. Yamamoto-Furusho et al. / Immunology Letters 149 (2013) 50–53
by the sample size. Previously, in the same population, the HLADRB1*0103 allele was associated with the presence of colectomy [29]. To the best of our knowledge, this is the first study that provides evidence about the association of IL-20 polymorphisms with the risk of developing UC. However considering that this gene is located in the genomic IL-10 region, the participation of other genes of this cluster in the genetic susceptibility to UC cannot be ruled out. According to this, our group reported the association of UC with IL-19 polymorphisms in the same group of patients [19]. There are some limitations to this study. The results were not replicated in an independent set of patients and controls. Considering that this is the first time that polymorphisms located within the IL-20 gene are associated with UC, replication in another cohort of patients is necessary to confirm these results. In our study was not possible to evaluate all the described polymorphisms in the IL-20 gene. We selected three polymorphisms that have been reported associated with psoriasis. In conclusion, our results suggest that the GG genotypes of the IL-20 polymorphisms (rs2981573 and rs2232360) might have an important role in the development of UC in the Mexican population. Additional studies in other populations could help to define the potential role of this marker as protective factor against UC development. Acknowledgments This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología and Fundación Gonzalo Rio Arronte, Mexico City, Mexico.
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immunorregulatory function in the mucosa of patients with ulcerative colitis. Rev Gastroenterol Mex 2011;76:113–9. Dumoutier L, Louahed J, Renauld JC. Cloning and characterization of IL-10-related T Cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9. J Immunol 2000;164: 1814–9. Gallagher G, Dickensheets H, Eskdale J, Izotova LS, Mirochnitchenko OV, Peat JD, et al. Cloning, expression and initial characterization of interleukin-19 (IL-19), a novel homologue of human interleukin-10 (IL-10). Genes Immun 2000;1:442–50. Kingo K, Koks S, Nicopensius T, Silm H, Vasar E. Polymorphisms in the interleukin 20 gene: relationships to different plaque-type psoriasis. Genes Immun 2004;5:117–21. Henriksen M, Jahnsen J, Lygren I, Sauar J, Kjellevold O, Schulz T, et al. IBSEN Study Group. Ulcerative colitis and clinical course: results of a 5year population-based follow-up study (the IBSEN study). Inflamm Bowel Dis 2006;12:543–50. Miller S, Dykes D, Polesky H. A simple salting-out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16: 1215–20. Juyal G, Prasad P, Senapati S, Midha V, Sood A, Amre D, et al. An investigation of genome-wide studies reported susceptibility loci for ulcerative colitis shows limited replication in north Indians. PLoS ONE 2011;6:e16565. Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, Ahmad T, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet 2010;42:1118–25. McGovern DP, Gardet A, Trkvist L, Goyette P, Essers J, Taylor KD, et al. Genomewide association identifies multiple ulcerative colitis susceptibility loci. Nat Genet 2010;42:332–7. Franke A, Balschun T, Sina C, Ellinghaus D, Håsler R, Mayr G, et al. Genome-wide association study for ulcerative colitis identifies risk loci at 7q22 and 22q13 (IL17REL). Nat Genet 2010;42:292–4. Franke A, Balschun T, Karlsen TH, Sventoraityte J, Nikolaus S, Mayr G, et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet 2008;40:1319–23. Yamamoto-Furusho JK. Álvarez-León E, Fragoso JM, Gozalishvilli A, Vallejo M, Vargas-Alarcón G. Protective role of interleukin-19 gene polymorphisms in patients with ulcerative colitis. Hum Immunol 2011;72: 1029–32. Koks S, Kingo K, Ratsep R, Karelson M, Silm H, Vasar E. Combined haplotype analysis of the interleukin-19 and -20 genes: relationship to plaque-type psoriasis. Genes Immun 2004;5:662–7. Kingo K, Mössner R, Kõks S, Rätsep R, Krüger U, Vasar E, et al. Association analysis of IL19, IL20 and IL24 genes in palmoplantar pustulosis. Br J Dermatol 2007;156:646–52. Truelove AL, Oleksyk TK, Shrestha S, Thio CL, Goedert JJ, Donfield SM, et al. Evaluation of IL10, IL19 and IL20 gene polymorphisms and chronic hepatitis B infection outcome. Int J Immunogenet 2008;35:255–64. Dumoutier L, Leemans C, Lejeune D, Kotenko SV, Renauld JC. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J Immunol 2001;167:3545–9. Wei CC, Hsun YH, Li HH, Wang YC, Hsieh MY, Chen WY, et al. IL-20: biological functions and clinical implications. J Biomed Sci 2006;13:601–12. Wei CC, Chen WY, Wang YC, Chen PJ, Lee JY, Wong TW, et al. Detection of IL-20 and its receptors on psoriatic skin. Clin Immunol 2005;117:65–72. Hsu YH, Li HH, Hsieh MY, Liu MF, Huang KY, Chin LS, et al. Function of interleukin-20 as a proinflammatory molecule in rheumatoid and experimental arthritis. Arthritis Rheum 2006;54:2722–33. Caligiuri G, Kaveri SV, Nicoletti A. IL-20 and atherosclerosis: another brick in the wall. Arterioscler Thromb Vasc Biol 2006;26:1929–30. Li HH, Hsu YH, Wei CC, Lee PT, Chen WC, Chang MS. Interleukin-20 induced cell death in renal epithelial cells and was associated with acute renal failure. Genes Immun 2008;9:395–404. Yamamoto-Furusho JK, Uscanga LF, Vargas-Alarcon G, Rodriguez-Perez JM, ˜ J, Granados J. Polymorphisms in the promoter region of tumor necrosis Zuniga factor alpha (TNF-alpha) and the HLA-DRB1 locus in Mexican Mestizo patients with ulcerative colitis. Immunol Lett 2004;95:31–5.
Contents lists available at SciVerse ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Genetic polymorphisms of interleukin 20 (IL-20) in patients with ulcerative colitis Jesús K. Yamamoto-Furusho a , Jorge L. De-León-Rendón a , Monica García de la Torre b , Edith Alvarez-León b , Gilberto Vargas-Alarcón b,∗ a b
Inflammatory Bowel Disease Clinic, Department of Gastroenterology. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico Department of Molecular Biology. Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
a r t i c l e
i n f o
Article history: Received 20 July 2012 Received in revised form 8 November 2012 Accepted 14 November 2012 Available online 23 November 2012 Keywords: Genetic susceptibility IL-20 polymorphisms Mexican Ulcerative colitis
a b s t r a c t Interleukin (IL)-20 belongs to the IL-10 family and is a potent immunomodulatory cytokine with implications for pathogenesis in the inflammatory bowel disease (IBD). The interleukin 20 gene is located within a 200 kb region of q31-32 locus of chromosome 1. No previous studies have reported this novel association between ulcerative colitis (UC) and IL-20 polymorphisms. In the present work, we evaluated the role of IL-20 gene polymorphisms as susceptibility markers for UC. Three polymorphisms of IL-20 gene (rs2981573, rs2232360, rs1518108) were genotyped by 5 exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real-Time PCR system in a group of 198 Mexican Mestizo patients with UC and 698 ethnically matched healthy unrelated individuals with no family history of UC. We found significant decreased frequencies of two IL-20 genotypes: GG (rs2981573) [10.6% vs. 17.6%, p = 0.017, OR = 0.55, 95% CI: 0.33–0.93] and GG (rs2232360) [10.6% vs. 17.6%, p = 0.017, OR = 0.55, 95% CI: 0.33–0.93] in UC patients as compared to healthy controls. No significant differences of gene frequencies were found between UC patients and healthy controls in the rs1518108 polymorphism. In the subgroup analysis, no differences were found between the IL-20 genotypes and the clinical characteristics of UC. The results suggest that the GG genotypes of the IL-20 polymorphisms (rs2981573 and rs2232360) might have an important role in the development of UC in the Mexican population. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ulcerative colitis (UC) is a multi-factorial disease; it means that several genes, together with environmental factors, contribute to the development of this disease. This disease is heterogeneous [1] and characterized by a chronic relapsing inflammation that is thought to result from an aberrant immune response to the intestinal flora in the context of genetic predisposition. Many common responses in the inflammatory bowel disease (IBD) are mediated by cytokines such as regulation of inflammatory mediators production, reactive oxygen metabolites, nitric oxide, leukotriens, platelet-activating factor, and prostaglandins, activation of the nuclear factor B (NF-B) and inhibition of apoptosis; how cytokines determine the nature of the immune response in IBD may be quite different among IBD forms [2].
∗ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez. Juan Badiano 1, Sección XVI, Tlalpan, Mexico City, Mexico. Tel.: +52 55 5573 2911x1460/1347; fax: +52 55 55135811. E-mail address: [email protected] (G. Vargas-Alarcón). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2012.11.008
Recently, new cytokines like IL-21, IL-22, and IL-24 have been implicated in the pathophysiology of UC [3–5]. Interleukin (IL)-20 belongs to the IL-10 family. IL-10 is an antiinflammatory cytokine that inhibits both antigen presentation and subsequent release of pro-inflammatory cytokines, thereby attenuating mucosal inflammation. Inflamed tissues and granulomas of Crohn disease (CD) show low IL-10 [6]. Melgar et al. [7] reported a highly significant increase in IL-10 mRNA levels in T lymphocytes and in IL-10-positive cells from colonic tissue of UC patients. A recent study found low expression of IL-10 gene in the colonic mucosa from patients with active UC compared to UC remission and healthy control groups [8]. IL-20 gene is located within a 200 kb region of chromosome 1 in q31–32 locus [6]. Linkage to several common autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis has been detected in this locus [9,10]. In a previous study, IL-20 polymorphisms (rs2981572, rs2981573, rs2232360 and rs1518108) were associated with the development of different types of psoriasis, such as familial, sporadic, early and late onset disease [11]. No previous studies have explored the role of IL-20 polymorphisms in patients with ulcerative colitis. Thus, the aim of the
J.K. Yamamoto-Furusho et al. / Immunology Letters 149 (2013) 50–53
present study was to establish the role of the IL-20 gene polymorphisms in the risk of developing UC in a well-characterized clinical cohort of Mexican Mestizo patients.
Table 1 Demographic and clinical characteristics of Mexican Mestizo patients with ulcerative colitis (UC). Gender (male/female)
2. Material and methods 2.1. Patients One hundred ninety eight Mexican Mestizo patients with diagnosis of ulcerative colitis confirmed by histology were studied, patients were from the Inflammatory Bowel Disease Clinic at the National Institute of Medical Sciences and Nutrition. Details of demographic and clinical characteristics of UC were obtained by a questionnaire, review of records, and personal interview. No patient had a family history for UC. Disease extension was defined by colonoscopy. Thus, disease was classified either as extensive colitis (inflammation proximal to the splenic flexure) and distal colitis. The clinical course of disease was defined as: Active then inactive (first episode with activity and then long-term remission for more than 5 years); intermittent activity (≥2 relapses in a year); chronic continual activity (persistent activity despite of medical conventional therapy) as previously described [12]. Their past and current therapy was assessed, as well as the clinical response to conventional medical therapy including 5-ASA agents, steroids and immune-modulator agents. Medical therapy failure was defined as lack of clinical response to maximal dose of 5-ASA (4.5 g/d), steroid resistance (persistent activity with prednisone above 40 mg/d), immune modulator resistance (persistent activity with azathioprine above 2.5 mg/kg/d at least for 6 months of therapy), and steroid dependence (relapse when tapering prednisone to less than 15 mg/d). The study complies with the Declaration of Helsinki. The Bioethics and Research Committee approved the present study and all study subjects signed an informed consent letter. 2.2. Controls Blood samples were obtained from 698 Mexican Mestizo healthy unrelated individuals with no family history of UC. All subjects included in the study (patients and controls) were ethnically matched, and we considered as Mexican Mestizos only those individuals who had been born in Mexico for three generations, including their own. A Mexican Mestizo is defined as someone born in Mexico, who is a descendant of the original autochthonous inhabitants of the region and of individuals, mainly Spaniards, of Caucasian and/or African origin, who came to America during the sixteenth century. 2.3. DNA extraction Genomic DNA from whole blood containing EDTA was isolated by standard technique [13]. 2.4. Determination of the IL-20 genotypes Polymorphisms (rs2981573, rs2232360, rs1518108) were genotyped using 5 exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real-Time PCR system according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). 2.5. Statistical analysis Gene frequencies of IL-20 polymorphisms in patients and controls were obtained by direct counting. The Hardy-Weinberg equilibrium was evaluated by chi-square test. Gene frequencies in patients were compared to those present in controls using a
51
Age at presentation (years) Duration of disease (years) Extent of UC (%) Pancolitis Distal Extraintestinal manifestations (%) Present No Clinical course of disease Active then inactive Intermitent activity Persistent activity Response to treatment Favorable Steroid dependant Steroid resistant Immumodulator resistant Colectomy (%) Yes No
91/107 31.5 ± 14 12.34 134 (67.6%) 64 (32.3%) 64 (32.3%) 134 (67.6%) 42% 30% 28% 68% 20.5% 7.5% 4% 29 (14.6%) 169 (85.3%)
2 × 2 contingency table and Chi-square. Odd ratios (OR) have been calculated for the disease in carriers of specific alleles. Comparisons of allele frequencies between subgroups were performed using the EPI-INFO statistical package (Version 5.0; USD incorporated 1990, Stone Mountain, Georgia). Statistical significance was considered as p < 0.05. Pair wise linkage disequilibrium (LD, D ) estimations between polymorphisms and haplotype reconstruction were performed with Haploview version 3:32 (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA). 3. Results 3.1. Clinical features Of the 198 patients, 107 (54%) were women and 91 (46%) men, with ages ranging from 20 to 78 (mean 31.5 years). The evolution of the disease was 12.34 years. Pancolitis was present in 134 patients (67.6%) and distal colitis in 64 (32.3%). Sixty-four patients presented extraintestinal manifestations (32.3%) that included arthropathy (18%), primary sclerosing cholangitis (8%), erythema nodosum (2%), sacroiliitis (1.5%), ankylosing spondilytis (1.5%), and aphthous ulceration (1.3%). According to the clinical course of the disease, 42% had a pattern of activity then inactivity, 30% intermittent activity, and 28% showed chronic continual activity. Regarding medical treatment, 68% had a favorable response; 20.5% were steroid-dependent, 7.5% steroid-resistant, and 4% were azathioprine resistant. None of the patients received treatment with cyclosporine or infliximab. In regard to surgical treatment, twenty-nine patients (14.6%) underwent radical colectomy due to refractory medical therapy, toxic megalon, perforation and uncontrolled bleeding (Table 1). 3.2. IL-20 gene polymorphisms All IL-20 gene polymorphisms were consistent with HWE (p > 0.05). No indication of genotype error was observed and results from duplicate samples (10%) were 100% concordant. We found significant decreased frequencies of two IL-20 genotypes: GG (rs2981573) [10.6% vs. 17.6%, p = 0.017, OR = 0.55 95% CI: 0.33–0.93] and GG (rs2232360) [10.6% vs. 17.6%, p = 0.017, OR = 0.55, 95% CI: 0.33–0.93] in UC patients as compared to healthy controls. Similar distribution of the rs1518108 polymorphism was observed in
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Table 2 Allele (af) and genotype (gf) frequency (%) of IL-20 gene polymorphisms (rs2981573, rs2232360, and rs1518108) in UC patients and healthy controls.
rs2981573 Allele A G Genotypes AA AG GG rs2232360 Allele A G Genotypes AA AG GG rs1518108 Allele T C Genotype TT TC CC
UC
Controls
(n = 198) n 253 143 n 76 101 21 (n = 198) n 253 143 n 76 101 21 (n = 180) n 178 182
gf 0.494 0.505
(n = 698) n 827 569 0.407 n 252 323 123 (n = 698) n 828 568 n 253 322 123 (n = 698) n 658 738
0.238 0.511 0.25
150 358 190
43 92 45
af 0.638 0.361 gf 0.383 0.51 0.106 af 0.638 0.362 gf 0.383 0.51 0.106
p
OR
95%CI
gf 0.361 0.462 0.176
0.017
0.55
0.33–0.93
af 0.593 0.406 gf 0.362 0.461 0.176
0.017
0.55
0.33–0.93
af 0.592
gf 0.471 0.528 0.214 0.521 0.272
All populations were in Hardy-Weinberg equilibrium. Abbreviations: UC = ulcerative colitis; OR = odds ratio; CI = confidence intervals. The rs1518108 polymorphism was determined in 180 patients.
UC patients and healthy controls (Table 2). In the subgroup analysis, no differences were found between IL-20 genotypes and the clinical characteristics of UC (data not shown). 3.3. IL-20 haplotype analysis The analysis of linkage disequilibrium of the IL-20 gene polymorphisms was done using the Haploview program. In this analysis only two polymorphisms (rs2981573 and rs2232360) were in high linkage disequilibrium. The distribution of the constructed haplotypes was similar in UC patients and healthy controls (data not shown). 4. Discussion Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis are chronic inflammatory disorders of the gastrointestinal tract. Several genetic factors contribute to genetic susceptibility to these diseases. Some genome wide association studies (GWAS) have identified several risk conferring loci, some of them have been confirmed in different populations [14–17]. Polymorphisms of the IL-10 gene have been associated with UC in these studies [14–16]. In a previous study, four polymorphisms were associated with UC in European individuals; these results were replicated in three independent case-control panels [18]. The rs3024505 SNP immediately flanking the IL-10 gene showed the most significant association in the combined samples. The association of this SNP was corroborated in a sample of Indian patients [14]. Although several studies have linked the IL-10 gene with UC, few studies in other genes related with IL-10 (IL-19, IL-20, IL-22 and IL-24) have been reported. Recently, our group reported that the IL-19 gene polymorphisms (rs224188 and rs2243193) might have an important role in the development of UC in Mexican individuals [19]. Considering the important role of IL-10 cluster genes in UC, in the present work, two polymorphisms (rs2981573 and rs2232360) in the IL-20 gene were studied in UC patients. The frequencies of the GG genotypes in both polymorphisms were decreased in the group of patients. The role of these polymorphisms in the development of
UC confirms in part its similar effect in patients with psoriasis [11]. In psoriasis, the IL-20 polymorphisms were associated with different forms of the disease such as early-onset, late-onset, familial and sporadic disease [11]. A combined haplotype analysis of the IL-19 and IL-20 gene polymorphisms in psoriasis demonstrates the susceptibility effect of the IL-20 gene [20]. Kingo et al. studied the IL-20 rs2981573 polymorphism in patients with palmoplantar pustulosis and reported a decreased frequency of the G allele in these patients [21]. This is the same allele (in genotype form) that was decreased in our group of UC patients. At the present time there is no information about the functional effect of this polymorphism. Another study by Truelove et al. [22] showed association of the IL-20 rs1400986, rs3024517 and rs1518108 polymorphisms with chronic hepatitis B infection outcome. Only the rs1518108 polymorphism was analyzed in our study and this polymorphism was not associated with UC. The distribution of the polymorphisms in our population was compared to those reported in other ethnic groups [21,22] and in the HapMap project. Similar distribution of the rs1518108 polymorphism was observed in African and European populations when compared to Mexicans [21,22]. According to the HapMap information, the distribution of the rs2981573 and rs2232360 polymorphisms was similar in Mexicans when compared to Africans and Europeans but different when compared to Asian populations. In this case, the “A” allele of both polymorphisms was more frequent in Mexicans, Europeans and Africans (59% to 87%) when compared to Asians (25% to 33%). The pleiotropic inflammatory cytokine IL-20 is expressed in monocytes, epithelial cells, and endothelial cells and exerts its biological functions on multiple cell types by activating IL-20R1/IL20R2 or IL-22R1/IL- 20R2 complexes [23]. IL-20 is involved in various inflammatory diseases [24], such as psoriasis [25], rheumatoid arthritis [26], atherosclerosis [27], and renal failure [28]. In our study, no association of IL-20 polymorphisms with clinical features was observed. In our previous study of IL-19 polymorphisms, lack of association with clinical characteristics was also observed [19]. In both studies, this lack of associations between interleukin polymorphisms and clinical features could be caused
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by the sample size. Previously, in the same population, the HLADRB1*0103 allele was associated with the presence of colectomy [29]. To the best of our knowledge, this is the first study that provides evidence about the association of IL-20 polymorphisms with the risk of developing UC. However considering that this gene is located in the genomic IL-10 region, the participation of other genes of this cluster in the genetic susceptibility to UC cannot be ruled out. According to this, our group reported the association of UC with IL-19 polymorphisms in the same group of patients [19]. There are some limitations to this study. The results were not replicated in an independent set of patients and controls. Considering that this is the first time that polymorphisms located within the IL-20 gene are associated with UC, replication in another cohort of patients is necessary to confirm these results. In our study was not possible to evaluate all the described polymorphisms in the IL-20 gene. We selected three polymorphisms that have been reported associated with psoriasis. In conclusion, our results suggest that the GG genotypes of the IL-20 polymorphisms (rs2981573 and rs2232360) might have an important role in the development of UC in the Mexican population. Additional studies in other populations could help to define the potential role of this marker as protective factor against UC development. Acknowledgments This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología and Fundación Gonzalo Rio Arronte, Mexico City, Mexico.
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