Abstract
The purpose of this study was to investigate whether common variants in inflammatory and immune response genes influence inflammatory bowel disease (IBD) risk among Moroccan patients. Using a candidate gene approach, 10 single-nucleotide polymorphisms mapping on six genes (MIF_rs755622, TNFA_rs1800629, IL6_rs2069840, IL6R_rs2228145, IL6ST_rs2228044, IL17A (rs2275913, rs4711998, rs7747909, rs8193036, rs3819024)) were assessed in 510 subjects grouped in 199 IBD and 311 healthy controls. Genotyping was performed with the TaqMan allelic discrimination technology. The results were analyzed using PLINK software. The frequency of allele A for TNFA rs1800629 was significantly higher in ulcerative colitis (UC) patients compared with controls (30.16 vs 16.72%; P=0.0005; odds ratio (OR)=2.15; 95% confidence interval (CI)=1.39–3.32). Statistically significant association to UC was also found under dominant AA+AG vs GG (OR=1.85, 95% CI=1.07–3.21; P=0.02) and recessive models (OR=8.38; 95% CI=2.86–24.53; P=0.0001). In the same way, an association of TNFA rs1800629 variant was observed with IBD under recessive model AA vs AG+GG (OR=4.10; 95% CI=1.56–10.76; P=0.004). No evidence of significant associations was found for the remaining investigated polymorphisms. Our data suggest that TNFA gene promoter polymorphism participates in determining IBD susceptibility in Moroccan patients.
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Introduction
Inflammatory bowel disease (IBD) is a group of chronic disorders that includes two main disease forms, Crohn’s disease (CD; MIM 266600) and ulcerative colitis (UC; MIM 191390). It represents an important and worldwide common health problem with a continually increasing incidence.1 The underlying etiology and pathogenesis mechanisms have not yet been fully determined, but remain a field of intensive research. Current knowledge defines IBD as a complex disease with interplay of multiple factors such as environment, intestinal microbial flora, aberrant immune response mechanisms and individual’s genetic predisposition.2
Both genome-wide association studies and candidate-gene analysis have revealed a major contribution of genetic susceptibility factors involved in different components of the immune system (cytokines, cytokine receptors, innate and adaptive immune response) in IBD pathogenesis.3 Analysis of candidate genes with known immunologic function include single-nucleotide polymorphisms (SNPs) in the Interleukin 6 (IL6), Interleukin 6 receptor (IL6R), Interleukin 6 signal transducer Glycoprotein 130 (IL6ST GP130), Macrophage migration inhibitory factor (MIF), Tumor necrosis factor-α (TNFA) and Interleukin 17A (IL17A) genes, as well as a number of other genes involved in pathways that have been reported to have a critical regulatory role in IBD, and that are essential to the regulation of intestinal immune responses.4
The overall balance of pro-inflammatory and anti-inflammatory cytokines production is believed to trigger disease onset and appears to be likely a contributor to the clinical outcome of IBD, implying mucosal inflammation and loss of intestinal function.5, 6
Increased spontaneous production of TNF-α, IL-6 as well as IL-1 by lamina propria mononuclear cells have been shown to partially induce functional changes in the intestinal mucosa of IBD patients.7 Analysis of cytokine profile provided evidence of a high level of the pro-inflammatory cytokine IL-17 in patients with IBD.8 Colonic MIF mRNA expression was increased during DSS-induced colitis.9 In addition, increased formation of IL-6/sIL-6R complexes, which interact with membrane-bound gp130 on T cells via trans-signaling, supported an essential role in the development and perpetuation of IBD.10
Therefore, the aim of the present study was to ascertain whether polymorphisms of genes encoding mediators of inflammation known to be involved in determining the level of the immune response in the inflammatory pathway are associated with the occurrence of IBD, CD and UC subsets among Moroccan patients.
Results
Baseline demographic and clinical characteristics of patients are reported in Table 1.
The success rates of genotyping assays were (93–99%) for controls and (91–99.5%) for patients. The statistical power for the different studied polymorphisms ranged between 56 and 88% to detect genotypic relative risk of Odds ratio (OR)=1.5. In both patients and controls, the genotype frequencies for all SNPs agree with those predicted under Hardy–Weinberg equilibrium and any deviation from the expected was not statistically significant.
TNFA rs1800629
Frequency distribution of TNFA rs1800629 genotypes and variant allele in patients and controls are shown in Tables 2 and 3. In the present study, the frequency of allele A for TNFA rs1800629 was significantly higher in UC patients compared with the controls (30.16 vs 16.72%; P=0.0005; OR=2.15; 95% confidence interval (CI)=1.39–3.32). When including all patients (IBD), a slightly increased rate of minor allele frequencies was encountered without being statistically significant (20.35 vs 16.72%; P=0.14; OR=1.27; 95% CI=0.92–1.76).
With regard to genotype frequencies, the homozygous variant genotype distribution was increased in all disease subgroups UC (14.29%), IBD (7.54%) and CD (4.41%), compared with controls (1.95%). Genotypic test disclosed a statistically significant difference for IBD (X2=9.83, P=0.007), UC (X2=21.45, P=2.20E-05), but not for CD patients (X2=3.93, P=0.14; Table 3).
When Genetic models were assessed for the effect of TNFA SNP on disease risk, the significant associations were observed under recessive genetic model AA vs AG+GG (OR=4.10; 95% CI=1.56–10.76; P=0.004) for IBD, and under dominant AA+AG vs GG (OR=1.85; 95% CI=1.07–3.21; P=0.02) and recessive genetic models (OR=8.38; 95% CI=2.86–24.53; P=0.0001) for UC.
IL17A, MIF, IL6, IL6R and IL6ST genes
All the studied polymorphisms in IL17A showed a lack of significant associations for CD, UC as well as IBD when considering minor allele frequencies distribution. A trend of a protective effect for the SNP rs3819024 was observed for UC patients (ORunadj=0.60, 95% CI=0.35–1.05, P=0.07; Table 2). Lower frequencies of both the homozygous and heterozygous variant genotypes of the latter polymorphism were observed in UC patients compared with controls (GG: 1.72% vs 6.92%; GA: 25.86% vs 30.45%). Taking into account these results, we performed genetic model analysis to account for the variation of risk effect, a non-significant reduced risk was observed for both dominant (OR=0.63; 95% CI=0.34–1.19; P=0.15) and recessive models (OR=0.23; 95% CI=0.03–1.79; P=0.16). The genetic model analysis for the remaining SNPs disclosed a significant association with CD under dominant genetic model for rs7747909 (OR=1.57; 95% CI=0.99–2.47; P=0.05), but this was not observed after FDR-BH correction.
The distribution of alleles and genotypes of MIF rs755622, IL6 rs2069840, IL6R rs2228145 and IL6ST (GP130) rs2228044 in the patients and control groups is shown in Tables 2 and 3. According to the obtained results, there was no statistically significant difference between the different groups when comparing allele and genotype frequencies of the investigated polymorphisms.
Regarding the possible effects of genetic models on disease risk, no association was seen with any of the studied polymorphisms, either for the IBD group as a whole or when dividing into CD and UC groups. Noteworthy that, the decreased frequency of mutant allele C and heterozygous variant genotype CG for IL6ST (GP130) rs2228044 in CD patients compared with controls, was suggestive of a protective effect. However, the difference was not statistically significant.
Discussion
This is the first study to examine the association of polymorphisms in genes encoding cytokines/cytokine receptors: MIF, TNFA, IL6, IL6R, IL6ST (GP130) and IL17A with the pathogenesis of IBD (CD and UC) in the Moroccan population. Our findings support a strong evidence of association between the functional TNFA rs1800629 polymorphism and the risk of UC and IBD. Genetic model testing demonstrated recessive inheritance (genotype AA vs AG+GG), to be the best-fit genetic model in the reported associations: 4.10 (95% CI, 1.56–10.76) for IBD and 8.38 (95% CI=2.86–24.53) for UC. The high relative risk of the TNF-α -308 polymorphism obtained in our study drives us to confirm the previously observed associations in other populations.
In line with our results, Song et al. reported significantly higher TNFA -308 genotypic and allelic frequencies (15.5% and 8.7% vs 4.1% and 2.0%, respectively; P<0.001), in patients with UC compared with controls. Similarly to our study, no significant difference was observed between patients with CD and the healthy controls.11
In the same way, variant allele A frequency of the TNFA rs1800629 was not different in CD compared with that in the control group in the study reported by Sykora et al.,12 whereas significant differences were found between the IBD group (P<0.05), the UC group (P<0.001) and controls. In addition, differences in the haplotype AG (−308A, −238 G) carrier frequency were noted between UC patients and the control group (OR=4.76; 95% CI=1.53–14.74; P<0.01).13 In contrast to our finding, no statistical differences in TNFA genotypes and allele distributions between the IBD groups and healthy controls were found in the Spanish population.14 In addition, the homozygous and the heterozygous variant genotypes of TNFA -308 G>A (rs1800629) (ORunadj=0.75; 95% CI=0.58–0.98; P=0.04) were associated with reduced risk of UC but not with CD in a Danish Cohort.15 Furthermore, Bouma et al.,16 observed a decreased -308A allele frequency in patients with UC compared with healthy controls (0.15 in UC vs 0.25 in HC, P=0.044). The same results were observed by Vatay et al.17 In the meta-analysis conducted by Fan W et al., the AA genotype significantly increased the risk of UC (OR=2.041; 95% CI=1.261–3.301) and CD (OR=1.730; 95% CI=1.168–2.564) in Europeans. Meanwhile in Asians, the GA genotype increased the risk of UC (OR=2.360; 95% CI=1.269 4.390).18
Several studies have reported the involvement of TNFA genetic variations on the enhancement of susceptibility to a large number of inflammatory and auto-immune diseases.19 Human TNF-α gene, spanning ~3 kb,20 has been mapped to the IBD3 region on chromosome 6p21.3, which is a good functional candidate for involvement in predisposition to IBD.21 SNPs in the promoter region of the TNFA, including the rs1800629 G>A located at position −308, have been associated with enhanced TNF-α expression both in vivo and in vitro.22, 23 The encoded protein is a key proinflammatory cytokine considered to have an essential role as a potent immuno-mediator in driving a wide variety of effector functions that are of major importance in IBD pathogenesis. This was evidenced by increased TNF-α levels found in the serum, mucosa, intestinal tissues, peripheral phagocytes and stool of patients with IBD.24, 25, 26 In addition, it should be noted that the infusion of monoclonal anti-TNF antibody is a highly efficacious IBD therapy.27
As for TNFA, an accumulating body of evidence has demonstrated the important pathological role played by genetic variations of IL-17 in the development of inflammatory and auto-immune diseases. The functional relevance of T helper 17 cells (Th17) and their signature cytokines (IL-17) have contributed to advance our understanding of mechanisms that regulate mucosal homeostasis and inflammation in the gut. Highly differentiated Th17 cells were abundantly found in the inflamed intestinal mucosa.28 Proinflammatory qualities of IL-17 were evidenced and are involved in several chronic inflammatory disorders, including IBD. IL-17A was abundantly found in inflamed IBD mucosa,29 its characterization represented a hallmark in IBD immunobiology by providing a new distinctive pathway for the communication between adaptive and innate immunity. Upregulation of the pleiotropic proinflammatory IL-17A cytokine has also been reported in active UC and CD.30 IL-17 contribute to perpetuation of intestinal inflammation through several mechanisms, including the proinflammatory effects mediated by the induction of TNF-α, IL-1b, IL-6, chemokines and metalloproteases,31 on intestinal epithelial cells, epithelial barrier, and other effectors (macrophages, lymphocytes and dendritic cells). We investigated the relevance of IL17A genetic variations in IBD pathogenesis. Our results showed that the genotype AA+AG vs GG of IL17A rs7747909 was associated with CD before correcting for multiple testing. However, no significant associations were found with regards to minor allele frequencies distributions. Several studies have indicated that the IL-17A G197A (rs2275913) is significantly linked to the development of UC,32, 33, 34 an association that was not confirmed in the present study.
In the other hand, studies on MIF, IL6, IL6R and IL6ST gene polymorphisms distribution in relation to IBD generated conflicting results in different populations. Data on association of these genes with IBD in the North African population are currently lacking. No significant difference was found at both allelic and genotypic levels between IBD, CD, UC patients and healthy controls in our population, suggesting that genetic variants of these genes have no effect on IBD pathogenesis.
We suggest that TNFA gene promoter polymorphism -308G/A participates in determining IBD susceptibility in Moroccan patients. The possible mechanism by which genetic variation in TNFA promoter region contribute to the development of IBD could be through alterations in the function of inflammatory pathways leading to enhancement of disease risk. Our finding requires further replication on larger study groups with additional functional investigations, evaluating TNFA transcriptional and cytokine production levels.
Material and methods
Study population
A total of 199 patients diagnosed with IBD (136 CD; 63 UC) at the CHU Ibn Rochd Hospital (Casablanca, Morocco) were included. Samples from 311 blood donors were used as ethnically matched controls. The diagnosis of CD or UC was established according to the conventional clinical, endoscopic, radiological and histological criteria as previously described.35, 36 CD phenotype was classified according to the Montreal classification.36 UC anatomic location was subgrouped using Paris classification.37 Patient’s clinical and demographic characteristics were collected in a case report form including questions on toxic behavior, disease location and phenotype, age at diagnosis and other clinical features. Informed written consent was obtained from all participants and approval from the local ethics committee was obtained.
Genotyping
Genomic DNA was isolated from peripheral blood using standard procedures and from Formalin Fixed Paraffin Embedded Tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA, USA). Ten SNPs located within the following genes were analyzed: MIF rs755622, TNFA rs1800629, IL6 rs2069840, IL6R rs2228145, IL6ST (GP130) rs2228044and IL17A (rs4711998; rs8193036; rs3819024; rs2275913; rs7747909). All the SNPs were genotyped using TaqMan allelic discrimination assays on the Light Cycler 480 System (Roche, Barcelona, Spain).
Statistical analysis
The overall statistical power of the present study was calculated using Power Calculator of Genetic Studies 2006 software (http://www.sph.umich.edu/csg/abecasis/CaTS/). Data analysis has been carried out using Plink software V1.07 (http://pngu.mgh.harvard.edu/purcell/plink/). The genotypes and alleles frequencies distribution were compared between patients and controls using the χ2 test or Fisher's test. ORs with a CI of 95% were calculated to measure the strength of association. The Hardy–Weinberg equilibrium was tested for all SNPs by χ2 analysis. Benjamini and Hochberg step-up false discovery rate control correction for multiple testing was applied to significant P-values of IL17A polymorphisms. A P-value <0.05 was considered statistically significant.
References
Xavier RJ, Podolsky DK . Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007; 448: 427–434.
Zhang YZ, Li YY . Inflammatory bowel disease: pathogenesis. World J Gastroenterol 2014; 20: 91–99.
Jostins L, Ripke S, Weersma RK, Duerr RH, McGovern DP, Hui KY et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012; 491: 119–124.
Senhaji N, Diakite B, Serbati N, Zaid Y, Badre W, Nadifi S . Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms: new data and a meta-analysis. BMC Gastroenterol 2014; 14: 206.
Hassig A, Kremer H, Liang WX, Stampfli K . The role of the Th-1 to Th-2 shift of the cytokine profiles of CD4 helper cells in the pathogenesis of autoimmune and hypercatabolic diseases. Med Hypotheses 1998; 51: 59–63.
Quaglietta L, te Velde A, Staiano A, Troncone R, Hommes DW . Functional consequences of NOD2/CARD15 mutations in Crohn disease. J Pediatr Gastroenterol Nutr 2007; 44: 529–539.
Reinecker HC, Steffen M, Witthoeft T, Pflueger I, Schreiber S, MacDermott RP et al. Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease. Clin Exp Immunol 1993; 94: 174–181.
Kobayashi T, Okamoto S, Hisamatsu T, Kamada N, Chinen H, Saito R et al. IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn's disease. Gut 2008; 57: 1682–1689.
Ohkawara T, Nishihira J, Takeda H, Hige S, Kato M, Sugiyama T et al. Amelioration of dextran sulfate sodium-induced colitis by anti-macrophage migration inhibitory factor antibody in mice. Gastroenterology 2002; 123: 256–270.
Atreya R, Mudter J, Finotto S, Mullberg J, Jostock T, Wirtz S et al. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med 2000; 6: 583–588.
Song Y, Wu KC, Zhang L, Hao ZM, Li HT, Zhang LX et al. Correlation between a gene polymorphism of tumor necrosis factor and inflammatory bowel disease. Chin J Dig Dis 2005; 6: 170–174.
Sykora J, Subrt I, Didek P, Siala K, Schwarz J, Machalova V et al. Cytokine tumor necrosis factor-alpha A promoter gene polymorphism at position -308 G—>A and pediatric inflammatory bowel disease: implications in ulcerative colitis and Crohn's disease. J Pediatr Gastroenterol Nutr 2006; 42: 479–487.
Sashio H, Tamura K, Ito R, Yamamoto Y, Bamba H, Kosaka T et al. Polymorphisms of the TNF gene and the TNF receptor superfamily member 1B gene are associated with susceptibility to ulcerative colitis and Crohn's disease, respectively. Immunogenetics 2002; 53: 1020–1027.
López-Hernández R, Valdés M, Campillo JA, Martínez-García P, Salama H, Bolarin JM et al. Pro- and anti-inflammatory cytokine gene single-nucleotide polymorphisms in inflammatory bowel disease. Int J Immunogenet 2015; 42: 38–45.
Bank S, Skytt Andersen P, Burisch J, Pedersen N, Roug S, Galsgaard J et al. Polymorphisms in the inflammatory pathway genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG are associated with susceptibility of inflammatory bowel disease in a Danish cohort. PLoS One 2014; 9: e98815.
Bouma G, Xia B, Crusius JB, Bioque G, Koutroubakis I, Von Blomberg BM et al. Distribution of four polymorphisms in the tumour necrosis factor (TNF) genes in patients with inflammatory bowel disease (IBD). Clin Exp Immunol 1996; 103: 391–396.
Vatay A, Bene L, Kovacs A, Prohaszka Z, Szalai C, Romics L et al. Relationship between the tumor necrosis factor alpha polymorphism and the serum C-reactive protein levels in inflammatory bowel disease. Immunogenetics 2003; 55: 247–252.
Fan W, Maoqing W, Wangyang C, Fulan H, Dandan L, Jiaojiao R et al. Relationship between the polymorphism of tumor necrosis factor-alpha-308 G>A and susceptibility to inflammatory bowel diseases and colorectal cancer: a meta-analysis. Eur J Hum Genet 2011; 19: 432–437.
Qidwai T, Khan F . Tumour necrosis factor gene polymorphism and disease prevalence. Scand J Immunol 2011; 74: 522–547.
Burke LE, Nerbonne MA . The influence of the guess factor on the speech reception threshold. J Am Aud Soc 1978; 4: 87–90.
Hampe J, Schreiber S, Shaw SH, Lau KF, Bridger S, Macpherson AJ et al. A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort. Am J Hum Genet 1999; 64: 808–816.
Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW . Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci USA 1997; 94: 3195–3199.
Elahi MM, Asotra K, Matata BM, Mastana SS . Tumor necrosis factor alpha -308 gene locus promoter polymorphism: an analysis of association with health and disease. Biochim Biophys Acta 2009; 1792: 163–172.
Komatsu M, Kobayashi D, Saito K, Furuya D, Yagihashi A, Araake H et al. Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR. Clin Chem 2001; 47: 1297–1301.
Braegger CP, Nicholls S, Murch SH, Stephens S, MacDonald TT . Tumour necrosis factor alpha in stool as a marker of intestinal inflammation. Lancet 1992; 339: 89–91.
Breese EJ, Michie CA, Nicholls SW, Murch SH, Williams CB, Domizio P et al. Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology 1994; 106: 1455–1466.
D'Haens G, Van Deventer S, Van Hogezand R, Chalmers D, Kothe C, Baert F et al. Endoscopic and histological healing with infliximab anti-tumor necrosis factor antibodies in Crohn's disease: a European multicenter trial. Gastroenterology 1999; 116: 1029–1034.
Abraham C, Cho J . Interleukin-23/Th17 pathways and inflammatory bowel disease. Inflamm Bowel Dis 2009; 15: 1090–1100.
Jiang W, Su J, Zhang X, Cheng X, Zhou J, Shi R, Zhang H . Elevated levels of Th17 cells and Th17-related cytokines are associated with disease activity in patients with inflammatory bowel disease. Inflammation Research. 2014; 63: 943–950.
Nielsen OH, Kirman I, Rudiger N, Hendel J, Vainer B . Upregulation of interleukin-12 and -17 in active inflammatory bowel disease. Scand J Gastroenterol 2003; 38: 180–185.
Jovanovic DV, Di Battista JA, Martel-Pelletier J, Jolicoeur FC, He Y, Zhang M et al. IL-17 stimulates the production and expression of proinflammatory cytokines, IL-beta and TNF-alpha, by human macrophages. J Immunol 1998; 160: 3513–3521.
Arisawa T, Tahara T, Shibata T, Nagasaka M, Nakamura M, Kamiya Y et al. The influence of polymorphisms of interleukin-17A and interleukin-17F genes on the susceptibility to ulcerative colitis. J Clin Immunol 2008; 28: 44–49.
Kim SW, Kim ES, Moon CM, Park JJ, Kim TI, Kim WH et al. Genetic polymorphisms of IL-23R and IL-17A and novel insights into their associations with inflammatory bowel disease. Gut 2011; 60: 1527–1536.
Hayashi R, Tahara T, Shiroeda H, Saito T, Nakamura M, Tsutsumi M et al. Influence of IL17A polymorphisms (rs2275913 and rs3748067) on the susceptibility to ulcerative colitis. Clin Exp Med 2013; 13: 239–244.
Serbati N, Senhaji N, Diakite B, Badre W, Nadifi S . IL23R and ATG16L1 variants in Moroccan patients with inflammatory bowel disease. BMC Res Notes 2014; 7: 570.
Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, Brant SR et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005; 19 (Suppl A): 5A–36A.
Levine A, Griffiths A, Markowitz J, Wilson DC, Turner D, Russell RK et al. Pediatric modification of the Montreal classification for inflammatory bowel disease: the Paris classification. Inflamm Bowel Dis 2011; 17: 1314–1321.
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We gratefully acknowledge all the members of LGMP and the Gastroenterology department.
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Senhaji, N., Serrano, A., Badre, W. et al. Association of inflammatory cytokine gene polymorphisms with inflammatory bowel disease in a Moroccan cohort. Genes Immun 17, 60–65 (2016). https://doi.org/10.1038/gene.2015.52
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DOI: https://doi.org/10.1038/gene.2015.52
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