Journal of Gastroenterology, Pancreatology & Liver Disorders

Background and Aim: Crohn’s disease (CD) and ulcerative colitis (UC) are promoted by a complex interaction of genetic, immunologic and environmental factors. Given the great efficacy of anti- TNF-α therapy, we aimed in this study to investigate the potential involvement of TNF-α polymorphisms for either pathogenesis or response to therapy.

Methods: 1221 patients, 586 CD (336 male, mean age 46±14 yrs), 635 UC (394 male, mean age 49±14 yrs), and 386 gender-matched healthy subjects were genotyped for G308A and C857T TNF-α polymorphisms. Allele-genotype frequencies and genotype/phenotype associations were evaluated by multivariate logistic regression.

Results: The frequency of -308A allele of the TNF-α gene was significantly increased in both CD (OR=1.6 CI=1.1-2.3; P=0.005), and UC patients (OR=1.6 CI=1.1-2.2; P=0.005), compared with controls (7%). Similarly, the homozygous and heterozygous carriers together, were significantly more frequent in both CD (20%; OR=1.7 CI=1.2- 2.5; P=0.003) and UC patients (19%; OR=1.6 CI=1.1-2.3; P=0.01), compared to healthy controls (11%). In contrast, for the -G857T polymorphism no significant difference was found. CD carriers of -308A risk allele displayed a more aggressive clinical course, with an increased frequency of surgical resection (42%; P=0.03) and steroid resistance (19%; P=0.007).At the stepwise logistic regression, the correlation between the TNF-α risk genotype and reduced efficacy of steroid therapy in patients with CD was confirmed (OR=2.7; CI=1.2– 6; P=0.015), also taking into account other potential confounders.

Conclusion: These observations, awaiting further confirms, leads us to hypothesize that carriers of the uncommon allele -308A, by producing more TNF-α, are more prone to became steroid-resistant.

Keywords: Inflammatory Bowel Disease (IBD), Ulcerative Colitis (UC), Crohn’s disease (CD), genetic predisposition, Tumour necrosis factor-alpha (TNF-α)

Ulcerative colitis (UC; MIM 191390) and Crohn’s disease (CD; MIM 266600) are two similar yet distinct conditions, collectively referred as inflammatory bowel disease (IBD). Although the key mechanisms underlying the development of IBD are still unknown, available evidences suggest that it results from a complex interaction of genetic, environmental, and immunologic factors.

In the last years, with improved genotyping technologies and the completion of the human genome sequence, several genomewide association studies (GWAS) have identified besides the known variants of CARD15 located at 16q12 [4,5] more than 150 loci that confer susceptibility or protection against IBD [6].

The IBD3 locus on chr 6p has been identified with consistence evidence in different genome wide scans [4-7] and metaanalyses [8,9]. This area encompasses the HLA region and the Tumour Necrosis Factor-alpha (TNF-α) gene. TNF-α encodes a pro-inflammatory citokine, involved in the imbalance between regulatory and effector cell immune response that was found increased in the mucosa, serum and stool of patients. The role of TNF-α in IBD has been largely demonstrated in animal, clinical, and functional studies. In the TNF^ΔARE model, deletion of 30 regulatory elements leads to increased TNF synthesis and a Crohn’s-like phenotype [10]. In contrast, TNF-/- mice show marked reduction to chemically induced intestinal inflammation compared with control mouse [11]. Convincing clinical evidence of the importance of TNF-α is seen in both CD and UC from the dramatic response following administration of anti-TNF-α monoclonal antibody [12,13]. Furthermore, TNF-α induces dysregulation of the intestinal barrier regulating the tight junctions leading to increased permeability [14].

A number of TNF promoter polymorphisms have been described raising the possibility that genetic factors might play a role in determining IBD susceptibility through altered TNF expression. Polymorphisms in the TNF-α gene and promoter region have been studied extensively on IBD susceptibility and/ or clinical manifestations, although with conflicting findings [15, 36].

For instance, the -G308A polymorphism in the promoter region has been associated with inducible levels of TNF-α in vitro and functional effect on gene transcription activity; in addition, carriers of the TNF-α risk allele -308A presented higher constitutive and inducible transcriptional levels than -G308 allele carriers [16,17]

Moreover, the promoter TNF-857 C-->T single nucleotide polymorphism (SNP) is functional through the binding to the transcription factor octamer transcription factor-1 (OCT-1), and has been frequently associated with CD [16, 19, 25, 31, 33, 38].

Given the potential interest of TNF-α polymorphisms for both pathogenesis and response to therapy in IBD patients, and the contradictory results available, we planned to investigate the contribution -C857T and -G308A polymorphisms in disease susceptibility and more specifically the response to medical therapy in a large population of Italian IBD patients.

IBD patients were consecutively recruited in three Italian referral centres: the IRCCS “Casa Sollievo della Sofferenza” Hospital of San Giovanni Rotondo, the University Hospital of Padua, and the Careggi University Hospital of Florence. Diagnosis of CD and UC was established according to accepted clinical, endoscopic, radiological, and histological criteria [18].

One thousand two hundred twenty-one IBD patients were included in the study; 586 were CD patients (336 male, mean age 46 ± 14 yrs), and 635 were UC patients (394 male, mean age 49 ± 14 yrs). Their clinical characteristics are summarized in Table 1. Three hundred eighty-six gender-matched healthy subjects were also evaluated. They were unrelated, asymptomatic subjects (blood donors, students, and staff members) recruited in the same centres, all of Caucasian and non-Jewish descent. Ethics approval was obtained for each of the participating centres. All the study participants were Caucasian, and gave their fully informed written consent

The Montreal classification was used [19]; for the evaluation of localisation, the largest extent of the disease based on x-ray, endoscopy, or surgical report was considered. A detailed clinical questionnaire concerning different features of the disease was used. In all patients the following clinical features were recorded: family history, age at diagnosis, duration of followup, localization of CD (ileum, ileocolonic, colonic, upper GI tract), disease ‘‘type’’ (penetrating, stricturing, inflammatory [non-penetrating and non-stricturing]), extent of UC (proctitis, left-sided colitis, pancolitis), presence of perianal fistulas, extraintestinal manifestations (presence or absence of any extra intestinal manifestations and more specifically arthropathy or skin lesions), abdominal surgery (colectomy in UC or bowel resection in CD) and smoking habit (at least 1 cigarette/day). To account for the known modification of clinical characteristics during the disease course, only patients with at least 3 years of follow-up from symptom onset and at least 1 year of follow-up from firm diagnosis were included in the genotype/phenotype analysis. The standard investigations used in these patients were upper GI endoscopy, ileocolonoscopy, barium follow-through and entero-MR. For the purpose of the study, particular attention was paid to medical therapy and, more specifically, to the response to mesalamine (5-ASA), corticosteroids (CS), azathioprine (AZT), 6-mercaptopurine (6-MP), methotrexate, cyclosporine A and anti-TNF agents. Patients were classified as responder or nonresponder on the basis of the review of medical records [20]. More specifically, for 5-ASA the clinical response was determined during the first course of treatment. First course was defined

as the period from the date that patients started therapy with 5-ASA to the date that the medication was discontinued or to the last follow-up if the medication was not discontinued. Response was defined as clinical remission or clear improvement (mild symptoms not requiring alternative therapies) by the initial 8 weeks of therapy [21]. Patients requiring oral/parenteral CS, immunosuppressive (IMS) drugs, or anti-TNFα during the disease course were considered non-responders to 5-ASA

Patients using CS were classified as CS responders (at least 1 course of systemic steroids with clinical remission reported in the medical history), CS dependent (according to accepted definition), or CS refractory (when an unsuccessful clinical response was achieved, leading to alternative therapies such as surgery, use of anti-TNFα or other IMS drugs) [22,23]. When a modification of response to therapy was recorded during the disease course (i.e., previous CS responder becoming CS dependent), only the most clinically relevant category was considered (i.e., refractory more relevant than dependent, more relevant than responder). Particular attention was paid to the dose and duration of the therapy used, according with current guidelines [24]. Patients with incomplete information or inadequate therapeutic schedule were excluded from analysis. Finally, patients were divided into anti-TNFα responder or non-responder according to short-term (12 weeks) response [25]. Many patients with IBD are currently treated with a combination of drugs during the disease course. A possible synergic effect of different drugs (i.e. 5-ASA and CS) was not assessed in this study; however, for the purpose of this analysis, the most clinically relevant category was chosen. For example, when a patient with a 5-year favourable response to 5-ASA eventually required steroid therapy, the patient was classified as 5-ASA non-responder, even if 5-ASA was continued. Accordingly, when the patient became steroid dependent and responsive to 6-MP after an effective course of steroid therapy, he or she was classified as a 5-ASA non-responder, steroid-dependent, 6-MP responder. Patients intolerant of any investigated drug were not included in this analysis. assay was used. The 380-bp PCR product was digested with HhaI (New England Biolabs), yielding 2 fragments of 138 and 242 bp in the presence of the C allele, and then visualized on 2% (wt/vol) agarose gel. One hundred random samples were also confirmed by sequencing on an ABI 310 DNA sequence (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations.

Genomic DNA samples were extracted from peripheral blood leukocytes, according to standard protocols [26] and genotyped at the Molecular Laboratory of the Unit of Gastroenterology of the San Giovanni Rotondo Hospital, Italy.

The TNF-α gene promoter polymorphisms G308A and C857T were genotyped by a polymerase chain reaction (PCR) restriction fragment length polymorphism procedure. In brief, after PCR (annealing temperature, 55°C, 35 cycles), the 170 base pairs (bp) for 308 and 154 bp for 857 products were digested by the restriction enzyme NcoI and HypCH4 IV (New England Biolabs, Ipswich, MA), respectively, and separated by agarose gel electrophoresis. The profile of the -G308A variant was characterised by 23 and 147 bp; for C857T, it was 21 and 128 bp. Genotyping for Arg702Trp and Leu1007 fsinsC common CARD15 variants was also performed by DHPLC, whereas for the Gly908Arg variant, a restriction fragment length polymorphism assay was used. The 380-bp PCR product was digested with HhaI (New England Biolabs), yielding 2 fragments of 138 and 242 bp in the presence of the C allele, and then visualized on 2% (wt/vol) agarose gel. One hundred random samples were also confirmed by sequencing on an ABI 310 DNA sequence (Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations.

Comparison of allele-genotype frequencies and genotype/ phenotype association was performed by the chi-square or Fisher exact tests, when appropriate; Student’s T test was used to compare means of continuous variables by the SPSS software ver. 11.5. Test of Hardy-Weinberg equilibrium and marker linkage disequilibrium analysis was performed by the Arlequin software ver 2.0.

Genotype-phenotype associations were analysed by means of univariate and multivariate logistic regression with SPSS software; this approach allowed us to take into account a dose-response effect (heterozygote or homozygote), the possible interactions between genes and the effect of potential confounding variables (e.g. disease behaviour, disease localisation).

All the investigated SNPs were found to be in Hardy- Weinberg equilibrium in the control group and in patients. Allele and genotype frequencies are presented in Table 2.

The frequency of -308A allele of the TNF-α gene was significantly increased in IBD (OR=1.6 CI=1.2-2.2; P=0.002), and in both CD (OR=1.6 CI=1.1-2.3; P=0.005), and UC patients (OR=1.6 CI=1.1-2.2; P=0.005), compared with controls (7%). Considering the homozygous and heterozygous states together, carriers were significantly more frequent in the whole IBD group (19%; OR=1.7 CI=1.2-2.3; P=0.003),in CD (20%; OR=1.7 CI=1.2-2.5; P=0.003) and UC patients (19%; OR=1.6 CI=1.1-2.3; P=0.01), compared to healthy controls (11%). In contrast, for either allele or genotype frequencies of the -G857T allele no significant difference in UC and CD patients compared with controls emerged.

When patients were stratified according to the presence or absence of at least 1 CARD15 variant to disclose possible gene– gene interaction, no significant difference in TNF-α was found (Table 3).

For the analysis of possible correlation between genotype and phenotype, patients were classified on the basis of presence (TNF+= -308AA or AG genotypes) or absence (TNF-= GG308 genotype) of the risk genotype for TNF-α. At the univariate analysis of clinical features of patients with CD (Table 4), an increased frequency of surgical resection in TNF+(42%) compared with TNF- carriers was found (31%; OR= 1.58; CI=1.0–2.4; P=0.03). No correlation was found for age at diagnosis, family history, disease behaviour, perianal fistulas, and extra-intestinal manifestations. When the efficacy of medical

therapy was investigated, patients TNF+were significantly more resistant to steroids (19%) compared with TNF- patients (9%; OR= 2.4; CI=1.26–4.8; P=0.007). No difference in the efficacy of mesalamine, IMS, and anti-TNFα was found.

In patients with UC (Table 4), no significant association of TNF-α polymorphisms with any clinical characteristics was demonstrated, with the exception of an increased frequency of surgical resection in TNF+(18%) compared with TNF- patients (12%; OR= 1.7; CI= 1–2.9; P=0.05).

At the stepwise logistic regression, the correlation between the TNF-α risk genotype and reduced efficacy of steroid therapy in patients with CD was confirmed (OR=2.7; CI=1.2–6; P=0.015), also taking into account other potential confounders such disease localisation, and disease behaviour. When patients with surgical resection were studied, taking into account potential confounders such as disease localisation, disease behaviour and TNF genotypes, the disease type (i.e. stenosing/fistulising) but not TNF genotype increased the risk of surgical treatment (OR=18.7; CI=8.8–39.8; P< 0.01) (Table 5).

Chronic inflammation in IBD is primarily due to an immunological imbalance between pro- and anti-inflammatory cytokines, together with a defective apoptosis of lamina propria T cells [41]. Amongst the pro-inflammatory cytokines, clinical, functional, and animal studies candidate TNF-α gene as an attractive player in the inflammatory process of IBD. TNF-α is a pro-apoptotic cytokine largely secreted by monocytes/ macrophages during innate immune responses and by effector Th1 cells.

Studies on TNF-α gene have pointed mainly on a limited number of promoter polymorphisms. Association of the C allele of the TNF -C857T polymorphism to IBD, with more significant association specifically to CD [17, 22, 28, 32] have been reported. In addition, carriers of the A risk allele of the TNF -G308A variant has been associated with stenosing and penetrating behaviour in CD [18,23,31,33] Moreover, by using a weighted linear combination of SNPs[27] the variation -308 was confirmed to be involved with CD to the same extent as markers of CARD15.

It has been also demonstrated that these variants are associated with increased constitutive and/or inducible levels of TNF protein production in vitro. However, whether this variant is itself functional or in LD with the actual causative allele is still unclear (41, 42). In addition, with the exception of our pilot study in children with IBD [40] no evidence of correlation of TNF-α polymorphism with response to medical therapy has been demonstrated.

In our large cohort of 1221, IBD patients, the two more studied polymorphisms, -G308A and-C857T, in the TNF-α gene promoter have been analysed.
The -308A allele was significantly associated with both CD (10%; P=0.005) and UC patients (10%; P=0.005). Carriers of combined homo or heterozygote genotypes were more frequently

CD (20%; P=0.003) and UC patients (19%; P=0.01) than healthy controls (11%). In contrast, for either allele or genotype frequencies of the -G857T allele no significant difference in patients with CD and patients with UC compared with controls emerged.

Moreover, CD carriers of -308A risk allele displayed a more aggressive clinical course, with an increased frequency of surgical resection (42%; P=0.03) and steroid resistance (19%; P=0.007). No correlation was found for age at diagnosis, family history, disease behaviour, perianal fistulas, extra-intestinal manifestations and the efficacy of mesalamine, IMS, and anti- TNFα. To better understand the contribution of the TNF-α variant to the clinical heterogeneity of CD with respect to other factors (i.e., disease localisation, disease behaviour and TNF) a logistic regression analysis has been performed. The correlation between the TNF-α risk genotype and reduced efficacy of steroid therapy in patients with CD was confirmed (OR=2.7; CI=1.2–6; P=0.015), also taking into account other potential confounders such disease localisation and behaviour.

In contrast, when patients with surgical resection were studied, the disease type (i.e. stenosing/fistulising), but not the TNF polymorphism, significantly increased the risk of surgical treatment (OR=18.7; CI=8.8–39.8; P< 0.01).

When considering the use of corticosteroids, 11% of our 407 CD patients did not respond to therapy; specifically, carriers of -308AA and AG genotypes were significantly more frequent into the resistant group (19%) compared to patient responders (9%; OR= 2.4; CI=1.26–4.8; P=0.007). The value remains significant after correcting for disease localisation and disease behaviour (P=0.015).

Corticosteroids are still effective as induction therapy in moderate to severe active UC and CD patients. Epidemiological data have shown that up to a quarter of patients became steroiddependent and 20% steroid-resistant within 1 year [28]. Although the mechanism of steroid resistance is unclear, it is known that TNF-α levels are increased in carriers of the uncommon -308A allele [26] and TNF-α was demonstrated to induce dysregulation of the intestinal barrier regulating the tight junctions (TJ) leading to increased permeability. A number of study have suggested that the elevated levels of TNF-α present in CD patients are responsible for the observed increases in intestinal permeability seen in these patients [27-29]. It was also proved that in vitro glucocorticoids inhibit the increase TJ permeability induced by TNF-α [28].

Taken together these observations leads us to speculate that carriers of the uncommon allele -308A, by producing more TNF-α, are more prone to became steroid-resistant probably because the drug does not exert the full activities to block the increased endogenous level of TNF-α of these patients. Therefore, pinpoint patients non-responsive to steroids based on TNF-α polymorphism might avoid prolonged and eventually ineffective therapy, reducing either the risks for the patient and a more prolonged exposure to drug.

Further studies are needed to confirm these data and discover the possible biological mechanism of steroid resistance and its correlation with TNF-α genotype and levels.

1. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature. 2001;411:599-603. doi:10.1038/35079107.
2.  Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature. 2001;411(6837):603-606.
3. 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(7422):119-124. doi: 10.1038/nature11582.
4. Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, McLeod RS, Griffiths AM, et al. Genome wide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci. AmJHumGenet. 2000;66(6):1863-1870.
5.   Hampe J, Shaw SH, Saiz R, Leysens N, Lantermann A, Mascheretti S, et al. Linkage of inflammatory bowel disease to human chromosome 6p. Am J Hum Genet. 1999;65(6):1647-1655.
6.    Dechairo B, Dimon C, van Heel D, Mackay I, Edwards M, Scambler P, et al. Replication and extension studies of inflammatory bowel disease susceptibility regions confirm linkage to chromosome 6p (IBD3). Eur J Hum Genet. 2001;9(8):627-633.
7.    Yang H, Plevy SE, Taylor K, Tyan D, Fischel-Ghodsian N, McElree C, et al. Linkage of Crohn's disease to the major histocompatibility complex region is detected by multiple non-parametric analyses. Gut. 1999;44(4):519-526.
8.    Van Heel DA, Fisher SA, Kirby A, Daly MJ, Rioux JD, Lewis CM et al. Inflammatory bowel disease susceptibility loci defined by genome scanmeta-analysis of 1952 affected relative pairs. Hum Mol Genet. 2004;13(7):763-770.
9.  Williams CN, Kocher K, Lander ES, Daly MJ, Rioux JD. Using a genome-wide scan and meta-analysis to identify a novel IBD locus and confirm previously identified IBD loci. Inflamm Bowel Dis. 2002;8(6):375-381.
10. Ahmad T, Marshall SE, Jewell D. Genetics of inflammatory bowel disease: the role of the HLA complex. World J Gastroenterol. 2006;12(23):3628-3635.
11. Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity. 1999;10(3):387-398.
12. Neurath MF, Fuss I, Pasparakis M, Alexopoulou L, Haralambous S, Meyer Z, et al. Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice. Eur J Immunol. 1997;27(7):1743-1750.
13. Present DH, Rutgeerts P, Targan S, Hanauer SB, Mayer L, van Hogezand RA, et al. Infliximab for the treatment of fistulas in patients with Crohn's disease. N Engl J Med. 1999;340(18):1398-1405.
14. Targan SR, Hanauer SB, van Deventer SJ, Mayer L, Present DH, Braakman T, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med. 1997;337(15):1029-1035..
15. Turner JR. Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application. Am J Pathol. 2006;169(6):1901-1909.
16. Negoro K, Kinouchi Y, Hiwatashi N, Takahashi S, Takagi S, Satoh J, et al. Crohn's disease is associated with novel polymorphisms in the 5'-flanking region of the tumor necrosis factor gene. Gastroenterology. 1999;117(5):1062-1068.
17. Hirv K, Seyfarth M, Uibo R, Kull K, Salupere R, Latza U, et al. Polymorphisms in tumour necrosis factor and adhesion molecule genes in patients with inflammatory bowel disease: associations with HLA-DR and -DQ alleles and subclinical markers. Scand J Gastroenterol. 1999;34(10):1025-1032. DOI: 10.1080/003655299750025147.
18. Koss K, Satsangi J, Fanning GC, Welsh KI, Jewell DP. Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies. Genes Immun. 2000;1(3):185-190.
19. Kawasaki A, Tsuchiya N, Hagiwara K, Takazoe M, Tokunaga K. Independent contribution of HLA-DRB1 and TNF alpha promoter polymorphisms to the susceptibility to Crohn's disease. Genes Immun. 2000;1(6):351-357.
20. Louis E, Peeters M, Franchimont D, Seidel L, Fontaine F, Demolin G, et al. Tumour necrosis factor (TNF) gene polymorphism in Crohn's disease (CD): influence on disease behaviour?Clin Exp Immunol. 2000;119(1):64-68.
21. van Heel DA, Udalova IA, De Silva AP, McGovern DP, Kinouchi Y, Hull J, et al. Inflammatory bowel disease is associated with a TNF polymorphism that affects an interaction between the OCT1 and NF(-kappa)B transcription factors. Hum Mol Genet. 2002;11(11):1281-1289.
22. 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(12):1020-1027.
23. Martin K, Radlmayr M, Borchers R, Heinzlmann M, Folwaczny C. Candidate genes colocalized to linkage regions in inflammatory bowel disease. Digestion. 2002;66(2):121-126.
24. Louis E, Vermeire S, Rutgeerts P, De Vos M, Van Gossum A, Pescatore P, et al. A positive response to infliximab in Crohn disease: association with a higher systemic inflammation before treatment but not with -308 TNF gene polymorphism. Scand J Gastroenterol. 2002;37(7):818-824. DOI :10.1080/713786515.
25. Mascheretti S, Hampe J, Kuhbacher T, Herfarth H, Krawczak M, Folsch UR, et al. Pharmacogenetic investigation of the TNF/TNF-receptor system in patients with chronic active Crohn's disease treated with infliximab. Pharmacogenomics J. 2002;2(2):127-136.
26. Gonzalez S, Rodrigo L, Martinez-Borra J, Lopez-Vazquez A, Fuentes D, Nino P, et al. TNF-alpha -308A promoter polymorphism is associated with enhanced TNF-alpha production and inflammatory activity in Crohn's patients with fistulizing disease. Am J Gastroenterol. 2003;98:1101-1106.
27. O'Callaghan NJ, Adams KE, van Heel DA, Cavanaugh JA. Association of TNF-alpha-857C with inflammatory bowel disease in the Australian population. Scand J Gastroenterol. 2003;38(5):533-534.
28. Kim TH, Kim BG, Shin HD, Kim JW, Kim CG, Kim JS, et al. Tumor necrosis factor-alpha and interleukin-10 gene polymorphisms in Korean patients with inflammatory bowel disease. Korean J Gastroenterol. 2003;42(5):377-386.
29. Ahmad T, Armuzzi A, Neville M, Bunce M, Ling KL, Welsh KI, et al. The contribution of human leucocyte antigen complex genes to disease phenotype in ulcerative colitis. Tissue Antigens. 2003;62(6):527-535.
30. Balding J, Livingstone WJ, Conroy J, Mynett-Johnson L, Weir DG, Mahmud N, et al. Inflammatory bowel disease: the role of inflammatory cytokine gene polymorphisms. Mediators Inflamm. 2004;13(3):181-187. doi:  10.1080/09511920410001713529.
31. Fowler EV, Eri R, Hume G, Johnstone S, Pandeya N, Lincoln D, et al. TNF alpha and IL10 SNPs act together to predict disease behaviour in Crohn's disease. J Med Genet. 2005;42(6):523-528.
32. 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(4):170-174.
33. Ferreira AC, Almeida S, Tavares M, Canedo P, Pereira F, Regalo G, et al. NOD2/CARD15 and TNFA, but not IL1B and IL1RN, are associated with Crohn's disease. Inflamm Bowel Dis. 2005;11(4):331-339.
34. Levine A, Shamir R, Wine E, Weiss B, Karban A, Shaoul RR, et al. TNF promoter polymorphisms and modulation of growth retardation and disease severity in pediatric Crohn's disease. Am J Gastroenterol. 2005;100(7):1598-1604.
35. Cantor MJ, Nickerson P, Bernstein CN. The role of cytokine gene polymorphisms in determining disease susceptibility and phenotype in inflammatory bowel disease. Am J Gastroenterol. 2005;100(5):1134-1142.
36. 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(5):479-487.
37. Fidder HH, Heijmans R, Chowers Y, Bar-Meir S, Avidan B, Pena AS, et al. TNF-857 polymorphism in Israeli Jewish patients with inflammatory bowel disease. Int J Immunogenet. 2006;33(2):81-85.
38. Tremelling M, Waller S, Bredin F, Greenfield S, Parkes M. Genetic variants in TNF-alpha but not DLG5 are associated with inflammatory bowel disease in a large United Kingdom cohort. Inflamm Bowel Dis. 2006;12(3):178-184.
39. Castro-Santos P, Suarez A, Lopez-Rivas L, Mozo L, Gutierrez C. TNFalpha and IL-10 gene polymorphisms in inflammatory bowel disease. Association of -1082 AA low producer IL-10 genotype with steroid dependency. Am J Gastroenterol. 2006;101:1039-1047. doi:10.1111/j.1572-0241.2006.00501.x.
40. Cucchiara S, Latiano A, Palmieri O, Canani RB, D'Inca R, Guariso G, et al. Polymorphisms of tumor necrosis factor-alpha but not MDR1 influence response to medical therapy in pediatric-onset inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2007;44(2):171-179.
41. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. Single base polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene detectable by NcoI restriction of PCR product. Hum Mol Genet. 1992;1(5):353.
42. 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 U S A. 1997;94(7):3195-3199.
43. Podolski DK. Inflammatory bowel disease. N Engl J Med 2002;347(6):417-429.
44. Silverberg MS, Satsangi J, Ahmad T, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: Reportof a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005;19(suppl A):5A–36A.
45. Palmieri O, Latiano A, Valvano R, D'Inca R, Vecchi M, Sturniolo GC, et al. MDR1 polymorphisms are not associated with inflammatory bowel disease and response to therapy in Italian patients. Aliment Pharmacol Ther 2005;22:1129–1138.
46. Ricart E, Taylor WR, Loftus EV, O'Kane D, Weinshilboum RM, Tremaine WJ, et al. N-Acetyltransferase 1 and 2 genotypes do not predict response or toxicity to treatment with mesalamine and sulfasalazine in patients with ulcerative colitis. Am J Gastroenterol 2002;97:1763–1768.  doi:10.1111/j.1572-0241.2002.05838.x.
47. Munkholm P, Langholz E, Davidsen M, Binder V. Frequency of glucocorticoid resistance and dependency in Crohn's disease. Gut. 1994;35(3):360–362.
48. Faubion WA Jr, Loftus EV Jr, Harmsen WS, Zinsmeister AR, Sandborn WJ. The natural history of corticosteroid therapy for inflammatory bowel disease: a population-based study. Gastroenterology 2001;121(2):255–260.
49. Orlando A, Armuzzi A, Papi C, Annese V, Ardizzone S, Biancone L, et al. The Italian Society of Gastroenterology (SIGE) and the Italian Group for the study of Inflammatory Bowel Disease (IG-IBD) Clinical Practice Guidelines: The use of tumor necrosis factor-alpha antagonist therapy in inflammatory bowel disease. Dig Liver Dis. 2011;43(1):1-20. doi: 10.1016/j.dld.2010.07.010.
50. Targan SR, Hanauer SB, van Deventer SJ, Mayer L, Present DH, Braakman T, et al. Short-term study of chimeric monoclonal antibody ca2 to tumor necrosis factor for Crohn's disease cA2 Study Group. N Engl J Med 1997;337(15):1029–1036.
51. Sambrook J, Fritsch EF, Maniatis F. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1989.
52. D'Addabbo A, Latiano A, Palmieri O, Creanza MT, Maglietta R, Annese V, et al. Association of genetic profiles to Crohn's disease by linear combinations of single nucleotide polymorphisms. Artif Intell Med. 2009;46(2):131-138. doi: 10.1016/j.artmed.2008.07.012. 
53. Vermeire S, Van Assche G, Rutgeerts P. Role of genetics in prediction of disease course and response to therapy. World J Gastroenterol. 2010;16(21):2609-2615.
54. 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 U S A. 1997;94(7):3195-3199.
55. Gibson PR. Increased gut permeability in Crohn's disease: is TNF the link? Gut. 2004;53(12):1724-1725.
56. Hollander D. Crohn's disease, TNF-alpha, and the leaky gut. The chicken or the egg? Am J Gastroenterol. 2002;97(8):1867-1868.
57. Ma TY, Iwamoto GK, Hoa NT, Akotia V, Pedram A, Boivin MA, et al. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation. Am J Physiol Gastrointest Liver Physiol. 2004;286(3):367-376.
58. Boivin MA, Ye D, Kennedy JC, Al-Sadi R, Shepela C, Ma TY. Mechanism of glucocorticoid regulation of the intestinal tight junction barrier. Am J Physiol Gastrointest Liver Physiol. 2007;292(2):590-598.