Ulcerative colitis (UC) and Crohn disease (CD) are 2 dominant forms of idiopathic inflammatory bowel disease (IBD). The natural course is characterized by unpredictable exacerbations, remissions and the development of serious complications, particularly in CD (1). Pediatric IBD incidence rates have changed over the second half of the 20th century, with a steady, gradual increase for both UC and CD (2,3). A recent study also confirmed an increase in incidence of CD in children younger than 15 years in the Czech Republic (4). Although UC and CD share some genetic predisposing factors and immunologic mechanisms, several lines of evidence suggest they are genetically and otherwise fundamentally distinct disease processes (5,6).
Although our knowledge of the initiating events of IBD is incomplete, both major clinical forms appear to represent polygenic conditions with significant variability in pathophysiology and clinical symptoms. Susceptibility to polygenic disorders may be provoked by a combination of common genetic variants, including single-nucleotide polymorphisms (SNPs) and immunologic and environmental factors (7). These may increase the risk for developing these disorders (8,9).
Cytokines are proteins encoded and often secreted by immunocompetent cells which influence immune activity within the cell or at a distance. Proinflammatory cytokines involved in IBD are part of a complex signaling network that is not completely understood. IBD is associated with increased tumor necrosis factor-alpha (TNF-α) secretion possibly leading to the initiation and propagation of the disease (10). In this cascade, TNF-α is produced first; interleukin (IL)-1β, second and IL-6, last. TNF-α and IL-1β stimulate each other, and both stimulate IL-6 (11). TNF-α mediates increased epithelial antigen uptake in the ileum of CD patients (12) and induces T-helper1 cytokines important in the onset and progression of IBD (13).
The grade of inflammatory response is reflected at a systemic level. C-reactive protein (CRP), an acute-phase protein, is expressed exclusively by the liver as part of the acute-phase response upon stimulation by cytokines originating at the site of inflammation (14). Laboratory testing is an essential element in the establishment of IBD. A short half-life makes CRP a valuable marker in clinical practice to detect and follow up disease activity in CD. In contrast and for reasons unknown, UC has a modest to absent CRP response despite active inflammation (15).
Although the cause of IBD is not entirely clear, abnormal immune responses based on genetics and environmental factors may play a role in its pathogenesis. IBD is characterized by unbalanced Th1 versus Th2 responses. This leads to dysregulated secretion of both proinflammatory (TNF-α, IL-6, IL-1β) and anti-inflammatory (IL-10) cytokines (16-18) in the lamina propria, and inflammatory cell infiltration in both CD and UC (19). TNF-α can potentiate production of interferon-γ (INF-γ) (20). Treatment of CD patients with anti-TNF-α antibodies (cA2) could inhibit production of TNF-α and INF-γ in mononuclear cells, as well as improve the CD activity index and reduce intestinal inflammation (21).
Molecular genetics has increased our understanding of the strong genetic component in IBD disease susceptibility (22,23), although little is known about the accountable genes. Cytokine genes are attractive candidate loci (24), but data regarding association studies in the pediatric population are largely unknown. To date, studies of polymorphisms in proinflammatory cytokines (IL-1, TNF-α, IL-6) or in regulatory cytokines (IL-2, INF-γ, IL-10) have not yet shown reproducible parallels with disease phenotype in IBD adults (25).
A region on chromosome 6p21, IBD3, has been identified as an IBD-susceptibility locus in linkage studies (26-28). IBD3 encompasses the TNF gene, a strong positional and functional candidate for IBD. The TNF-α gene is located in the major histocompatibility complex region, and a large number of polymorphisms of its promoter have been described (29). The regulation of TNF expression is in part genetically determined because the polymorphisms −238, −308, −863, −857, and −1031 found in the promoter region are associated with increased TNF production (30). The polymorphisms at position −308 G→A in the TNF-α promoter region (G allele, TNF*1, and A allele, TNF*2) are associated with inducible levels of TNF-α in vitro (31). A nucleotide change at −308 of the human TNF-α gene promoter might influence transcriptional activation of the cytokine (32). A growing body of data supports the association of TNF-α polymorphism at position −308 with susceptibility and outcome of various autoimmune and infectious diseases (33). However, limited information is available on the TNF-α G→A polymorphism with the G to A transition at position −308 in the pediatric population with IBD. There are 2 relevant reports on the TNF-α −308 polymorphism in pediatric CD (34,35). Confirmed data are not readily available relating to TNF-α 308 G→A polymorphism, disease activity, nor are objective biochemical markers of inflammation in IBD in children.
To address this issue, we conducted this pilot study to determine the association of TNF-α G→A promoter SNP at position −308 in subgroups of IBD stratified according to disease phenotype and in healthy controls and the relationship between gene polymorphism, CRP levels, and disease severity in a pediatric population.
MATERIAL AND METHODS
A total of 164 children were included in the study. Eighty-two subjects with established diagnosis of IBD (46 boys, 36 girls; age range, 8-18 years) were recruited from 2 teaching hospital-based practices, (Department of Pediatrics, Division of Gastroenterology, Charles University Hospital, Pilsen and Hradec Králové). All of the patients were unrelated and belonged to the same ethnic group (Czech). A reference material consisting of 82 healthy individuals was also analyzed. Normal controls consisted of subjects without apparent abnormal findings on medical examination and were drawn from the same geographical area and social standing as the study group. All examined subjects were born in the Czech Republic. Demographic and clinical data were recorded. Investigators obtaining phenotype were unaware of genotype; conversely, genotype was analyzed independently while blinded to clinical data.
Diagnostic Criteria for IBD
The definitive diagnosis of IBD (CD and UC) was based on a combination of clinical characteristics, laboratory assessment, ultrasonography, colonoscopy, histopathology, and computed tomography scan. For upper gastrointestinal tract disease, upper endoscopy, push-enteroscopy, computed tomography-clysis or 99Tc-labeled white cell scanning (36,37) was used. Individuals whose IBD diagnosis was not confirmed were excluded. The total Pediatric Crohn's Disease Activity Index score (PCDAI) (38), and the Truelove index (39) were used to assess disease activity for CD and UC, respectively. All patients were screened for complications at follow-up management. UC patients were grouped according to extent of the lesion: proctitis, proctosigmoiditis, left-sided colitis (involvement limited to splenic flexure), sub(-total) colitis. Furthermore, the number of recurrent exacerbations was specified. CD subjects were registered following the Vienna classification (36). This included the anatomic location of CD involvement (only small bowel, only large bowel, small and large bowel, upper gastrointestinal tract) and the phenotypic behavior. The latter was defined as stenosing when the luminal narrowing or bowel occlusion was demonstrated with prestenotic dilatation and/or obstructive symptoms; penetrating, defined as the occurrence of intra-abdominal or perianal fistulas, inflammatory masses and/or abscesses; or simply inflammatory without the previous criteria. Furthermore, the need for surgical intervention was recorded. Extraintestinal manifestations included musculoskeletal, dermatologic, ophthalmologic, thromboembolic, hepatobiliary and pancreatic involvement.
For the determination of TNF-α G→A promoter gene polymorphism at position −308, genomic DNA was extracted from peripheral blood leukocytes using the Qiagen Blood DNA kit in accordance with the manufacturer's instructions. The TNF-α G→A polymorphism was determined by polymerase chain reaction with subsequent and respective restriction fragment length polymorphism (40). This method was developed to determine the G to A transition at position −308. A 107-base pair (bp) region was amplified using polymerase chain reaction with both positive and negative controls included in each run. Amplification products were digested with NcoI. The TNF-α −308 A allele remained undigested (107 bp), whereas the wild type allele TNF-α −308 G produced 2 fragments (87 bp and 20 bp). The DNA fragments were analyzed on a 3% metaphor agarose gel and visualized by ethidium-bromide staining.
Measurement of CRP
C-reactive protein serum levels were determined by immunoturbidimetric method using Olympus AU 2700 analyzer by K-assay set (Kamiya Biomedical Company). CRP was measured by nephelometry (340 nm). The normal range was considered 0 to 10 mg/L. CRP results were matched with disease activity and the promoter SNP at TNF-α −308.
All data were entered into the central database. All values were presented as means with SD and as proportions of the entire sample (%). Measurable data were compared using the t test, Wilcoxon test or Kruskal-Wallis test, as appropriate. Subgroups (percentages) in the 2 populations were compared using χ2 test or when appropriate, Fisher exact test. Correlation coefficients were calculated by the Pearson formula. We calculated the odds ratios (ORs). For each OR, the 95% CIs and P value were estimated. Multivariate logistic regression was carried out to determine which variables were independently associated with CD complications. Variables included TNF-α G→A polymorphism, age, sex, disease duration, age at onset, age at inclusion, the PCDAI, extent of disease, number of previous flares, number of hospitalizations, bowel surgery and CRP. Each variable was transformed for univariate and multivariate analyses. All factors that were at least marginally associated with CD complications were tested by multivariate logistic regression analysis. The statistical package for social sciences software for Windows, Version 10, (SPSS Inc, Chicago, IL) was used to analyze the data. The level of significance was defined as P < 0.05 if not otherwise stated.
The Local Research Ethics Committee approved the recruitment protocol. Informed consent was obtained from all of the patients' parents before enrollment in this study. The study was performed in accordance with the principles stated in the Declaration of Helsinki and its later revision.
Distribution of TNF-α −308 G→A Polymorphism in UC, CD Patients and Healthy Controls
Of the 164 subjects analyzed, genotype results were available for all subjects. Overall, 8.5% of patients (7/82) had been given the diagnosis before the age of 10 years. All others had the disease diagnosed after age of 10 years. Only 2.5% patients (2/82) had a positive family history of IBD. Of all the 82 IBD diagnoses in our study, 46 patients (56.1%) were diagnosed as having CD and 36 (43.9%) with UC. Twenty-two patients with IBD (26.8%) and 11 (13.4%) of the 82 controls carried at least one copy of −308 A (GA, AA) compared with −308 GG patients. Of note, there were 2 (5.5%) homozygous subjects for the −308 A allele in UC group carrying 2 copies of −308 A (AA). One control (1.2%) was also homozygous for −308 A polymorphism. When all patients with IBD regardless of the disease phenotype were analyzed together, significant differences were found comparing the distribution of the TNF-α 308 A polymorphism between IBD patients and control subjects (P < 0.05). Comparison of the TNF-α 308 A polymorphism between the UC patients and the control group showed a statistically significant difference (P < 0.001). The TNF-α 308 A polymorphism was not significantly overrepresented in CD patients compared with healthy controls. Results of this analysis are detailed in Table 1.
Clinical Parameters in IBD
Table 2 shows characteristics of the study subjects.
At the time of diagnosis, clinical activity was mild in 58.3% patients (21/36), and severe in 41.7% patients (15/36). Fifteen (41.7%) of 36 patients developed extradigestive tract complications. Disease involved proctitis in 11.1%, proctosigmoiditis in 13.8%, left-sided colitis in 44.4% and sub (-total) colitis in 30.5%. None of the UC patients underwent surgical intervention before inclusion into the study. Thirty-five (97.2%) of 36 patients were receiving treatment with 5-aminosalicylic acid, 33.3% (12/36) with steroids, 22.2% (8/36) with azathioprine and 13.8% (5/36) with ursodeoxycholic acid. This therapy continued throughout the study course. No significant differences were found in sex, age, age at diagnosis, disease location and extraintestinal complications with respect to TNF-α −308 G→A polymorphism. A trend toward a higher frequency of left-sided colitis was observed in −308 GG patients, compared with −308 GA and AA children, but these differences did not reach statistically significant level.
No significant differences were found in age at diagnosis and sex in both CD and UC. Thirty (65.2%) of 46 patients demonstrated an inflammatory disease phenotype. Sixteen (34.8%) of 46 CD patients had complex disease that was classified as either stricturing in 26.1% or penetrating in 13.1%. Among them, 2 patients had concurrent fibrostenosing and perforating complications. Disease was localized to the small bowel in 21.7%, combined small and large bowel in 67.4% and large bowel in 10.9%. Upper tract disease (with or without disease involvement elsewhere) was present in 10.9%. One patient with stenosing and penetrating behavior had experienced a life-threatening thromboembolic event. Thirteen (28.3%) of forty-six patients had extraintestinal manifestations. Nine (19.6%) of forty-six patients had required resections of the involved bowel segments before inclusion. At inclusion, 97.8% (45/46) patients were given 5-aminosalicylic acid, 60.9% (28/46) steroids, 32.6% (15/46) azathioprine, 2.2% (1/46) methotrexate and 13.1% (6/46) infliximab. All subjects with CD were given enteral nutrition.
Factors Influencing CD Behavior
Effect of TNF-α 308 G→A polymorphism on CD behavior
At the time of diagnosis, 97.8% of newly diagnosed children with CD had simple inflammatory disease. Of note, only one (2.2%) already had stenotic behavior at initial diagnosis. Sixteen (34.8%) of 46 patients were diagnosed as having stenotic and/or penetrating complications at inclusion based on the aforementioned defined criteria. All patients except one carrying TNF-α A polymorphism developed stenosing and penetrating disease, whereas only 9 of the 38 patients with normal GG allele developed complications of CD (21.6%). With regard to genotyping, stenosing/penetrating complications were associated with the TNF genotype. The allele frequencies of the TNF-α A polymorphism were significantly higher in CD patients with complications (stenosing/penetrating) as a whole than in the remaining CD children without complications (P < 0.001). Moreover, in our CD patients, a significant difference was seen in the occurrence of TNF-α A polymorphism between CD patients predominantly with complications and normal control subjects (P < 0.01).
Clinical Features of CD Related to Strictures and Penetrating Complications
Comparative data of different clinical patterns between CD patients with and without complications
Data are shown in Tables 3 and 4. No significant differences in any of the background clinical features of CD were observed between patients with stenosing/penetrating behavior and those without, except for disease duration (years) (P < 0.05), disease duration as a percentage of total life (%) (P < 0.01) and the number of hospitalizations due to CD (P < 0.01), Table 3. Stenosing/penetrating complications were significantly associated with a higher PCDAI index (P < 0.001). There were no significant differences between the location of disease and morphology in CD patients with complications as shown in Table 4.
Relationship Between TNF-α 308 G→A Polymorphism, Laboratory Inflammatory Activity
CRP and disease activity of both CD and UC
Mean ± SD CRP serum levels (Fig. 1) were 74.9 ± 54.9 mg/L versus 35.9 ± 40.1 mg/L (P < 0.05), for IBD patients carrying at least one mutation −308 A, compared with those without the polymorphism.
CRP and the PCDAI in CD
Mean ± SD CRP serum levels in CD patients were 50.1 ± 49.3 mg/L. CRP serum levels were significantly higher in patients with CD intestinal inflammation carrying the −308 A polymorphism (94.9 ± 62.8mg/L) compared with 308 GG children (40.7 ± 40.5 mg/L) (P < 0.05), Figure 1. The PCDAI score was slightly but significantly increased in CD patients with TNF-α 308 A polymorphism with CD patients with the GG genotype, 40.4 ± 7.5 versus 29.7 ± 12.6 (P < 0.05). Spearman rank order correlation analysis clearly revealed a significant linear correlation between CRP and the PCDAI (n = 46, r = 0.615, P < 0.001), Figure 2. Significant differences in CRP serum levels with respect to CD complications (P < 0.001) were observed, as shown in Table 3. However, our study did not prove a significant correlation between disease extent and both the PCDAI and CRP.
CRP and disease activity in UC
Mean ± SD CRP serum levels in the UC group were 43.2 ± 46.4 mg/L. CRP serum levels were significantly higher in patients carrying the −308 A polymorphism compared with those with −308 GG (64.3 ± 42.2 mg/L and 26.4 ± 36.2 mg/L, respectively) (P < 0.05), Figure 1. CRP levels in UC children with mild activity (19.2 ± 27.4 mg/L) were significantly lower than in patients with severe UC activity (81.8 ± 45.1 mg/L) as expressed by the Truelove index (P < 0.001). In addition, a significant association was found between TNF-α 308 G→A polymorphism and disease activity: mild 2/22 (9.1%) and severe 12/14 (85.7%) (P < 0.001). No significant differences were observed with regard to extent of disease, age and number of male patients.
Univariate comparisons were made with χ2 and Fisher exact test, and multivariate analysis was performed using a multiple logistic regression model. Where comparisons could be made, this multiple analysis revealed that only TNF-α 308 A allele distribution (OR, 12.9; CI, 1.18-140.81, P < 0.001) and CRP serum levels (OR, 1.020; CI, 1.00-1.04, P < 0.001) were independently associated with the development of CD complications. No other clinical or analytical findings were predictive for the risk of development of CD complications. Validation of this logistic regression analysis was confirmed by examining the association of predicted probabilities and the observed mode of behavior. The percent concordance was found to be 76.1%, thus demonstrating a high degree of predictability of mode of behavior. None of the other clinical or analytical findings were found to be useful for the prediction of complications.
The present study demonstrated that there is a significant association between TNF-α 308 G→A polymorphism and IBD in our established pediatric population. An interesting observation was that TNF-α 308 G→A polymorphism influences the IBD phenotype and may play an important role in IBD onset. Differences were clearly observed in the carriage rate for TNF-α 308 G→A between UC and control-group children. The study of Koss et al (41) showed that patients with distal colitis had a significantly higher occurrence of TNF-α 308 A allele compared with the control group. Sashio et al (10) who investigated UC and CD adult patients compared with controls also observed the same trend where TNF-α 308 G→A and −238 G→A polymorphisms were found more frequently in UC patients. However, our study has not proven any link between TNF-α 308 G→A polymorphism and the extent of disease and other clinical variables in the UC group.
Crohn disease patients can be assigned into 2 distinct groups, as follows: firstly, a stenotic and/or penetrating disease and secondly, a subset of patients with an inflammatory phenotype (36). The inflammatory process of CD appears to destabilize over time and may evolve into strictures and penetrating complications. Characteristics of CD complications in our study were no sex predominance and no relationship to therapy but substantial occurrence of complications several years after initial diagnosis. Another relevant finding of our study was the demonstration of TNF-α 308 G→A polymorphism in the 2 clearly defined subgroups of CD patients with and without complications: low frequency of 308 G→A allelic variations in CD patients presenting with inflammatory disease and a significantly higher frequency in those with evidence of complications. These findings may substantiate a common genetic predisposition underlying both processes. Kim et al (42) reported in Korean patients that the TNF-α gene polymorphisms at positions −308 and −238 have influences on the susceptibility to CD or the behavior of CD. Bouma et al (43) showed that TNF −308 polymorphism did not confer protection against fistulizing CD. Interestingly, as for CD our findings clearly demonstrated that TNF-α 308 G→A gene polymorphism appears to have a greater effect on modifying the CD phenotype than overall susceptibility to CD. Levine at al (34) made similar observations that the TNF-α 308 G→A polymorphism was not significantly more frequent in pediatric onset of CD.
Despite extensive investigation, it remains incompletely understood that the above-mentioned designations are rooted in distinct pathogenetic mechanisms (44). The number of reference CD patients with complications in our study was not large enough to clearly define the relationship between TNF-α 308 G→A and patients with either stricturing or perforating behavior. We therefore analyzed all CD patients with complications as a whole group. Based on these findings, we came to the conclusion that pediatric CD patients presenting with complications may be a genetically distinct subgroup of CD. This is an additional piece of evidence that may explain different pathogenetic mechanisms in CD phenotype. This hypothesis is in keeping with our observations that the occurrence of TNF-α 308 G→A was higher in CD patients with complications than that in the normal controls. Louis et al (45) reported that TNF gene polymorphism could be associated with certain CD phenotypes. González et al (46) did not find a significant association between the distributions of the 308 G→A polymorphism and the susceptibility of CD patients developing fistulas. Other studies related to TNF-α association with CD showed conflicting data (41,47). Several reasons could explain this discrepancy on the effect of the genotypic distribution on CD behavior: genetic heterogeneity of the clinical populations examined, different definitions of CD phenotypes among different populations and perhaps environmental factors may also contribute to CD behavior. Furthermore, results can also be influenced by the ethnic differences in the frequency of TNF-α 308 G→A polymorphism. Of note, our study sample did not include subjects from different ethnic backgrounds, thus preventing ethnic bias on TNF-α polymorphism. With the larger number of subjects to be attained, attempts should be made in the future not to miss certain clinical and pathogenetic associations. Further investigation among different ethnic populations is clearly warranted.
The promoter SNP at TNF-α −308 A was related to TNF-α production (31). Some patients with IBD have been found to have large amounts of TNF-α in the colonic mucosa and the stool, although relatively low levels in serum (48). Vatay et al (49) studied the relationship between TNF-α polymorphism and CRP serum levels in CD and UC. They reported that CRP levels in the active phase were similar to those in the inactive phase when patients were homozygous carriers at position −308 GG. Mazlam and Hodgson (50) showed that TNF-α release correlated significantly with CRP serum. It is notable that TNF-α also appears to be stimulated by CRP (51). Koss et al (41) found in adults that increased TNF-α production in UC patients was not observed despite significantly higher occurrences of TNF-α 308 A polymorphism in patients compared with healthy controls. Van Heel et al (52) reported that IBD is associated with a TNF polymorphism that can affect interplay between the OCT l and nuclear factor-κB transcription factors. It is interesting to observe in our subjects that TNF-α 308 A polymorphism significantly correlated with serum CRP in CD and UC. We have shown that the PCDAI score points and CRP may be related to TNF-α −308 G→A promoter gene polymorphism in CD patients. In our series, not only changes in CRP serum levels but also disease activity, as reflected by the PCDAI score, were comparable with TNF-α −308 G→A promoter gene polymorphism. Although a clear reason for the higher PCDAI activity, the Truelove index and CRP remains unclear, but it might be, in part, accounted for by −308 A allele carriers. Our study supports the routine use of CRP and evaluation of disease activity markers in IBD. It has been suggested that adult patients who carry −308 A present with more intense inflammation than −308 G carriers and that the polymorphism at −308 G→A of TNF-α may play an essential role in modifying the production of TNF-α and the phenotypic expression of CD (46). Thus, it appears that a long period of subclinical inflammation unregulated by TNF-α may progress to fibrotic stenosis and/or fistulas. One explanation may lie in the potential role of the TNF-α 308 A polymorphism that may favor and promote complications of CD by inducing more aggressive inflammatory activity at the mucosal level (45). This elucidation of the development of CD-related complications might also be supported by our observations as the PCDAI and CRP were significantly higher in children with complications compared with children without such behavior.
The present data confirm that TNF-α −308 G→A polymorphism may participate in defining the biological basis of IBD in children. Indeed, our preliminary observations and the strength of associations clarified the association of the polymorphism with the UC group as well as the CD patients with complications. These results may contribute to the explanation of disease heterogeneity. However, it is reasonable to speculate that the effect of the 308 A polymorphism in the TNF-α promoter is directly or indirectly mediated by the increase in TNF-α production. Whether the TNF-α 308 A polymorphism is directly involved in regulating cytokine production or serves merely as a marker in the linkage disequilibrium should be further investigated.
In summary, although the promoter SNP at TNF-α 308 may not necessarily dictate IBD initiation, the available evidence suggests that TNF-α 308 A polymorphism may be associated with subsets of IBD patients after stratification for the disease phenotype. A higher incidence of this polymorphism was found in UC patients compared with those with controls and CD, where TNF-α 308 A polymorphism may play a role in modifying the CD phenotype. TNF-α 308 A polymorphism may participate in determining disease activity as well as more intense inflammatory activity in both forms of IBD and may modify the progression of chronic digestive tract inflammation. Future research will be necessary regarding IBD genes and the potency of biological therapy (chimeric anti-TNF antibody).
The authors thank Mrs Martina Šafrová for skillful technical assistance.
1. Murch SH, Baldassano R, Buller H, et al. Inflammatory bowel disease: Working Group Report of the Second World Congress of Pediatric Gastronterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr
2004;39:S647-54. 2 (suppl).
2. Feeney MA, Murphy F, Clegg AJ, et al. A case-control study of childhood environmental risk factors for the development of inflammatory bowel disease. Eur J Gastroenterol Hepatol
3. van der Zaag-Loonen HJ, Casparie M, Taminiau JAJ, et al. The incidence of pediatric inflammatory bowel disease in the Netherlands: 1999-2001. J Pediatr Gastroenterol Nutr
4. Pozler O, Malý J, Bónová O, et al, for the Czech Collaborative Group for the Study of Chronic Illnesses of the Gastrointestinal Tract in Children. Incidence of Crohn disease
in the Czech Republic in the years 1990 to 2001 and assessment of pediatric population with inflammatory bowel disease. J Pediatr Gastroenterol Nutr
5. Podolsky DK. Inflammatory bowel disease. N Engl J Med
6. Elitsur Y, Lawrence Z, Tolaymat N. The diagnostic accuracy of serologic markers in children with IBD: the West Virginia Experience. J Clin Gastroenterol
7. Risch N, Merikangas K. The future of genetic studies of complex human disease. Science
8. Karlinger K, Gyorke T, Mako E, et al. The epidemiology and the pathogenesis of inflammatory bowel disease. Eur J Radiol
9. Sýkora J, Varvaøovská J, Pomahaèová R, et al. Simultaneous presentation of celiac disease, ulcerative colitis
, and autoimmune thyroiditis in childhood. J Clin Gastroenterol
10. Sashio H, Tamura K, Ito R, et al. Polymorphisms of TNF gene and TNF receptor super-family member lB gene are associated with susceptibility to ulcerative colitis
and Crohn's disease, respectively. Immunogenetics
11. Ceciliani F, Giordano A, Spagnolo V. The systematic reaction during inflammation: The acute-phase proteins. Protein and Pept Lett
12. Soderholm JD, Streutker C, Yang PC, et al. Increased epithelial uptake of protein antigens in the ileum of Crohn's disease mediated by tumor necrosis factor alpha. Gut
13. Plevy SE, Landers CJ, Prehn J, et al. A role for TNF-alpha and mucosal T helper-1 cytokines in the pathogenesis of Crohn's disease. J Immunol
14. Volanakis JE. Human C-reactive protein: expression, structure, and function. Mol Immunol
15. Vermeire S, Van Assche G, Rutgeerts P. C-reactive protein as a marker for inflammatory bowel disease. Inflamm Bowel Dis
16. Camoglio L, Te Velde AA, Tigges AJ, et al. Altered expression of interferon-gamma and interleukin-4 in inflammatory bowel disease. Inflamm Bowel Dis
17. Breese EJ, Michie CA, Nicholls SW, et al. Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology
18. Reimund JM, Wittersheim C, Dumont S, et al. Mucosal inflammatory cytokine production by intestinal biopsies in patients with ulcerative colitis
and Crohn's disease. J Clin Immunol
19. Woywodt A, Ludwig D, Neustock P, et al. Mucosal cytokine expression, cellular markers and adhesion molecules in inflammatory bowel disease. Eur J Gastroenterol Hepatol
20. Prehn JL, Mehdizadeh S, Landers CJ, et al. Potential role for TL1A, the new family member and potent costimulator of IFN-gamma, in mucosal inflammation. Clin Immunol
21. Agnholt J, Kaltoft K. Infliximab downregulates interferon-gamma production in activated gut T-lymphocytes from patients with Crohn's disease. Cytokine
22. Kinouchi Y, Negoro K, Takagi S, et al. Genotype and phenotype relation in inflammatory bowel disease. J Gastroenterol
23. Bonen DK, Cho JH. The genetics of inflammatory bowel disease. Gastroenterology
24. Balding J, Livingstone WJ, Conroy J, et al. Inflammatory bowel disease: the role of inflammatory cytokine gene polymorphisms. Mediators Inflamm
25. Louis E, Satsangi J, Roussomoustakaki M, et al. Cytokine gene polymorphisms in inflammatory bowel disease. Gut
26. Hampe J, Shaw SH, Saiz R, et al. Linkage of inflammatory bowel disease to human chromosome 6p. Am J Hum Genet
27. Rioux JD, Silverberg MS, Daly MJ, et al. Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci. Am J Hum Genet
28. Dechairo B, Dimon C, van Heel D, et al. Replication and extension studies of inflammatory bowel disease susceptibility regions confirm linkage to chromosome 6p (IBD3). Eur J Hum Genet
29. Ruuls SR, Sedgwick JD. Unlinking tumor necrosis factor biology from the major histocompatibility complex: lessons from human genetic and animal model. Am J Hum Genet
30. Yamamoto-Furusho JK. Immunogenetics of chronic ulcerative colitis
. Rev Invest Clin
31. Kroeger KM, Carville KS, Abraham L. The −308 tumor necrosis factor-alpha promoter polymorphisms effects transcription. Mol Immunol
32. Wilson AG, Symons JA, Mc Dowell TL, et al. Effects of a polymorphism in the human tumor necrosis factor α promotor on transcriptional activation. Proc Natl Acad Sci USA
33. Ruuls SR, Sedgwick JD. Unlinking tumor necrosis factor biology from the major histocompatibility complex: lessons from human genetics and animal model. Am J Hum Genet
34. Levine A, Karban A, Eliakim R, et al. A polymorphism in the TNF-α promoter gene is associated with pediatric onset and colonic location of Crohn's disease. Am J Gastroenterol
35. Levine A, Shamir R, Wine E, et al. TNF promoter polymorphisms and modulation of growth retardation and disease severity in pediatric Crohn's disease. Am J Gastroenterol
36. Gasche C, Scholmerich J, Brynskov J, et al. A simple classification of Crohn's disease: report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998. Inflamm Bowel Dis
37. IBD Working Group of the European Society for Pediatrics Gastroenterology, Hepatology and Nutrition (ESPGHAN). Inflammatory bowel disease in children and adolescents: Recommendations for diagnosis-the Porto criteria. J Pediatr Gastroenterol Nutr
38. Hyams J, Markowitz J, Otley A, et al, for the Pediatric Inflammatory Bowel Disease Collaborative Research Group. Evaluation of the Pediatric Crohn disease
activity index: a prospective multicenter experience. J Pediatr Gastroenterol Nutr
39. Herrlinger KR, Dittmann R, Weitz G, et al. Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis. Inflamm Bowel Dis
40. Wilson AG, di Giovine FS, Blakemore AIF, et al. Single base polymorphism in human tumour necrosis factor alpha gene detectable by NcoI restriction of the PCR product. Hum Mol Genet
41. Koss K, Satsangi J, Fanning GC, et al. Cytokine (TNF alpha, LT alpha, and IL-l0) polymorphisms in inflammatory bowel disease and normal controls: differential effects on production and allele frequencies. Genes Immun
42. Kim TH, Kim BG, Shin HD, et al. Tumor necrosis factor-alpha and interleukin-10 gene polymorphisms in Korean patients with inflammatory bowel disease. Korean J Gastroenterol
43. Bouma G, Poen AC, Garcia-Gonzales MA, et al. HLA-DRBl*03, but not the TNFA-308 promoter gene polymorphisms, confers protection against fistulising Crohn's disease. Immunogenetics
44. Abreu MT, Yang H. Defining subtypes of Crohn's disease patients: The ground work for translational research in inflammatory bowel disease. J Clin Gastroenterol
45. Louis E, Peeters M, Franchimont D, et al. Tumour necrosis factor (TNF) gene polymorphisms in Crohn's disease (CD): influence on disease behaviour? Clin Exp Immunol
46. González S, Rodrigo L, Martínez-Borra J, et al. TNF-α-308A promoter polymorphism is associated with enhanced TNF-α production and inflammatory activity in Crohn's disease patients with fistulising disease. Am J Gastroenterol
47. Kawasaki A, Tsuchiya N, Hagiwara K, et al. Independent contribution of HLA-DRBl and TNF alpha promoter polymorphisms to the susceptibility to Crohn's disease. Genes Immun
48. Brynskov J, Foegh P, Pedersen G, et al. Tumour necrosis factor alpha converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease. Gut
49. Vatay A, Bene L, Kovacs A, et al. Relationship between the tumor necrosis factor alpha polymorphism and the serum C-reactive protein levels in inflammatory bowel disease. Immunogenetics
50. Mazlam MZ, Hodgson HJ. Peripheral blood monocytes cytokine production and acute phase response in inflammatory bowel disease. Gut
51. Ballou SO, Lozanski G. Induction of inflammatory cytokine release from cultured human monocytes. Cytokine
52. Van Heel DA, Udalova IA, De Silva AP, 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
Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
TNF-α 308 G→A; ulcerative colitis; Crohn disease; stenotic and fistulizing complications; CRP; disease activity