Inflammatory bowel disorders (IBDs) are a heterogeneous group of chronic gastrointestinal (GI) diseases of unknown origin. Over the past 50 years, they have become 1 of the major chronic GI diseases among children and adolescents in the Western world. Approximately 10% of IBD patients are younger than 18 years of age, with 30% of them being diagnosed before 21 years of age (1). A complex set of interactions between genetic and environmental factors plays a key role in the pathogenesis of IBD (2–4).
The recent application of complementary strategies of candidate gene analysis and genome-wide scanning has led to the discovery of the first gene underlying susceptibility to Crohn disease (CD), the CARD15 gene (5,6). Genotype/phenotype analyses have consistently demonstrated that CARD15 major variants are mostly associated with ileal disease and stricturing behaviour (7). In a pediatric series, correlations with ileal localisation (8–12), stricturing behaviour (9,10,13), early surgery (9,10), growth delay (10,11), and greater disease activity (11,14) have also been reported, although with conflicting findings (15–17).
Because CARD15 variants account only for ≈20% of genetic predisposition to CD, additional genetic factors should be investigated. Indeed, 11 genome-wide studies and 2 meta-analyses have pointed to several other candidate loci in a number of susceptibility regions on chromosomes 1, 3, 4, 5, 6, 7, 10, 12, 14, 16, 19, and X (18).
More recently, variants of the OCTN1 and OCTN2 genes (19) within the previously identified IBD5 risk haplotype (5q31) (20) and the 113G→A variant of the DLG5 gene (10q23) (21) have been proposed as susceptibility genes. However, replication studies are conflicting (22), especially for the DLG5 gene, and data on a pediatric population are scarce.
A significant clinical heterogeneity has been recognized within IBD, suggesting the existence of phenotypic subsets based on features such an anatomic location, disease behaviour, and natural history. It has been hypothesised that this clinical heterogeneity is influenced by genetic heterogeneity, with some genetic factors involved in disease predisposition and others influencing natural history and response to therapy (23). The latter aspect is particularly relevant in pediatric-onset IBD patients who are conceivably less influenced by some environmental factors (ie, smoking) compared with the adult population and, in contrast, more exposed to possible deleterious effect of drugs because of the ongoing growth and longer life expectancy. On the basis of these findings, we aimed to investigate candidate genes possibly involved in response to therapy.
Tumour necrosis factor-α (TNF-α) is a potent proinflammatory cytokine, and its important role in IBD is apparent from clinical, functional and animal studies (24). Few reports are available on TNF-α polymorphism in pediatric-onset CD. Levine et al. (25,26) demonstrated a significant correlation of the −863A variant with colonic disease and greater height at diagnosis. Sykora et al. (27) found a correlation of the −308A polymorphism with stricturing/penetrating behaviour in patients with CD and higher disease activity in both patients with CD and patients with ulcerative colitis (UC). The MDR1 gene is also an attractive candidate gene for the pathogenesis of IBD at both the functional and genetic levels (28). However, data on a correlation with IBD predisposition and response to therapy are conflicting in adults and completely lacking in pediatrics.
In this study, we investigated the contribution of variants of the TNF-α and MDR1 genes in IBD predisposition and response to medical therapy in a large Italian pediatric cohort of patients with IBD patients. We also examined genotype/phenotype relationships and gene–gene interaction with the CARD15 variants.
PATIENTS AND METHODS
Patients under age 18 years at diagnosis were recruited to the study from 16 tertiary pediatric and gastroenterologic centres in Italy (see Appendix) in a multicenter study endorsed by the Italian Society for Pediatric Gastroenterology and Nutrition. Ethics approval was obtained for each of the participating centres. All of the study participants (including healthy controls) were white, and their parents gave written informed consent. The diagnosis of CD or UC was established by conventional clinical, radiological, endoscopic, and histological criteria (29). Cases with indeterminate colitis were not included.
The recruited population consisted of 200 patients with CD, 186 patients with UC, and 347 adult healthy unrelated blood donors who served as controls (200 male subjects; mean age, 25 y; range, 16–32 y). All had peripheral blood samples collected. In addition, blood samples were taken from 434 parents, providing 217 complete family trios (108 CD and 109 UC) for analysis.
A detailed clinical questionnaire concerning different features of the disease was used. The Vienna classification was used for CD disease type (30); for classifying localisation, the largest extent of the disease based on x-ray, endoscopy, or surgical reports was considered. In all of the patients, the following clinical features were recorded: family history, age at diagnosis, duration of follow-up, localization of CD (ileum, ileocolonic, colonic, upper GI tract), disease “type” (penetrating, stricturing, inflammatory [nonpenetrating and nonstricturing]), extent of UC (rectosigmoid, left-sided colitis, pancolitis), presence of perianal fistulas, extraintestinal manifestations (presence or absence of any extraintestinal manifestations and, more specifically, arthropathy or skin lesions), previous abdominal surgery (colectomy in UC or bowel resection in CD) and smoking habit (at least 1 cigarette/d, including passive smoking). In all of the patients, weight and height percentiles were calculated at diagnosis. Growth retardation was defined as a reduction to the fifth percentile or below for weight, height, or both. 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, and barium follow-through.
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), and immunosuppressive (IMS) drugs, namely azathioprine (AZT), 6-mercaptopurine (6-MP), methotrexate, cyclosporine A and infliximab.
Patients were classified as responder or nonresponder on the basis of a review of medical records (31). 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 (32). Patients requiring oral/parenteral CS, IMS drugs, or infliximab during the disease course were considered nonresponders 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 previously accepted definition) (33,34), or CS refractory (when an unsuccessful clinical response was achieved, leading to alternative therapies such as surgery or use of infliximab or other IMS drugs) (33,34). When a modification of response to therapy was recorded during the disease course (ie, previous CS responder becoming CS dependent), only the most clinically relevant category was considered (ie, refractory more relevant than dependent, more relevant than responder). Particular attention was paid to the dose and duration of the therapy used, which should comply with current guidelines (35). Patients with incomplete or unclear information were excluded from analysis. Furthermore, patients were globally classified as IMS user or IMS nonuser. The concept behind this subgrouping was to evaluate the possible effect of MDR1 and TNF-α polymorphisms in these difficult-to-treat patients with dependency or refractoriness to CS therapy, although IMS drugs may have different effects and pharmacogenetics. Patients with incomplete information or inadequate therapeutic schedule were excluded. Finally, patients were divided into infliximab responder or nonresponder according to short-term (12 weeks) response (36).
Many patients with IBD are currently treated with a combination of drugs during the disease course. A possible synergic effect of different drugs (ie, 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 nonresponder, 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 nonresponder, steroid-dependent 6-MP responder. Patients intolerant of any investigated drug were not included in this analysis; however, because MDR1 could conceivably contribute to drug intolerance, it was evaluated separately.
Genomic DNA was extracted from peripheral blood leucocytes according to standard protocols (37) and genotyped in the laboratory of San Giovanni Rotondo Hospital. 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. For the MDR1 gene, the C3435T single nucleotide polymorphism (SNP) was evaluated by denaturing high-performance liquid chromatography [DHPLC], Wave System, Transgenomic Ltd, UK) as previously described (31).
Genotyping for Arg702Trp and Leu1007fsinsC 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 sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. A summary of primer sequences and reaction conditions is given in Table 1.
Allele and genotype frequency comparisons were performed by χ2 and Fisher exact tests when appropriate; Student t test was used to compare means of continuous variables with SPSS software version 11.5 (SPSS, Chicago, IL). Tests for Hardy-Weinberg equilibrium, linkage disequilibrium, and transmission disequilibrium were performed by the Haploview software version 3.2 (http://www.broad.mit.edu/personal/jvbarret/haploview).
Power sample calculation was performed with PS software (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize). 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 (eg, duration of follow-up, disease localisation).
The main clinical characteristics of patients included in the study are summarized in Table 2; data regarding the medical therapy also are given. The 200 patients with CD had a mean age at diagnosis of 12 ± 4 years (range, 1–18 y) with a slight male predominance (male/female = 115/85). The 186 patients with UC had a mean age at diagnosis of 11 ± 5 years (range, 1–18 y) with a female predominance (73/113; P = 0.0003 vs patients with CD). Mean age at diagnosis and duration of follow-up was similar in the 2 groups of patients. Eighteen percent of the patients with CD had upper GI involvement. Compared with patients with UC, the frequency of resective surgery (26% vs 7%; P < 0.01), extraintestinal manifestations (40% vs 25%; P = 0.002), growth delay (42% vs 14%; P = <0.001) and perianal fistulas (18% vs 0.5%; P < 0.001) was significantly higher in patients with CD. In the UC series, almost half of the patients had pancolitis (47%). The frequency of anti-Saccharomyces cerevisiae antibody (ASCA) and anti-neutrophilic cytoplasmic antibody (ANCA) was 71% and 17% in patients with CD and 24% and 57% in patients with UC, respectively, which was significantly different (P = <0.0001 and P = <0.0001, respectively).
Of note, 66% and 74% of patients with CD and patients with UC, respectively, had used steroids; moreover, significantly more patients with CD (50%) compared with patients with UC (27%) were using immunosuppressors, mainly AZT or 6-MP (P < 0.001). Only 7 and 13 patients had used methotrexate and cyclosporine, respectively. Infliximab was used in 31 patients with CD and 4 patients with UC. Intolerance to AZT/6-MP, methotrexate, and mesalamine was reported in 19, 3, and 10 patients, respectively.
The success rate of genotyping (ie, the percentage of samples that could be analysed) was 94%; failures were mainly the result of old and denatured DNA. The error rate (defined as the percentage of disagreement in genotypes among 200 random samples tested in duplicate) was <1% for all SNPs. All of 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 3.
The −308A allele of the TNF-α gene was significantly increased in both patients with CD (15%; odds ratio [OR] = 2.79; 95% confidence interval [CI] = 1.82–4.26; P < 0.01) and patients with UC (11%; OR = 1.96; 95% CI = 1.24–3.12; P < 0.003) compared with controls (6%). Considering the homozygous and heterozygous states together, carriers were significantly more frequent in patients with CD (27%; OR = 2.94; 95% CI = 1.85–4.67; P < 0.01) and patients with UC (19%; OR = 1.86; 95% CI = 1.12–3.08; P = 0.015) than in healthy controls (11%). In contrast, no significant difference was found for the −857 allele. For either allele or genotype frequencies of the MDR1 SNP, no significant difference in patients with CD and patients with UC compared with controls emerged.
At least 1 variant of CARD15 was found in 38% of patients with CD (P < 0.001), 20% of patients with UC, and 15% of controls. Two variants of CARD15 (either homozygous or compound heterozygous) were present in 15% of patients with CD (P < 0.001) and 3% of patients with UC compared with 1% of healthy controls. After patients were stratified on the basis of the presence or absence of at least 1 CARD15 variant to disclose possible gene–gene interaction, no significant difference in TNF-α and MDR1 genotype frequencies was found (Table 4).
In the family-based analysis, a trend toward an overtransmission of the C allele of the MDR1 gene was found in the whole group of patients with IBD (transmitted/undertransmitted, 116/90; P = 0.07); however, the difference was less pronounced in the CD and UC subgroups (P = 0.19 and P = 0.28, respectively). No significant distortion of transmission was found for both TNF-α SNPs (data not shown but available on request).
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. For the MDR1 gene, given the lack of association in this study and the conflicting reports in the literature, a possible correlation with specific clinical features was examined for all 3 genotypes.
In the univariate analysis of clinical features of patients with CD (Table 5), the localization at the upper GI tract was significantly less frequent in carriers of the TNF-α risk genotype (8%) compared with noncarriers (22%; OR = 0.30; 95% CI = 0.10–0.91; P < 0.03). Conversely, an increased frequency of surgical resection in TNF+ (35%) compared with TNF− was found (20%; OR = 2.1; 95% CI, 1.05–4.3; P = 0.035). No correlation was found for age at diagnosis, family history, disease behaviour, presence of growth delay, perianal fistulas, and extraintestinal manifestations. When the efficacy of medical therapy was investigated, patients with TNF+ were significantly more resistant to steroids (22%) compared to patients with TNF− (8%, OR = 0.29, CI = 0.09–0.95, P = 0.032). In contrast, no difference was found in patients with steroid dependency. No difference in the efficacy of mesalamine, AZT, and infliximab was disclosed. Furthermore, no significant correlations of MDR1 genotype with either clinical features of patients with CD or efficacy of medical therapy were found.
In patients with UC (Table 6), no significant association of both TNF-α and MDR1 polymorphisms with any clinical characteristics was demonstrated. Only a trend toward an increased frequency of steroid resistance (20%) was found in carriers of the TNF-α risk genotype compared with noncarriers (12%).
In 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 = 0.29; 95% CI, 0.08–0.98; P = 0.04), also taking into account other potential confounders such as age at diagnosis, duration of follow-up, disease extension and localisation, disease behaviour and MDR1 and CARD15 status. The correlation of TNF-α risk genotype with surgical resection, however, also was negatively influenced by age at diagnosis (OR = 0.89; 95% CI, 0.82–0.97; P = 0.01).
This study reports a large cohort of pediatric patients with IBD investigated for possible influence of TNF-α and MDR1 genes on disease predisposition, clinical characteristics, and response to medical therapy. The −308A promoter polymorphism of TNF-α, known to enhance TNF-α production, was correlated with predisposition to both CD and UC. More interestingly, the same polymorphism significantly reduced the efficacy of steroid therapy, especially in patients with CD, and was correlated with increased frequency of surgical resection. In contrast, no apparent correlation with the MDR1 polymorphism was disclosed.
The role of TNF-α in IBD is apparent from clinical, functional, and animal studies (24). TNF-α levels are elevated in serum, stools, and inflamed bowel mucosa of patients with IBD (38–42). NF-κB translocation to the nucleus and TNF-α production are increased in mononuclear cells of patients with CD (43). Genetically engineered mice with an elevated TNF-α production have a CD-like phenotype, whereas TNF-α knockout mice develop a lower amount of bowel inflammation after chemical induction of colitis compared with control mice (44,45). The TNF-α gene maps at the IBD3 locus within the major histocompatibility complex region, the same region where several studies have pointed out a susceptibility locus for IBD (46–48). Polymorphisms in the TNF-α gene and promoter region have been studied extensively in adult patients with IBD with conflicting findings (49–60). For instance, the polymorphism −308A in the promoter region has been associated with inducible levels of TNF-α in vitro. Preliminary evidence has reported this polymorphism more frequently in steroid-dependent patients with CD (55), with earlier age at onset, colonic localisation, and fistulising behaviour (24). Moreover, certain TNF-α polymorphisms (−308A, −857C) appear to increase TNF-α production or inflammation, raising the possibility of correlation with different disease course or response to therapy (24).
Reports in pediatric-onset patients with IBD are scarce and limited by the small sample size. Sykora et al. (27), investigating the same −308A polymorphism in 82 patients with IBD, found a correlation with stricturing/penetrating behaviour in patients with CD (P < 0.001) and higher disease activity in both patients with CD and patients with UC (P < 0.05). Our study expands this observation in a larger data set, confirming the role in disease predisposition in both patients with UC and patients with CD. Moreover, CD carriers of the risk allele display a more aggressive clinical course, with steroid resistance and need for surgical resection. These associations also were confirmed by stepwise logistic regression and may advocate a specific therapeutic intervention such as earlier use of infliximab in this subset of patients (61). The lack of association with the other TNF-α polymorphism (−857C) is not surprising because the 2 SNPs were not in linkage disequilibrium (D′ = 0.267 in controls). The inverse correlation of the −308A TNF-α polymorphism with upper GI tract involvement needs to be verified in further studies.
The product of the MDR1 gene, the P-glycoprotein (P-gp), functions as a transmembrane efflux pump (62), influencing the disposition and response of many drugs, some of which (ie, CS) are central to IBD therapy (63). In addition, P-gp is highly expressed in many epithelial surfaces, including the GI tract (64), with a putative role in decreasing the absorption of endogenous or exogenous toxins and perhaps host–bacteria interaction. Many genetic variations of the MDR1 gene have been described, and in some instances (C3435T) evidence for different P-gp expression and drug metabolism has been provided (65). Likely, the greatest evidence of the physiological importance of P-gp in the GI tract has come from the mdr1 knockout mice model, which develops a spontaneous colitis in a specific pathogen-free environment (66). Studies investigating MDR1 gene polymorphism and predisposition to IBD, however, have shown conflicting results (67–70). In a recent meta-analysis (71), a weak but significant association of the 3435T allele and the 3435TT genotype, leading to a reduced expression of Pg-p, was found in patients with UC (OR = 1.17, 95% CI = 1.06–1.31, P = 0.003; and OR = 1.36, 95% CI = 1.05–1.76, P = 0.017, respectively). In the present study, we could not confirm a correlation between the C3435T polymorphism of MDR1 and either IBD predisposition or response to medical therapy, similar to our previous findings in adult patients with IBD (31). Reasons for the discrepancies with other studies lie in the known difficulties encountered in complex diseases, especially when the (supposed) genetic contribution is weak (72). Given the frequency of the (supposed) susceptibility allele in our control population (7%), >1000 patients would be needed to disclose a significant difference (72). Moreover, the implication of other variants cannot be excluded (73).
In summary, our study shows in a large pediatric cohort of patients with IBD that a TNF-α polymorphism, namely the −308A in the promoter region of the gene known to increase TNF-α production, is significantly involved in predisposition to both CD and UC. More important, this polymorphism carries a significant reduction in response to steroid therapy, probably leading to a more frequent need for surgical resection.
We thank Ermelinda De Santo, Tiziana Latiano, Carla Zagaria, and Giuseppe Corritore for their skillful technical support.
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The following physicians contributed to the study by providing DNA samples and clinical information of their patients (number of probands): S. Giovanni Rotondo: A. Andriulli, V. Annese, F. Bossa, M. Pastore, M. D'Altilia (75); Roma: S. Cucchiara, O. Borrelli, C. Bascietto (69); Napoli: E. Berni Canani, A.M. Staiano (42); Padova: R. D'incà, G.C. Sturniolo (36); Padova: G. Guariso, V. Lodde (33); Messina: G. Vieni, C. Sferlazzas (31); Bari: V. Rutigliano, D. De Venuto (15); Milano: M. Vecchi, S. Saibeni (15); Napoli: G. Riegler, A. de Leone, S. Giaquinto (14); Parma: G.L. de'Angelis (13); Roma: R. Caprilli, D. Guagnozzi (10); Reggio Calabria: C. Romano (9); Palermo: S. Accomando (8); Foggia: A. Campanozzi (6); Firenze: F. Tonelli (6); and Napoli: F. Castiglione (4).
73. Ho GT, Soranzo N, Nimmo ER, et al. ABCB1/MDT1
gene determines susceptibility and phenotype in ulcerative colitis: discrimination of critical variants using a gene-wide haplotype tagging approach. Hum Mol Gen 2006; 15:707–805.