Dig Dis Sci (2008) 53:622–629 DOI 10.1007/s10620-007-9925-y
ORIGINAL PAPER
Interleukin-10 (819 C/T) and Tumor Necrosis Factor-a (308 G/A) Gene Variants Influence Gastritis and Lymphoid Follicle Development B. R. Achyut Æ Priya Tripathi Æ Uday Chand Ghoshal Æ Nikhil Moorchung Æ Balraj Mittal
Received: 27 December 2006 / Accepted: 4 July 2007 / Published online: 24 August 2007 Springer Science+Business Media, LLC 2007
Abstract Helicobacter pylori (H. pylori) causes gastritis, development of lymphoid follicles and later monoclonal mucosa-associated lymphoid tissue (MALT) lymphoma. We evaluated the association of tumor necrosis factor (TNF)-a (308 G/A) and IL-10 (819 C/T) gene polymorphisms with gastritis and lymphoid follicle formation. H. pylori infection was detected using modified Giemsa staining and IgG anti-CagA enzyme-linked immunosorbent assay (ELISA). One hundred and thirty patients with nonulcer dyspepsia (NUD) and 200 healthy age-matched controls were genotyped for TNF-a and IL-10 polymorphisms using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). Subjects with IL-10 819 T allele [patients (46.5%) versus controls (35.7%), p = 0.006, OR = 1.56, 95% CI = 1.14–2.15] were at risk of gastritis. Infection with H. pylori was more often associated with lymphoid follicles formation than its absence (46% versus 22%, p = 0.009). TNF-a polymorphism did not influence gastritis but patients with TNF-a 308 A allele carriers showed >2 fold risk of lymphoid follicle formation [presence (26%) versus absence (11.25%), p = 0.029, OR = 2.8; 95% CI = 1.09–7.08]. There was a trend towards association of lymphoid follicles and TNF-a 308 A allele carriers with H. pylori infection than without (58.5% versus 22.2%; p = 0.064). IL-10 819 T and TNF-a 308 A alleles may increase risk of gastritis and lymphoid follicle formation. B. R. Achyut P. Tripathi N. Moorchung B. Mittal (&) Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India e-mail:
[email protected];
[email protected] U. C. Ghoshal Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India
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Keywords Cytokine Polymorphism Gastritis Lymphoid follicle Risk
Introduction Helicobacter pylori causes gastritis, peptic ulcer, gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma [1]. Despite the high prevalence of H. pylori infection, the incidence of gastric malignancies is paradoxically low in Asian countries (the Asian enigma) [2]. In India, the prevalence of H. pylori infection in the general population is *84%. Frequency of gastric neoplasm, however, is somewhat low in India despite high seroprevalence of H. pylori (the Indian enigma) [3–6]. The normal gastric mucosa contains very few lymphocytes in the lamina propria. A subset of patients with gastritis develops lymphoid follicles in the gastric mucosa. Frequency of Lymphoid follicle varies from 27 to 100% in gastric mucosa from patients with H. pylori-associated gastritis [7–10], some of whom later develop MALT lymphoma. Six percent of gastric neoplasms in India [11] and 8.7% in the west [12] are contributed by primary gastric lymphoma. The relationship among lymphoid follicles, inflammatory parameters and H. pylori has been defined in several studies [10, 13]. The immunological interaction between the host and H. pylori seems to play a critical role in the pathophysiology of lymphoid follicle formation. Cytokine secretion and function are influenced by gene polymorphisms [14, 15]. Although, a few studies have focused on influence of cytokine gene polymorphisms in gastritis development [16–18], to date no study has evaluated the role of cytokine gene polymorphisms
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in the development of lymphoid follicles in patients with gastritis. Pro and anti-inflammatory cytokines such as TNF-a and IL-10 play role in initiation of the B-cell-mediated immune response [19]. TNF-a is involved in the up-regulation of inflammatory reactions and has been suggested to play role in gastric mucosal damage induced by H. pylori infection. TNF-a 308 A variant is supposed to induce more-efficient transcription of TNF-a and other pro-inflammatory cytokines, amplifying the inflammatory cascade against the infection; excessive inflammatory response in gastric mucosa may be associated with inhibition of gastric acid secretion and higher susceptibility to gastric cancer [20]. Cytokine IL-10 is involved in the down-regulation of the inflammatory response. It inhibits the formation of proinflammatory cytokines such as TNF-a. Several studies have reported functional effect of IL-10 promoter polymorphisms on transcriptional activation which may alter IL-10 protein production in vitro [21, 22]. Many features of inflammation, including neutrophilic and lymphocytic infiltration, gastric atrophy, intestinal metaplasia, and the development of gastric lymphoid follicles, can be explained by cytokine gene polymorphisms. In the present study, we aimed to evaluate the genetic association of TNF-a (308 G/A) and IL-10 (819 C/T) polymorphisms with gastritis and cellular features of gastric mucosa such as neutrophilic and lymphocytic infiltration, precancerous lesions like atrophy, and intestinal metaplasia. We also aimed to evaluate presence of lymphoid follicles in gastric mucosa in relation to genetic polymorphisms in IL-10 and TNF-a genes and presence of serum anti-CagA antibody.
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the local ethics committee had approved the protocol. Two hundred age-matched healthy blood donors without dyspeptic symptoms were included as controls.
Endoscopy and histopathology Patients underwent upper gastrointestinal endoscopy with standard endoscopes (Olympus Optical Co Ltd., Tokyo, Japan). After an overnight fast, four biopsies (antrum and corpus) were taken from each of 130 patients for histological examination. The biopsies were processed by standard techniques. Sections were stained using the haematoxylin and eosin and modified Giemsa stain for the detection of H. pylori. Histological characteristics were assessed according to the updated Sydney system [23]. The scoring system proposed by Wotherspoon et al. [24] was used to assess lymphoid follicles.
Serology and DNA isolation After informed consent a blood sample (5 ml) was taken in ethylenediamine tetraacetic acid (EDTA) as well as in plain vials. The plasma obtained from peripheral blood was tested for anti-CagA antibody using a commercial ELISA kit (Genesis diagnostics, UK). Positive and negative results were based on the cut-off values as per the manufacturer’s instructions. The genomic DNA was extracted from peripheral blood leukocytes using the salting out method [25].
Genotyping of TNF-a (308 G/A) polymorphism Methods Subjects One hundred and thirty patients with NUD who underwent upper gastrointestinal endoscopy from August 2004 to August 2006 were included in present study. Dyspeptic symptoms included epigastric pain or discomfort, anorexia, nausea, and need of antacids. Demographic data and clinical details were obtained through personal interviews using a standard clinical proforma. Exclusion criteria were: present or past history of gastric neoplasm or gastric surgery, long-term treatment with nonsteroidal anti-inflammatory drugs, liver disease, and previous treatment with antibiotics or bismuth salts. History of tobacco and alcohol consumption was recorded. Dietary habits included a vegetarian or nonvegetarian diet. All subjects had given informed consent for the study and
Polymerase chain reaction (PCR) was performed in a total volume of 50 ll with 25 pmol of each primer, genomic DNA (100–150 ng), 10 mM deoxynucleotide triphosphates, 10· buffer containing Tris–HCl, pH 8.6, 50 mM KCl, 1.5 mM MgCl2, and 1.5 units of Taq polymerase (Bangalore Genei, India), with an initial denaturation at 94C for 2 min followed by 35 cycles of 94C for 45 s, 55C for 1 min and 30 s, 72C for 1 min, and a final extension step for 1 min at 72C. The forward primer was 50 -GAGGCAATAGGTTTTGAGGGCCAT-30 and the reverse primer was and 50 -GGGACACACAAGCAT CAAG-30 [26]. The amplified product (107 bp) was electrophoresed on a 2% agarose gel. The restriction digestion was carried out in a 10 ll reaction containing 2.5 ll of PCR reaction and two units of NcoI in the buffer supplied by the vendor (New England Biolabs; http://www.neb.com) at 37C overnight. The reaction was electrophoresed on a 10% polyacrylamide gel. The wild type (G at nucleotide
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308) was cut by NcoI to produce two smaller fragments (87 bp, 20 bp), while the variant (A at nucleotide 308) remained uncut by NcoI (107 bp).
Genotyping of IL-10 (819 C/T) polymorphism PCR was performed with initial denaturation at 95C for 10 min followed by 35 cycles of 95C for 1 min, 63C for 1 min, 72C for 1 min, and a final extension step for 7 min at 72C. The forward primer was 50 -GAC TCC AGC CAC AGA A GC TTA C-30 and the reverse primer was and 50 -ACTAAGGC CCAGAGACCT ACTAAGGC CCAGAGACCT-30 [17]. The amplified product (386 bp) was electrophoresed in a 2% agarose gel. The restriction digestion was carried out in a 10 ll reaction containing 3 ll of PCR product and 10 units of RsaI in the buffer supplied by the vendor (Fermentas, Inc., USA) at 37C overnight. The digested products were electrophoresed on a 15 % polyacrylamide gel. Gel analysis was performed under an ultraviolet (UV) illuminator after staining with ethidium bromide. Gel documentation was done by AlphaimagerTM 1220, Alpha Innotech Corporation, USA.
Quality control Twenty percent of samples from patients and controls including samples of each genotype were regenotyped by other laboratory personnel. No discrepancy was found after sequencing the randomly selected 5% samples.
Statistical analysis
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Results Demographic profile and H. pylori status There was no significant difference in the mean age of patients (36.22 ± 12.37 years) and controls (34.12 ± 7.93 years). H. pylori seropositivity among patients with NUD was 68.5%, however, 59.2% showed presence of H. pylori infection histologically. Eighty patients (61.54%) showed absence and 50 (38.46%) showed different grades (II, III, and IV) of lymphoid follicles (Table 1).
TNF-a (308 G/A) gene polymorphism In controls, all the frequencies of tested genotypes and alleles were in Hardy–Weinberg equilibrium. There was no association of TNF-a (308 G/A) polymorphism with gastritis, either with genotypes or allele (Table 2) as the frequencies of genotypes were not significantly different between patients and controls. Frequencies of genotypes and alleles were 308 GG genotype: 108/130, 83.1% versus 164/200, 82.0%; GA genotype: 18/130, 13.8% versus 29/200, 14.5%; AA genotype: 4/130, 3.1% versus 43/200, 3.5%; 308 G allele: 234/260, 90% versus 357/ 400, 89.3% and A allele: 26/260, 10% versus 43/400, 10.7% between patients and controls, respectively.
Table 1 Demographic profile and H. pylori status in gastritis patients Demographic profile
Patients
Controls
No. of subjects
130
200
Age ± SD (years)
36.22 ± 12.37
34.12 ± 7.93
Male
74 (56.9%)
105 (52.5%)
Female
56 (43.1%)
95 (47.5%)
Gender
Sample size was calculated using the QUANTO Version 1 program (http://hydra.use.edu/gxe). The desired power of our study was set at 80% with a significance level of 0.05 in a two-sided test. We chose the log-additive inheritance model, which is the most suitable for polygenic diseases. By means of the QUANTO program, our sample was considered adequate to study both polymorphisms. Data was analyzed using the statistical software SPSS 11.5 (Chicago, IL, USA). The v2-test or Fisher’s exact test was used to determine differences in frequencies. Logistic regression models were used to calculate odds ratio. p-value < 0.05 was considered significant. Any deviation from Hardy–Weinberg equilibrium was calculated by a v2 goodness-of-fit test. The age variable was expressed as mean ± standard deviation (SD).
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Anti-CagA antibody Negative
41 (31.5%)
Positive
89 (68.5%)
H. pylori Giemsa staining Nil
53 (40.8%)
Mild Moderate
44 (33.8%) 12 (9.2%)
Marked
21 (16.2%)
Lymphoid follicles Nil
80 (61.54%)
Grade II
30 (23.07%)
Grade III
18(13.85%)
Grade IV
2 (1.54%)
2.35 (1.19–4.63)
0.006
1.56 (1.14–2.15)
Allele C
139 (53.5)
257 (64.3)
T
121 (46.5)
143 (35.7)
Reference [1]
* Total number of patients 130 and controls 200 (for genotypes) and total number of chromosomes patients 260 and controls 400 (for alleles)
IL-10 (819 C/T) gene polymorphism After comparing genotype and allele frequencies between patients and controls (Table 3), increased risk for gastritis was observed with IL-10 819 CT genotype (71/130, 54.6% versus 91/200, 45.5%, p = 0.012, OR = 1.91, 95% CI = 1.15–3.16), TT genotype (25/130, 19.2% versus 26/ 200, 13.0%, p = 0.014, OR = 2.35, 95% CI = 1.19–4.65) and T allele (121/260, 46.5% versus 143/400, 35.7%, p = 0.006, OR = 1.56, 95% CI = 1.14–2.15).
Association of gene polymorphisms with histological features Higher neutrophilic infiltration (p > 0.05, OR = 1.3, 95% CI = 0.44–3.86), and glandular atrophy (p > 0.05, OR = 2.0, 95% CI = 0.78–4.94) was observed in TNF-a 308 A allele carriers. IL-10 819 T allele carriers were at risk of increased neutrophilic infiltration (p > 0.05, OR = 1.4, 95% CI = 0.58–3.17), glandular atrophy (p > 0.05, OR = 1.2, 95% CI = 0.55–2.73), and intestinal metaplasia (p > 0.05, OR = 1.2, 95% CI = 0.44–3.33) (Table 4) in patients with gastritis.
1.2 (0.44–3.33) 19 (20%)
4 (18.2%) 0.9 (0.28–3.0)
6 (17.1%) 1.0 (Ref) 29 (82.9%)
87 (80.6%) 21 (19.4%) 1.0 (Ref) 1.0 (Ref)
Ref, reference. N0, nil; N1, mild; N2, moderate; N3, marked neutrophilic infiltration. L1, mild; L2, moderate; L3, marked lymphocytic infiltration. G0, nil; G1, mild; G2, moderate; G3, marked glandular atrophy * Sum of genotypes GA + AA and CT + TT for TNF-a and IL-10 polymorphisms, respectively
1.91 (1.15–3.16)
0.014
T allele carriers* 24 (25.3%) 71 (74.7%) 1.4 (0.58–3.17) 24 (25.3%) 71 (74.7%) 0.61 (0.23–1.65) 55 (57.9%) 40 (42.1%) 1.2 (0.55–2.73) 76 (80%)
0.012
26 (13.0)
22 (62.9%) 13 (37.1%) 1.0 (Ref)
91 (45.5)
25 (19.2)
1.0 (Ref)
71 (54.6)
TT
6 (17.1%) 29 (82.9%)
CT
11 (31.4%) 24 (68.6%) 1.0 (Ref)
Reference [1]
CC
83 (41.5)
41 (38%)
CC 34 (26.2)
67 (62%)
Genotype
1.0 (Ref)
Patients n* (%) Controls n* (%) p-value OR (95% CI)
5 (22.7%) 17 (77.3%) 1.02 (0.34–3.05) 10 (45.5%) 12 (54.5%) 2.0 (0.78–4.94) 18 (81.8%)
Table 3 Genotype and allele frequency distribution of IL-10 (819 C/T) polymorphism in patients and controls
25 (23.1%) 83 (76.9%)
* Total gastritis patients 130 and controls 200 (genotypes) and total chromosome number patients 260 and controls 400 (alleles)
5 (22.7%) 17 (77.3%) 1.3 (0.44–3.86)
0.758
30 (27.8%) 78 (72.2%) 1.0 (Ref)
43 (10.7%)
A allele carriers*
26 (10.0%)
GG
A
IM (n = 25)
0.758
Non-IM (n = 105)
357 (89.3%)
OR (95%CI)
234 (90.0%)
G2/G3 (n = 53)
G
G0/G1 (n = 77)
0.824
Alleles
OR (95%CI)
7 (3.5%)
L2/L3 (n = 100)
4 (3.1%)
L1 (n = 30)
0.855
AA
OR (95%CI)
0.962
29 (14.5%)
N2/N3 (n = 95)
164 (82.0%)
18 (13.8%)
N0/N1 (n = 35)
108 (83.1)
GA
Intestinal metaplasia
GG
Genotype
Glandular atrophy
p-value
Lymphocytic infiltration
Controls n* (%)
Neutrophilic infiltration
Patients n* (%)
Genotype
Table 2 Genotype and allele frequency distribution of TNF-a (308 G/A) polymorphism in gastritis patients and controls
OR (95%CI)
625 Table 4 Variant allele carriers in patients with different degrees of neutrophilic (N), lymphocytic (L) infiltration, and glandular atrophy (G), as well as the presence or absence of intestinal metaplasia (IM)
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Table 5 Association of lymphoid follicles with presence and absence of H. pylori infection
Table 7 Association of variant allele carriers with lymphoid follicles in H. pylori-positive patients
H. pylori infection Lymphoid follicles
Genotype
Absent (n = 80)
Present (n = 50)
p-value OR (95%CI)
Lymphoid follicles and H. pylori infection Absence (n = 9)
Presence (n = 41)
TNF-a (308 G/A) Negative
32 (40%)
9 (18%)
Positive
48 (60%) 41 (82%) 0.009
1.0 (reference)
GG
7 (77.8%)
17 (41.5%)
3.0 (1.3–7.1)
A allele carriers* IL-10 (819 C/T)
2 (22.2%)
24 (58.5%)**
CC
1 (11.1%)
12 (29.3%)
T allele carriers*
8 (88.9%)
29 (70.7%)
Association between H. pylori and presence of lymphoid follicles Patients with H. pylori had a higher risk of lymphoid follicle formation than those without [(41/50, 82%) versus (48/80, 60%); p = 0.009; OR = 3.0, 95% CI = 1.3–7.1] (Table 5).
* Sum of genotypes (GA + AA and CT + TT respectively) ** Frequency difference (p = 0.064) of 308 A allele carriers between absence and presence groups of lymphoid follicle in H. pylori-positive patients
higher frequency of lymphoid follicles than non-carriers (58.5% versus 22.2%, p = 0.064) (Table 7). Association of gene polymorphisms with presence of lymphoid follicles Discussion Frequency of TNF-a 308 A allele was higher in patients with lymphoid follicles than patients without it (26% versus 11.25%) (Table 6). Presence of 308 A allele was significantly (p = 0.029) associated with high risk (OR = 2.8, 95% CI = 1.09–7.08) of presence of lymphoid follicles. However, IL-10 819 T allele showed no effect on lymphoid follicle development. Frequency was comparable in patients with and without lymphoid follicles (74% versus 72.5%).
Association of gene polymorphisms with presence of lymphoid follicles in H. pylori-positive patients There was no influence of H. pylori infection on lymphoid follicle development in the presence of IL-10 819 T allele. However, TNF-a 308 A allele carriers showed Table 6 Association of variant allele carriers of TNF-a (308 G/A) and IL-10 (819 C/T) polymorphisms in patients with presence or absence of lymphoid follicles Genotype
Lymphoid follicles Absent (n = 80)
Present (n = 50)
p-value OR (95%CI)
TNF-a (308 G/A) GG A allele carriers*
71 (88.75%) 37 (74%) 9 (11.25%) 13 (26%) 0.029
1.0 (reference) 2.8 (1.09–7.08)
IL-10 (819 C/T) CC
22 (27.5%)
T allele carriers* 58 (72.5%)
13 (26%)
1.0 (Reference)
37 (74%) 0.851
1.1 (0.49–2.40)
* Sum of genotypes (GA + AA and CT + TT, respectively)
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In the present study, we hypothesized that variability in secretion of TNF-a and IL-10 cytokines, probably influenced by promoter polymorphisms in their genes, might determine the degree of mucosal inflammation (gastritis) and development of lymphoid follicles, a precursor of MALT lymphoma. In this case-control study, IL-10 (819 C/T) polymorphism was significantly associated with increased risk of gastritis. Concisely, patients showed high frequency of 819 T allele, known to be a low secreter of IL-10 cytokine (anti-inflammatory). Hence, this allele promotes inflammation and the risk of gastritis development. Our results corroborates earlier findings. Rad et al. [17] reported that subjects with 819 T allele in the ATA haplotype had lower mucosal IL-10 mRNA levels than 819 C allele in the GCC haplotype. Another study reported an association between carcinoma and the anti-inflammatory (high IL-10-secreting) haplotype GCC [27]. However, some studies also showed contradictory results, e.g., one report found a higher prevalence of gastric cancer in patients with pro-inflammatory (low IL-10-secreting) haplotype ATA [28]. Our study provides some explanation for this paradox. In the case-only study, IL-10 (819 C/T) polymorphism indicated marginal risk of chronic cellular infiltration, glandular atrophy, intestinal metaplasia, and lymphoid follicle formation. H. pylori infection contributes to recruitment of neutrophils and lymphocytes, which in turn increases epithelial damage through the release of cytokines such as TNF-a. Chronic inflammation in later stages causes glandular atrophy and intestinal metaplasia. In a mouse model, IL-10 has been shown to protect from the
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development of severe gastritis and mucosal damage through down-regulation of H. pylori-induced Th1 response [29, 30]. G to A polymorphism at position 308 of the TNF-a gene is considered to be functionally important [31, 32]. Studies have found association between polymorphism and chronic atrophic gastritis or gastric cancer [18, 28], suggesting that 308 AA genotype favors development of a hypochlorhydric, atrophic response to gastric infection with H. pylori, but negative association also exist [33]. We also did not find significant association of TNF-a (308 G/ A) polymorphism with gastritis. IL-10 and TNF-a cytokines have complex and predominantly opposing roles in inflammation and immunity [34, 35]. Functional polymorphisms at the promoter region are known to influence disease outcome. Ethnic variability and differences in allele/genotype frequency distribution among different ethnic population is considered to be the key factor in association studies. TNF-a 308 A allele has a prevalence of 7% in China and Korea [33, 36]. Rad et al. [17] reported a 15% frequency of 308 A allele in Germany. Perri et al. [37] found an 11% frequency in normal Italian subjects. However, the Portuguese [18], Finnish [38] and US population [28] have 13%, 14%, and 15% 308 A allele, respectively. In India, TNF-a 308 A allele frequency falls between the values for China/Korea and Italy. In Caucasian populations, H. pylori strains with CagA pathogenic island showed more-severe disease than strains that lacked CagA [39, 40]. There was a strong association between the presence of serum antibodies to CagA and the isolation of CagA-positive strains from a patient [41]. H. pylori infection was found to influence MALT lymphoma development [24]. Although in Asian populations the association of CagA positivity and disease risk is different than in the Western population [42], our study showed that presence of anti-CagA antibody was significantly associated with risk of lymphoid follicle formation. We investigated 38.5% positivity of lymphoid follicles in three antral specimens from 130 patients with gastritis, higher than the 27.4% positivity reported by Wyatt and Rathbone [7]. Other authors have also reported higher prevalence of lymphoid follicles in H. pylori-infected gastric mucosa; i.e., Eidt and Stolte (54%) [8], Genta and Hammer (63.8% in gastritis and 100% in patients with H. pylori infection) [9] and Chen et al. (76%) [10]. TNF-a is involved in the pathogenesis of lymphoproliferative disorders such as non-Hodgkin lymphoma, and serum levels are valuable prognostic markers [43]. TNF-a and its receptors are involved in constructing the germinal centers of Peyer’s patches, spleen, or peripheral lymph nodes [44]. In vitro studies have shown that TNF-a production may promote the growth of lymphoid cells [45].
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Wu et al. [46] showed an inverse relationship between TNF-a -857 T allele and risk of lymphoma. A pathogenic role of TNF-a (308 G/A) polymorphism had not yet been studied in relation to lymphoma. In a case-only analysis, we found significant association (p = 0.029) between TNF-a polymorphism and presence of lymphoid follicles. The frequency of 308 A allele carriers was higher in the lymphoid-follicle-positive group with high risk (odds ratio = 2.8). Recently, one study from Germany has also reported the functional role of TNF-a gene polymorphisms in primary gastric B-cell lymphoma [47]. We also analyzed whether this association was related to H. pylori positivity. We found a trend towards association (p = 0.064) between 308 A allele carriers and presence of lymphoid follicles in H. pylori-infected patients. To the best of our knowledge, this is the first study showing association between pro-inflammatory cytokine polymorphisms and lymphoid follicles in India. Further studies are needed to see if this association is peculiar to the Indian subcontinent or is a universal phenomenon. In conclusion, IL-10 819 C/T polymorphism was significantly associated with gastritis. TNF-a (308 G/A) polymorphism did not influence gastritis but patients with TNF-a 308 A allele were at risk of lymphoid follicle development. Genetic polymorphisms could not favor lymphoid follicle development in addition to H. pylori infection. Our study highlighted the pathogenic role of TNF-a in lymphoid follicle formation. H. pylori infection is very high in India and TNF-a genotyping in patients with gastritis may be potentially helpful in identifying subjects at risk of lymphoid follicle development and thus may help in strategies to prevent MALT lymphoma. Acknowledgements CSIR (New Delhi, India) is greatly acknowledged for providing fellowship to Mr. B.R. Achyut.
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