- Email: [email protected]
A) as a biomarker in chronic pancreatitis
Gene 539 (2014) 186–189
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
A single-nucleotide polymorphism in tumor necrosis factor-α (− 308 G/A) as a biomarker in chronic pancreatitis K. Sri Manjari a, A. Jyothy a, P. Shravan Kumar b, B. Prabhakar c, M. Uma Devi b, M. Ramanna b, Pratibha Nallari d, A. Venkateshwari a,⁎ a
Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, India Department of Gastroenterology, Gandhi General Hospital, Secunderabad, India Department of Gastroenterology, Osmania General Hospital, Hyderabad, India d Department of Genetics, Osmania University, Hyderabad, India b c
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 18 November 2013 Accepted 7 February 2014 Available online 18 February 2014 Keywords: Inflammation Genotypes Pancreatic fibrosis Pancreatic stellate cells
a b s t r a c t Objective: Chronic pancreatitis is a gradual, long-term inflammation of the pancreas that results in alteration of its normal structure and function. The study aims to investigate the role of −308 (G/A) polymorphism of TNF-α gene in chronic pancreatitis. Material and methods: A total of 200 subjects were included in this case–control study. A total of 100 in patients admitted in the Gastroenterology Unit of Gandhi Hospital and Osmania General Hospital, Hyderabad were included in the present study. An equal number of healthy control subjects were randomly selected for the study. The genotyping of TNF-α gene was carried out by tetra-primer ARMS PCR followed by gel electrophoresis. The TNF-α levels were assayed by enzyme-linked immunosorbent assay. Results: A significant variation with respect to the genotypic and allelic distribution in the disease group when compared to control subjects [OR = 2.001 (1.33–3.005), p b 0.0001**] was observed. Subjects homozygous for the A allele had higher TNF-α levels compared to G allele. Conclusion: The present study revealed a significant association of the TNF-α gene promoter polymorphism with chronic pancreatitis. Thus, TNF-α genotype can be considered as one of the biological markers in the etiology of chronic pancreatitis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chronic pancreatitis (CP) is an ongoing inflammatory condition in which pancreatic secretory parenchyma is destroyed and replaced by fibrous tissue, ultimately leading to malnutrition and diabetes. The prevalence of chronic pancreatitis in France is 26 per 100,000 people. This prevalence is not unsimilar to the middle of three estimates from Japan, but considerably lower than the figure of 114–200 per 100,000 in South India (Braganza et al., 2011). The main symptom of chronic pancreatitis is usually pain, identical to that of acute pancreatitis which is usually a constant and disabling pain.
Abbreviations: TNF, tumor necrosis factor; ARMS PCR, amplification refractory mutation system polymerase chain reaction; CP, chronic pancreatitis; CT scan, computed tomography scan; ERCP, endoscopic retrograde cholangiopancreatography; EDTA, ethylenediaminetetraacetic acid; DNA, deoxyribonucleic acid; OR, Odds ratio; CI, class intervals; ELISA, enzyme-linked immunosorbent assay. ⁎ Corresponding author at: Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad 500 016, India. E-mail address: [email protected] (A. Venkateshwari).
http://dx.doi.org/10.1016/j.gene.2014.02.014 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Recently, chemokines have been contemplated as essential factors in the development of chronic pancreatitis (Ito, 2007). They are elucidated as important factors that affect inflammation by increasing fibrosis which is an important feature of chronic pancreatitis. Thus, inflammatory chemokines are evidently associated with the chemo-attraction of leukocytes in early-stage chronic pancreatitis, leading to the movement of monocytes into the pancreas thereby resulting in the formation of pancreatic fibrosis (Grady et al., 1997; Inoue et al., 2002; Saurer et al., 2000). Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine with strong immune-stimulatory activity. It is involved in the inflammatory responses by stimulating the cytokine production, increasing the expression of adhesion molecules, and neutrophil activation, and it also acts as a co-stimulator for T-cell activation and antibody production by B-cells (Vassalli, 1992). TNF-α secretion is controlled primarily at the transcription level, and inter-individual differences in TNF-α secretion are associated with TNF-α promoter variant (Beranek et al., 2003). Single-nucleotide polymorphisms positioned within the gene promoters have been shown to affect transcription factor binding and hence gene expression. One of the common G/A polymorphisms has
K. Sri Manjari et al. / Gene 539 (2014) 186–189
been described at position −308 (TNF-308 G/A). The −308 polymorphism could probably affect the cell-type and stimulus specific control of TNF synthesis at the transcriptional level. A genetic predisposition to produce elevated TNF levels, due to the presence of the − 308A polymorphism, may change the course of an immune response such that an individual is more susceptible to the disease (Abraham and Kroeger, 1999). Given the local and systemic immune-stimulating effects of TNF-α, we hypothesized that promoter polymorphism of this gene may be a genetic modifying factor for chronic pancreatitis (Beranek et al., 2003). Hence, the aim of the present study was to investigate the qualitative and quantitative variation of TNF-α in the etiology of chronic pancreatitis. 2. Materials and methods 2.1. Study population The study design was a prospective case control study conducted with a total of 200 unrelated individuals. One hundred clinically evaluated chronic pancreatitis (CP) patients referred and admitted in the Gastroenterology Unit of Gandhi Hospital and Osmania General Hospital, Hyderabad between March 2008 and March 2010 were involved in the present study. The diagnosis of chronic pancreatitis was established on the findings of pancreatic duct dilation or pancreatic calcifications, or on the histological findings of chronic pancreatitis of various diagnostic tests like X-ray, CT scan, ERCP, etc. (Abraham and Kroeger, 1999). An alcoholic origin of chronic pancreatitis was assumed when the alcohol intake was more than 80 g/day in men for at least two years. For smoking status, a person who had smoked at least once a day for N 1 year in his or her lifetime was regarded as a smoker (Özhan et al., 2010). An equal number of healthy control subjects were randomly selected for the study from the individuals visiting our institute for regular health checkup. As male preponderance was observed in the patient group, more number of male controls was included in the study for better comparative analysis. Among these controls, 40% of them were found to be alcoholics at random. A structured proforma was used to seek information on dietary habits, smoking, alcohol consumption, family history, etc. Written informed consent was obtained from all the subjects, included for the study. The study was also approved by the Institutional Ethical Committee. 2.2. DNA isolation 5 ml of venous blood was drawn from each individual in vacutainers with and without EDTA for the separation of plasma and serum respectively and stored at −70 °C until use. Genomic DNA was isolated from whole blood by following the salting out procedure of Lahiri and Nurnberger (1991). 2.3. Genotyping of the TNF-α gene
2.4. Estimation of TNF-α level Concentrations of TNF-α (Invitrogen®) in plasma were measured using a commercial ELISA kit according to the manufacturer's protocols. In brief, 50 μl incubation buffer was added to different wells designated as samples and standards. 100 μl of standard or control samples was added to each well. After 2 h of incubation at room temperature on a constant shaker (500 ± 50 rpm), the reaction solution was aspirated and the wells were washed 4 times with wash buffer; 100 μl of biotinylated anti-TNF-α conjugate was then added to each well and incubated for another 1 h on the shaker at room temperature. Then the aspiration/wash steps were repeated as mentioned above, followed by adding of 100 μl of streptavidin–HRP solution to each well. The microplate was allowed to stand for 30 min at room temperature. The aspiration steps were repeated. Then stabilized chromogen (100 μl) was added to each well followed by 30 min of incubation at room temperature in the dark. The optical density of each sample was determined at 450 nm and represented in ng/ml. The TNF-α concentration for each sample was calculated from the standard curve obtained. 2.5. Statistical analysis Statistical analysis was done by using the computer software Statistical Package for Social Sciences-SPSS for windows (version 19.0) (Chicago, IL). ORs and 95% CIs were estimated by conditional logistic regression analyses based on the comparison of genotypes between patients with the disease and healthy controls, and by adjusting the potential confounders such as age, sex, smoking and alcoholism. Hardy–Weinberg equilibrium was conducted to compare the observed and expected genotype frequencies among cases and controls, respectively. A two-sided P-value b 0.05 was considered to be statistically significant. For analyses of genotype frequencies, the wild-type category was the reference group. 3. Results The study population (Table 1) consisted of 100 patients with CP with an age range of 13–70 years, as well as 100 control subjects with an age range of 20–70. CP group consisted of 95.0% males and 5.0% females whereas control group consisted of 94.0% males and 6.0% females. Patients and control subjects were derived from the same geographic location and are representative of South Indian population from Andhra Pradesh. A significant difference was observed with respect to mean age (b 0.000**) smokers (b0.000**) and alcoholics (b 0.000**) in patients compared to control subjects. Table 2 gives the genotype distribution of TNF-α (−308 G/A) gene in control subjects and patient group. The distribution of the TNF-α Table 1 Demographic and clinical parameters of the study groups (CP). Parameter
The A and G alleles at position − 308 in the promoter region of the TNF-α gene were determined using the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) methodology (Ye et al., 2001). Each PCR reaction was carried out in a total volume of 10 μl, containing 30 ng of template DNA, 10 pmol of each inner primer, 1 pmol of each outer primer, 200 μM dNTP, 2.5 mM of MgCl2, 20 mM Tris–HCl pH 8.4, 50 mM KCl and 0.5 U Taq polymerase (B. Genei). The cycling conditions were as follows: an initial denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 1 min, 61 °C for 1 min and 72 °C for 1 min. The final extension step was at 72 °C for 2 min. The amplified products were: 323 bp internal control, 224 bp G allele and 154 bp A allele which were separated by electrophoresis on a 2% agarose gel stained with ethidium bromide. The gel was visualized under ultraviolet light with a 100-base pair ladder.
187
Controls
CP
N
%
N
%
Gender Males Females
94 6
94 6
95 5
95 5
0.82 (0.22–2.92)
0.378⁎
Age b40 years ≥40 years
78 22
78 22
52 48
52 48
3.25 (1.77–6.10)
b0.000⁎⁎
Addictions Smokers Non-smokers Alcoholics Non-alcoholics
22 78 40 60
22 78 40 60
49 51 76 24
49 51 76 24
0.29 (0.15–0.56)
b0.000⁎⁎
0.21 (0.11–0.38)
b0.000⁎⁎
⁎ p b 0.05. ⁎⁎ p b 0.0001.
OD (CI)
p value
188
K. Sri Manjari et al. / Gene 539 (2014) 186–189
Table 2 Genotypic and allelic frequencies of TNF-α genotypes between controls and CP. Study group
Controls CP
G/G
G/A
A/A
Total
N
(%)
n
(%)
N
(%)
50 35
(50) (35)
36 33
(36) (33)
14 32
(14) (32)
Allelic frequencies
100 100
p
q
0.68 (136) 0.52 (103)
0.32 (64) 0.49 (97)
[OD = 2.001 (1.33–3.005), p b 0.0001**].
(− 308 G/A) genotypes in controls was in Hardy–Weinberg equilibrium. Among the controls the genotype distribution was: 50% (G/G), 36% (A/G), and 14% (A/A), whereas the genotypes in chronic pancreatitis patients: 35% (G/G), 33% (A/G) and 32% (A/A) were not in Hardy– Weinberg equilibrium. A significant variation was observed in the distribution of A allele in the patient group (49%) compared to control subjects (32%) [Odds ratio = 2.001 (1.33–3.005), p b 0.0001**]. *p b 0.05, **p b 0.0001. Comparison of genotypes was made with regard to the smoking habit and alcoholism. There was an increased frequency of A allele in smokers compared to control subjects, indicating an association between TNF-α (A-308 GA + AA) containing genotypes and smoking (OR = 0.305, 95% CI (0.094–0.976), p = 0.035). However, there was no significant association between TNF and alcoholism (OR = 0.648, 95% CI (0.270–1.555), p = 0.648). Further patients were subgrouped into smokers and non-smokers among alcoholic and non-alcoholics to test for the association of TNF A allele by multivariate regression analysis. We did not find any significant result with smoking as a modifying factor. Comparison of mean levels of TNF-α with respect to the genotypes (Table 3) revealed elevated levels in the disease compared to control subjects. However, a significant increase was observed in CP with GG and AA genotype (111.29 ± 57.94) compared to control subjects (80.35 ± 43.34) (p b 0.0001**). There was no significant difference in the mean levels of TNF-α with respect to smoking and alcoholism. Further, comparison was also made within the disease group in different clinical manifestations like steatorrhoea (13%), calcification (31%), atrophy (10%), diabetes (7%) and we did not found any association between the A allele and subgroups. A number of groups have carried out investigations into the functional significance of the TNF-α (− 308 G/A) polymorphism in acute and chronic pancreatitis. We have considered only CP cases for the present study and a total of six publications were reported on the genotype frequency of TNF-α (− 308 G/A) in chronic pancreatitis and controls (Table 4). Data from each publication included the following details: year of publication, first author's name, country in which the study was conducted, study design, ethnicity, sample size of CP patients, controls and the association of the genotype. Our study is in agreement with the study of Abdulrazeg et al. (2001), wherein higher expressive allele A is associated with the disease. However, other studies showed no such association with the disease indicating the variation in the distribution of genotypes in different ethnic groups. 4. Discussion Chronic pancreatitis, an irreversible inflammatory disease of the pancreas, is marked by chronic inflammatory cell infiltration, acinar Table 3 Mean serum levels (in ng/ml) of TNF-α in chronic pancreatitis patients and control subjects with respect to the genotypes.*p b 0.05, **p b0.0001. TNF-α
Patients (N)
Genotype: G/G 35 G/A 33 A/A 32 Total 100
Mean ± SD
Controls (N)
Mean ± SD
t-Value
p-Value
77.00 98.30 162.19 111.29
50 36 14 100
52.60 89.72 155.36 80.35
3.5086 1.0656 0.4246 4.2761
0.0007* 0.2904 0.6732 0.000**
± ± ± ±
42.83 38.75 54.58 57.94
± ± ± ±
20.34 27.64 37.75 43.34
cell degeneration, and development of fibrosis, which lead to impairment of pancreatic exocrine and endocrine function (Ito, 2007). Irrespective of the etiologies of pancreatic injury, pancreatic fibrosis is caused commonly by a chronic and uncontrolled inflammatory/repair process leading to excessive deposition of collagen and other components of extracellular matrix (Whitcomb, 2004). Abnormal deposition of fibrous tissue is a characteristic histological feature of chronic pancreatitis, a progressive necro-inflammatory condition of the pancreas that often results in exocrine and endocrine insufficiency (Apte et al., 2011). Tumor necrosis factor-α is a multifunctional pro-inflammatory cytokine implicated in modulating the progression of chronic pancreatitis by inducing pancreatic stellate cells activation as indicated by increased proliferation, α-SMA expression, and/or collagen synthesis leading to fibrosis (Berberat et al., 2000). Hutchinson et al. (1998) demonstrated that individuals differ in their capacity to synthesize various cytokines and attributed such differences to the existence of allelic polymorphism in the promoter region of cytokines. The transcription regulatory region of TNF-α (− 308 G/A) polymorphism has been associated with differential expression and different serum concentrations of TNF-α. The recent finding of an increased gene expression of TNF-α and its receptors in blood leucocytes of patients with late stage chronic alcoholic pancreatitis implies a pathogenic importance of TNF-α in the clinical features of chronic pancreatitis (Beranek et al., 2003; Hanck et al., 1999). In addition to several autoimmune diseases, studies have shown a close association of the TNF-308A allele to primary sclerosing cholangitis (Mitchell et al., 2001), alcoholic steato-hepatitis (Grove et al., 1997), extended ulcerative colitis (Koss et al., 2000) and coeliac disease (de la Concha et al., 2000). Studies have highlighted that small difference at cytokine levels as a result of genetic variants may have an important effect on inflammatory response and may influence the pathophysiology. The less common −308A allele is strongly associated with the MHC haplotype HLA-A1-B8 and DR3, which is in turn associated with high TNF-α production and autoimmune disease. This genetic propensity to produce elevated TNF-α levels, due to the presence of the −308A polymorphism, may alter the course of an immune response (Scardapane et al., 2012). TNF-alpha polymorphism is associated with clinical features of chronic obstructive pulmonary disease including progression. There is clear evidence of TNF-alpha overexpression and bioactivity with neutrophilic inflammation (Sapey et al., 2010). SNP located at nucleotides − 308 have been extensively researched on the susceptibility to a range of autoimmune disorders, including rheumatoid arthritis, exfoliation glaucoma, and many kinds of cancers. Carriage of the TNF308 G allele correlates with disease severity and hepatic fibrosis, which may contribute to a higher risk for HCC (Jeng et al., 2007). The present study was undertaken to investigate the association of TNF-α −308 allelic polymorphism and CP as no such data is available for the Indian population. In the present study we found a positive association between TNF-α (−308 G/A) promoter polymorphism and CP. The polymorphic studies of TNF-α on various ethnic groups have yielded conflicting results in association with CP. The preliminary report by Abdulrazeg et al. (2001) supported our suggestion that the TNF-α (−308 G/A) promoter polymorphism may be associated with chronic pancreatitis. However, other studies from Beranek et al. (2003), Schneider et al. (2003, 2004), Bendicho et al. (2005) and Farkas et al. (2007) showed no association between this polymorphism and CP. Zhang et al. (2003) did not find any correlation between the polymorphism on position − 308 of the TNF-α gene and acute pancreatitis. The contradictory outcomes of various groups indicate a complex association between TNF-α (−308 G/A) polymorphism, CP and ethnicity. We did find a significant association of (GA + AA) genotype with smoking but not with alcoholism. We did not find any association of TNF-α (−308 G/A) polymorphism in different clinical manifestations of chronic pancreatitis which is in confirmation with Schneider et al. (2003).
K. Sri Manjari et al. / Gene 539 (2014) 186–189
189
Table 4 Characteristics of earlier publications on TNF-α-308 polymorphism and chronic pancreatitis susceptibility. Reference
Country
Ethnicity
Source of cases
Source of controls
Association of TNF-α
Abdulrazeg et al. (2001) Beranek et al. (2003) Schneider et al. (2003) Schneider et al. (2004) Bendicho et al. (2005) Farkas et al. (2007) Present study
United Kingdom Germany USA USA Brazil Hungary India
Caucasian German Caucasian Caucasian Caucasian Hungarian Indian
University study (64) Hospital patients (335) University study (54) Disease center (42) Hospital outpatient clinic (28) Hospital patients (83) Hospital patients
Healthy controls (250) Healthy blood donors (116) and (25) healthy carriers Random individuals (94) Random individuals (94) Blood donors (94) Healthy blood donors (75) Healthy controls
Yes No No No No No Yes
Thus, in the present study the positive association between TNF-α (−308 G/A) promoter polymorphism and CP may be due to ethnic difference in the population. Mews et al. (2002) showed that the quiescent stellate pancreatic cells, observed on normal pancreas, could be activated by cytokines such as TNF-α, IL-1, IL-6, and IL-10 and suggested that the persistent activation of these cells could be a factor involved in the progression of CP. Vaccaro et al. (2000) verified that the TNF-α expression on acinar pancreas cells is not typical but occurs at the beginning of pancreatitis. In most of the studies performed on chronic pancreatitis, higher TNF-α levels were observed which is correlated to the exacerbation severity (Kıyıcı et al., 2009; Zhukov et al., 2003, 2004). Thus, our findings agree with the previous studies that higher level of TNF-α leads to the activation of the pancreatic stellate cells which are the main source for increased deposition and disorganization of extracellular matrix proteins resulting in the fibrosis and chronic inflammation of the pancreas. Further, our results suggest that a single dose of A allele may contribute two fold increased risk than GG genotype to the disease. Thus excess production of TNF-α seems to be a typical feature of CP. The inflammatory response is dependent on the level of TNF-α which the pancreas is exposed to and may result in increased pancreatic stellate cell proliferation. Therefore, polymorphic variation of the TNF-α gene leading to variable TNF-α levels may influence TNF-α-dependent inflammation and damage of the pancreas in CP. In conclusion, the present study focuses the role of TNF-α gene promoter polymorphism and their corresponding levels in chronic pancreatitis and indicates it as one of the biomarkers in the etiology of the disease. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgment The authors acknowledge the financial support from the University Grants Commission (UGC), New Delhi. Authors are also thankful to the Department of Biotechnology, New Delhi for the financial assistance. References Abdulrazeg, E.M., et al., 2001. TNF-alpha promoter region gene polymorphisms in patients with alcohol-induced chronic pancreatitis. Gastroenterology 120, A32. Abraham, L.J., Kroeger, K.M., 1999. Impact of the −308 TNF promoter polymorphism on the transcriptional regulation of the TNF gene: relevance to disease. Journal of Leukocyte Biology 66, 562–566. Apte, M., et al., 2011. The fibrosis of chronic pancreatitis: new insights into the role of pancreatic stellate cells. Antioxidants and Redox Signaling 15, 2711–2722. Bendicho, M.T., et al., 2005. Polymorphism of cytokine genes (TGF-beta1, IFN-gamma, IL-6, IL-10, and TNF-alpha) in patients with chronic pancreatitis. Pancreas 30, 333–336. Beranek, H., et al., 2003. Analysis of tumour necrosis factor alpha and interleukin 10 promotor variants in patients with chronic pancreatitis. European Journal of Gastroenterology and Hepatology 15, 1223–1227. Berberat, P.O., et al., 2000. Chronic pancreatitis—new pathophysiological concepts. Swiss Surgery 6, 227–230. Braganza, J.M., et al., 2011. Chronic pancreatitis. Lancet 377, 1184–1197.
de la Concha, E.G., et al., 2000. Celiac disease and TNF promoter polymorphisms. Human Immunology 61, 513–517. Farkas Jr., G., et al., 2007. Relevance of transforming growth factor-beta1, interleukin-8, and tumor necrosis factor-alpha polymorphisms in patients with chronic pancreatitis. European Cytokine Network 18, 31–37. Grady, T., et al., 1997. Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis. Gastroenterology 113, 1966–1975. Grove, J., et al., 1997. Association of a tumor necrosis factor promoter polymorphism with susceptibility to alcoholic steatohepatitis. Hepatology 26, 143–146. Hanck, C., et al., 1999. Cytokine gene expression in peripheral blood mononuclear cells reflects a systemic immune response in alcoholic chronic pancreatitis. International Journal of Pancreatology 26, 137–145. Hutchinson, I.V., et al., 1998. Genetic regulation of cytokine synthesis: consequences for acute and chronic allograft rejection. Graft 1, 186–192. Inoue, M., et al., 2002. The role of monocyte chemoattractant protein-1 in experimental chronic pancreatitis model induced by dibutyltin dichloride in rats. Pancreas 25, e64–e70. Ito, T., 2007. Can measurement of chemokines become useful biological and functional markers of early-stage chronic pancreatitis? Journal of Gastroenterology 42, 72–77. Jeng, J.E., et al., 2007. Tumor necrosis factor-alpha 308.2 polymorphism is associated with advanced hepatic fibrosis and higher risk for hepatocellular carcinoma. Neoplasia 9, 987–992. Kıyıcı, A., et al., 2009. Serum TNF-alpha levels in acute and chronic pancreatitis. European Journal of General Medicine 6, 103–107. Koss, K., et al., 2000. Cytokine (TNF alpha, LT alpha and IL10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies. Genes and Immunity 1, 185–190. Lahiri, D.K., Nurnberger, J.I., 1991. A rapid non-enzymatic method for the preparation of HMW DNA from blood. Nucleic Acids Research 19, 5444. Mews, P., et al., 2002. Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut 50, 535–541. Mitchell, S.A., et al., 2001. Association of the tumour necrosis factor alpha-308 but not the interleukin 10-627 promoter polymorphism with genetic susceptibility to primary sclerosing cholangitis. Gut 49, 288–294. Özhan, G., et al., 2010. Polymorphisms in tumour necrosis factor alpha (TNFα) gene in patients with acute pancreatitis. Mediators of Inflammation 2010, 1–6. Sapey, E., et al., 2010. Tumor necrosis factor-{alpha} rs361525 polymorphism is associated with increased local production and downstream inflammation in chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 15, 192–199. Saurer, L., et al., 2000. Differential expression of chemokines in normal pancreas and in chronic pancreatitis. Gastroenterology 118, 356–367. Scardapane, A., et al., 2012. TNF-α polymorphisms in juvenile idiopathic arthritis: which potential clinical implications? International Journal of Rheumatology 756291, 1–16. Schneider, A., et al., 2003. Hereditary, familial, and idiopathic chronic pancreatitis are not associated with polymorphisms in the tumor necrosis factor alpha (TNF-alpha) promoter region or the TNF receptor 1 (TNFR1) gene. Genetics in Medicine 5, 120–125. Schneider, A., et al., 2004. Analysis of tumor necrosis factor-alpha, transforming growth factor-beta 1, interleukin-10, and interferon-gamma polymorphisms in patients with alcoholic chronic pancreatitis. Alcohol 32, 19–24. Vaccaro, M.I., et al., 2000. Pancreatic acinar cells submitted to stress activate TNF-a gene expression. Biochemical and Biophysical Research Communications 268, 485–490. Vassalli, P., 1992. The pathophysiology of tumor necrosis factors. Annual Review of Immunology 10, 411–452. Whitcomb, D.C., 2004. Mechanisms of disease: advances in understanding the mechanisms leading to chronic pancreatitis. Nature Clinical Practice. Gastroenterology & Hepatology 1, 46–52. Ye, S., et al., 2001. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Research 29, E88–E. Zhang, D., et al., 2003. Association of two polymorphisms of tumor necrosis factor gene with acute severe pancreatitis. Journal of Surgical Research 112, 138–143. Zhukov, N.A., et al., 2003. Dynamics of expression of cytokines and lactoferrin in patients with chronic alcohol pancreatitis and chronic relapsing pancreatitis. Eksperimental'naia i Klinicheskaia Gastroenterologiia 5, 67–71. Zhukov, N.A., et al., 2004. Implication of cytokines and role of biliary-pancreatic reflux in mechanisms of exacerbation and chronicity of recurrent pancreatitis. Terapevticheskiĭ Arkhiv 76, 11–14.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
A single-nucleotide polymorphism in tumor necrosis factor-α (− 308 G/A) as a biomarker in chronic pancreatitis K. Sri Manjari a, A. Jyothy a, P. Shravan Kumar b, B. Prabhakar c, M. Uma Devi b, M. Ramanna b, Pratibha Nallari d, A. Venkateshwari a,⁎ a
Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, India Department of Gastroenterology, Gandhi General Hospital, Secunderabad, India Department of Gastroenterology, Osmania General Hospital, Hyderabad, India d Department of Genetics, Osmania University, Hyderabad, India b c
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 18 November 2013 Accepted 7 February 2014 Available online 18 February 2014 Keywords: Inflammation Genotypes Pancreatic fibrosis Pancreatic stellate cells
a b s t r a c t Objective: Chronic pancreatitis is a gradual, long-term inflammation of the pancreas that results in alteration of its normal structure and function. The study aims to investigate the role of −308 (G/A) polymorphism of TNF-α gene in chronic pancreatitis. Material and methods: A total of 200 subjects were included in this case–control study. A total of 100 in patients admitted in the Gastroenterology Unit of Gandhi Hospital and Osmania General Hospital, Hyderabad were included in the present study. An equal number of healthy control subjects were randomly selected for the study. The genotyping of TNF-α gene was carried out by tetra-primer ARMS PCR followed by gel electrophoresis. The TNF-α levels were assayed by enzyme-linked immunosorbent assay. Results: A significant variation with respect to the genotypic and allelic distribution in the disease group when compared to control subjects [OR = 2.001 (1.33–3.005), p b 0.0001**] was observed. Subjects homozygous for the A allele had higher TNF-α levels compared to G allele. Conclusion: The present study revealed a significant association of the TNF-α gene promoter polymorphism with chronic pancreatitis. Thus, TNF-α genotype can be considered as one of the biological markers in the etiology of chronic pancreatitis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chronic pancreatitis (CP) is an ongoing inflammatory condition in which pancreatic secretory parenchyma is destroyed and replaced by fibrous tissue, ultimately leading to malnutrition and diabetes. The prevalence of chronic pancreatitis in France is 26 per 100,000 people. This prevalence is not unsimilar to the middle of three estimates from Japan, but considerably lower than the figure of 114–200 per 100,000 in South India (Braganza et al., 2011). The main symptom of chronic pancreatitis is usually pain, identical to that of acute pancreatitis which is usually a constant and disabling pain.
Abbreviations: TNF, tumor necrosis factor; ARMS PCR, amplification refractory mutation system polymerase chain reaction; CP, chronic pancreatitis; CT scan, computed tomography scan; ERCP, endoscopic retrograde cholangiopancreatography; EDTA, ethylenediaminetetraacetic acid; DNA, deoxyribonucleic acid; OR, Odds ratio; CI, class intervals; ELISA, enzyme-linked immunosorbent assay. ⁎ Corresponding author at: Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad 500 016, India. E-mail address: [email protected] (A. Venkateshwari).
http://dx.doi.org/10.1016/j.gene.2014.02.014 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Recently, chemokines have been contemplated as essential factors in the development of chronic pancreatitis (Ito, 2007). They are elucidated as important factors that affect inflammation by increasing fibrosis which is an important feature of chronic pancreatitis. Thus, inflammatory chemokines are evidently associated with the chemo-attraction of leukocytes in early-stage chronic pancreatitis, leading to the movement of monocytes into the pancreas thereby resulting in the formation of pancreatic fibrosis (Grady et al., 1997; Inoue et al., 2002; Saurer et al., 2000). Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine with strong immune-stimulatory activity. It is involved in the inflammatory responses by stimulating the cytokine production, increasing the expression of adhesion molecules, and neutrophil activation, and it also acts as a co-stimulator for T-cell activation and antibody production by B-cells (Vassalli, 1992). TNF-α secretion is controlled primarily at the transcription level, and inter-individual differences in TNF-α secretion are associated with TNF-α promoter variant (Beranek et al., 2003). Single-nucleotide polymorphisms positioned within the gene promoters have been shown to affect transcription factor binding and hence gene expression. One of the common G/A polymorphisms has
K. Sri Manjari et al. / Gene 539 (2014) 186–189
been described at position −308 (TNF-308 G/A). The −308 polymorphism could probably affect the cell-type and stimulus specific control of TNF synthesis at the transcriptional level. A genetic predisposition to produce elevated TNF levels, due to the presence of the − 308A polymorphism, may change the course of an immune response such that an individual is more susceptible to the disease (Abraham and Kroeger, 1999). Given the local and systemic immune-stimulating effects of TNF-α, we hypothesized that promoter polymorphism of this gene may be a genetic modifying factor for chronic pancreatitis (Beranek et al., 2003). Hence, the aim of the present study was to investigate the qualitative and quantitative variation of TNF-α in the etiology of chronic pancreatitis. 2. Materials and methods 2.1. Study population The study design was a prospective case control study conducted with a total of 200 unrelated individuals. One hundred clinically evaluated chronic pancreatitis (CP) patients referred and admitted in the Gastroenterology Unit of Gandhi Hospital and Osmania General Hospital, Hyderabad between March 2008 and March 2010 were involved in the present study. The diagnosis of chronic pancreatitis was established on the findings of pancreatic duct dilation or pancreatic calcifications, or on the histological findings of chronic pancreatitis of various diagnostic tests like X-ray, CT scan, ERCP, etc. (Abraham and Kroeger, 1999). An alcoholic origin of chronic pancreatitis was assumed when the alcohol intake was more than 80 g/day in men for at least two years. For smoking status, a person who had smoked at least once a day for N 1 year in his or her lifetime was regarded as a smoker (Özhan et al., 2010). An equal number of healthy control subjects were randomly selected for the study from the individuals visiting our institute for regular health checkup. As male preponderance was observed in the patient group, more number of male controls was included in the study for better comparative analysis. Among these controls, 40% of them were found to be alcoholics at random. A structured proforma was used to seek information on dietary habits, smoking, alcohol consumption, family history, etc. Written informed consent was obtained from all the subjects, included for the study. The study was also approved by the Institutional Ethical Committee. 2.2. DNA isolation 5 ml of venous blood was drawn from each individual in vacutainers with and without EDTA for the separation of plasma and serum respectively and stored at −70 °C until use. Genomic DNA was isolated from whole blood by following the salting out procedure of Lahiri and Nurnberger (1991). 2.3. Genotyping of the TNF-α gene
2.4. Estimation of TNF-α level Concentrations of TNF-α (Invitrogen®) in plasma were measured using a commercial ELISA kit according to the manufacturer's protocols. In brief, 50 μl incubation buffer was added to different wells designated as samples and standards. 100 μl of standard or control samples was added to each well. After 2 h of incubation at room temperature on a constant shaker (500 ± 50 rpm), the reaction solution was aspirated and the wells were washed 4 times with wash buffer; 100 μl of biotinylated anti-TNF-α conjugate was then added to each well and incubated for another 1 h on the shaker at room temperature. Then the aspiration/wash steps were repeated as mentioned above, followed by adding of 100 μl of streptavidin–HRP solution to each well. The microplate was allowed to stand for 30 min at room temperature. The aspiration steps were repeated. Then stabilized chromogen (100 μl) was added to each well followed by 30 min of incubation at room temperature in the dark. The optical density of each sample was determined at 450 nm and represented in ng/ml. The TNF-α concentration for each sample was calculated from the standard curve obtained. 2.5. Statistical analysis Statistical analysis was done by using the computer software Statistical Package for Social Sciences-SPSS for windows (version 19.0) (Chicago, IL). ORs and 95% CIs were estimated by conditional logistic regression analyses based on the comparison of genotypes between patients with the disease and healthy controls, and by adjusting the potential confounders such as age, sex, smoking and alcoholism. Hardy–Weinberg equilibrium was conducted to compare the observed and expected genotype frequencies among cases and controls, respectively. A two-sided P-value b 0.05 was considered to be statistically significant. For analyses of genotype frequencies, the wild-type category was the reference group. 3. Results The study population (Table 1) consisted of 100 patients with CP with an age range of 13–70 years, as well as 100 control subjects with an age range of 20–70. CP group consisted of 95.0% males and 5.0% females whereas control group consisted of 94.0% males and 6.0% females. Patients and control subjects were derived from the same geographic location and are representative of South Indian population from Andhra Pradesh. A significant difference was observed with respect to mean age (b 0.000**) smokers (b0.000**) and alcoholics (b 0.000**) in patients compared to control subjects. Table 2 gives the genotype distribution of TNF-α (−308 G/A) gene in control subjects and patient group. The distribution of the TNF-α Table 1 Demographic and clinical parameters of the study groups (CP). Parameter
The A and G alleles at position − 308 in the promoter region of the TNF-α gene were determined using the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) methodology (Ye et al., 2001). Each PCR reaction was carried out in a total volume of 10 μl, containing 30 ng of template DNA, 10 pmol of each inner primer, 1 pmol of each outer primer, 200 μM dNTP, 2.5 mM of MgCl2, 20 mM Tris–HCl pH 8.4, 50 mM KCl and 0.5 U Taq polymerase (B. Genei). The cycling conditions were as follows: an initial denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 1 min, 61 °C for 1 min and 72 °C for 1 min. The final extension step was at 72 °C for 2 min. The amplified products were: 323 bp internal control, 224 bp G allele and 154 bp A allele which were separated by electrophoresis on a 2% agarose gel stained with ethidium bromide. The gel was visualized under ultraviolet light with a 100-base pair ladder.
187
Controls
CP
N
%
N
%
Gender Males Females
94 6
94 6
95 5
95 5
0.82 (0.22–2.92)
0.378⁎
Age b40 years ≥40 years
78 22
78 22
52 48
52 48
3.25 (1.77–6.10)
b0.000⁎⁎
Addictions Smokers Non-smokers Alcoholics Non-alcoholics
22 78 40 60
22 78 40 60
49 51 76 24
49 51 76 24
0.29 (0.15–0.56)
b0.000⁎⁎
0.21 (0.11–0.38)
b0.000⁎⁎
⁎ p b 0.05. ⁎⁎ p b 0.0001.
OD (CI)
p value
188
K. Sri Manjari et al. / Gene 539 (2014) 186–189
Table 2 Genotypic and allelic frequencies of TNF-α genotypes between controls and CP. Study group
Controls CP
G/G
G/A
A/A
Total
N
(%)
n
(%)
N
(%)
50 35
(50) (35)
36 33
(36) (33)
14 32
(14) (32)
Allelic frequencies
100 100
p
q
0.68 (136) 0.52 (103)
0.32 (64) 0.49 (97)
[OD = 2.001 (1.33–3.005), p b 0.0001**].
(− 308 G/A) genotypes in controls was in Hardy–Weinberg equilibrium. Among the controls the genotype distribution was: 50% (G/G), 36% (A/G), and 14% (A/A), whereas the genotypes in chronic pancreatitis patients: 35% (G/G), 33% (A/G) and 32% (A/A) were not in Hardy– Weinberg equilibrium. A significant variation was observed in the distribution of A allele in the patient group (49%) compared to control subjects (32%) [Odds ratio = 2.001 (1.33–3.005), p b 0.0001**]. *p b 0.05, **p b 0.0001. Comparison of genotypes was made with regard to the smoking habit and alcoholism. There was an increased frequency of A allele in smokers compared to control subjects, indicating an association between TNF-α (A-308 GA + AA) containing genotypes and smoking (OR = 0.305, 95% CI (0.094–0.976), p = 0.035). However, there was no significant association between TNF and alcoholism (OR = 0.648, 95% CI (0.270–1.555), p = 0.648). Further patients were subgrouped into smokers and non-smokers among alcoholic and non-alcoholics to test for the association of TNF A allele by multivariate regression analysis. We did not find any significant result with smoking as a modifying factor. Comparison of mean levels of TNF-α with respect to the genotypes (Table 3) revealed elevated levels in the disease compared to control subjects. However, a significant increase was observed in CP with GG and AA genotype (111.29 ± 57.94) compared to control subjects (80.35 ± 43.34) (p b 0.0001**). There was no significant difference in the mean levels of TNF-α with respect to smoking and alcoholism. Further, comparison was also made within the disease group in different clinical manifestations like steatorrhoea (13%), calcification (31%), atrophy (10%), diabetes (7%) and we did not found any association between the A allele and subgroups. A number of groups have carried out investigations into the functional significance of the TNF-α (− 308 G/A) polymorphism in acute and chronic pancreatitis. We have considered only CP cases for the present study and a total of six publications were reported on the genotype frequency of TNF-α (− 308 G/A) in chronic pancreatitis and controls (Table 4). Data from each publication included the following details: year of publication, first author's name, country in which the study was conducted, study design, ethnicity, sample size of CP patients, controls and the association of the genotype. Our study is in agreement with the study of Abdulrazeg et al. (2001), wherein higher expressive allele A is associated with the disease. However, other studies showed no such association with the disease indicating the variation in the distribution of genotypes in different ethnic groups. 4. Discussion Chronic pancreatitis, an irreversible inflammatory disease of the pancreas, is marked by chronic inflammatory cell infiltration, acinar Table 3 Mean serum levels (in ng/ml) of TNF-α in chronic pancreatitis patients and control subjects with respect to the genotypes.*p b 0.05, **p b0.0001. TNF-α
Patients (N)
Genotype: G/G 35 G/A 33 A/A 32 Total 100
Mean ± SD
Controls (N)
Mean ± SD
t-Value
p-Value
77.00 98.30 162.19 111.29
50 36 14 100
52.60 89.72 155.36 80.35
3.5086 1.0656 0.4246 4.2761
0.0007* 0.2904 0.6732 0.000**
± ± ± ±
42.83 38.75 54.58 57.94
± ± ± ±
20.34 27.64 37.75 43.34
cell degeneration, and development of fibrosis, which lead to impairment of pancreatic exocrine and endocrine function (Ito, 2007). Irrespective of the etiologies of pancreatic injury, pancreatic fibrosis is caused commonly by a chronic and uncontrolled inflammatory/repair process leading to excessive deposition of collagen and other components of extracellular matrix (Whitcomb, 2004). Abnormal deposition of fibrous tissue is a characteristic histological feature of chronic pancreatitis, a progressive necro-inflammatory condition of the pancreas that often results in exocrine and endocrine insufficiency (Apte et al., 2011). Tumor necrosis factor-α is a multifunctional pro-inflammatory cytokine implicated in modulating the progression of chronic pancreatitis by inducing pancreatic stellate cells activation as indicated by increased proliferation, α-SMA expression, and/or collagen synthesis leading to fibrosis (Berberat et al., 2000). Hutchinson et al. (1998) demonstrated that individuals differ in their capacity to synthesize various cytokines and attributed such differences to the existence of allelic polymorphism in the promoter region of cytokines. The transcription regulatory region of TNF-α (− 308 G/A) polymorphism has been associated with differential expression and different serum concentrations of TNF-α. The recent finding of an increased gene expression of TNF-α and its receptors in blood leucocytes of patients with late stage chronic alcoholic pancreatitis implies a pathogenic importance of TNF-α in the clinical features of chronic pancreatitis (Beranek et al., 2003; Hanck et al., 1999). In addition to several autoimmune diseases, studies have shown a close association of the TNF-308A allele to primary sclerosing cholangitis (Mitchell et al., 2001), alcoholic steato-hepatitis (Grove et al., 1997), extended ulcerative colitis (Koss et al., 2000) and coeliac disease (de la Concha et al., 2000). Studies have highlighted that small difference at cytokine levels as a result of genetic variants may have an important effect on inflammatory response and may influence the pathophysiology. The less common −308A allele is strongly associated with the MHC haplotype HLA-A1-B8 and DR3, which is in turn associated with high TNF-α production and autoimmune disease. This genetic propensity to produce elevated TNF-α levels, due to the presence of the −308A polymorphism, may alter the course of an immune response (Scardapane et al., 2012). TNF-alpha polymorphism is associated with clinical features of chronic obstructive pulmonary disease including progression. There is clear evidence of TNF-alpha overexpression and bioactivity with neutrophilic inflammation (Sapey et al., 2010). SNP located at nucleotides − 308 have been extensively researched on the susceptibility to a range of autoimmune disorders, including rheumatoid arthritis, exfoliation glaucoma, and many kinds of cancers. Carriage of the TNF308 G allele correlates with disease severity and hepatic fibrosis, which may contribute to a higher risk for HCC (Jeng et al., 2007). The present study was undertaken to investigate the association of TNF-α −308 allelic polymorphism and CP as no such data is available for the Indian population. In the present study we found a positive association between TNF-α (−308 G/A) promoter polymorphism and CP. The polymorphic studies of TNF-α on various ethnic groups have yielded conflicting results in association with CP. The preliminary report by Abdulrazeg et al. (2001) supported our suggestion that the TNF-α (−308 G/A) promoter polymorphism may be associated with chronic pancreatitis. However, other studies from Beranek et al. (2003), Schneider et al. (2003, 2004), Bendicho et al. (2005) and Farkas et al. (2007) showed no association between this polymorphism and CP. Zhang et al. (2003) did not find any correlation between the polymorphism on position − 308 of the TNF-α gene and acute pancreatitis. The contradictory outcomes of various groups indicate a complex association between TNF-α (−308 G/A) polymorphism, CP and ethnicity. We did find a significant association of (GA + AA) genotype with smoking but not with alcoholism. We did not find any association of TNF-α (−308 G/A) polymorphism in different clinical manifestations of chronic pancreatitis which is in confirmation with Schneider et al. (2003).
K. Sri Manjari et al. / Gene 539 (2014) 186–189
189
Table 4 Characteristics of earlier publications on TNF-α-308 polymorphism and chronic pancreatitis susceptibility. Reference
Country
Ethnicity
Source of cases
Source of controls
Association of TNF-α
Abdulrazeg et al. (2001) Beranek et al. (2003) Schneider et al. (2003) Schneider et al. (2004) Bendicho et al. (2005) Farkas et al. (2007) Present study
United Kingdom Germany USA USA Brazil Hungary India
Caucasian German Caucasian Caucasian Caucasian Hungarian Indian
University study (64) Hospital patients (335) University study (54) Disease center (42) Hospital outpatient clinic (28) Hospital patients (83) Hospital patients
Healthy controls (250) Healthy blood donors (116) and (25) healthy carriers Random individuals (94) Random individuals (94) Blood donors (94) Healthy blood donors (75) Healthy controls
Yes No No No No No Yes
Thus, in the present study the positive association between TNF-α (−308 G/A) promoter polymorphism and CP may be due to ethnic difference in the population. Mews et al. (2002) showed that the quiescent stellate pancreatic cells, observed on normal pancreas, could be activated by cytokines such as TNF-α, IL-1, IL-6, and IL-10 and suggested that the persistent activation of these cells could be a factor involved in the progression of CP. Vaccaro et al. (2000) verified that the TNF-α expression on acinar pancreas cells is not typical but occurs at the beginning of pancreatitis. In most of the studies performed on chronic pancreatitis, higher TNF-α levels were observed which is correlated to the exacerbation severity (Kıyıcı et al., 2009; Zhukov et al., 2003, 2004). Thus, our findings agree with the previous studies that higher level of TNF-α leads to the activation of the pancreatic stellate cells which are the main source for increased deposition and disorganization of extracellular matrix proteins resulting in the fibrosis and chronic inflammation of the pancreas. Further, our results suggest that a single dose of A allele may contribute two fold increased risk than GG genotype to the disease. Thus excess production of TNF-α seems to be a typical feature of CP. The inflammatory response is dependent on the level of TNF-α which the pancreas is exposed to and may result in increased pancreatic stellate cell proliferation. Therefore, polymorphic variation of the TNF-α gene leading to variable TNF-α levels may influence TNF-α-dependent inflammation and damage of the pancreas in CP. In conclusion, the present study focuses the role of TNF-α gene promoter polymorphism and their corresponding levels in chronic pancreatitis and indicates it as one of the biomarkers in the etiology of the disease. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgment The authors acknowledge the financial support from the University Grants Commission (UGC), New Delhi. Authors are also thankful to the Department of Biotechnology, New Delhi for the financial assistance. References Abdulrazeg, E.M., et al., 2001. TNF-alpha promoter region gene polymorphisms in patients with alcohol-induced chronic pancreatitis. Gastroenterology 120, A32. Abraham, L.J., Kroeger, K.M., 1999. Impact of the −308 TNF promoter polymorphism on the transcriptional regulation of the TNF gene: relevance to disease. Journal of Leukocyte Biology 66, 562–566. Apte, M., et al., 2011. The fibrosis of chronic pancreatitis: new insights into the role of pancreatic stellate cells. Antioxidants and Redox Signaling 15, 2711–2722. Bendicho, M.T., et al., 2005. Polymorphism of cytokine genes (TGF-beta1, IFN-gamma, IL-6, IL-10, and TNF-alpha) in patients with chronic pancreatitis. Pancreas 30, 333–336. Beranek, H., et al., 2003. Analysis of tumour necrosis factor alpha and interleukin 10 promotor variants in patients with chronic pancreatitis. European Journal of Gastroenterology and Hepatology 15, 1223–1227. Berberat, P.O., et al., 2000. Chronic pancreatitis—new pathophysiological concepts. Swiss Surgery 6, 227–230. Braganza, J.M., et al., 2011. Chronic pancreatitis. Lancet 377, 1184–1197.
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