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Annals of Oncology Advance Access published online on March 27, 2008
Annals of Oncology, doi:10.1093/annonc/mdn055
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© The Author 2008. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Analysis of Pro12Ala PPAR gamma polymorphism and Helicobacter pylori infection in gastric adenocarcinoma and peptic ulcer disease

K. N. Prasad1,*, A. Saxena1, U. C. Ghoshal2, M. R. Bhagat4 and N. Krishnani3

1 Department of Microbiology
2 Department of Gastroenterology
3 Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow
4 Department of Gastroenterology, Central Command Hospital, Lucknow, India

* Correspondence to: Dr K. N. Prasad, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, India. Tel: +91-522-2668631; Fax: +91-522-2668017; E-mail: knprasad{at}sgpgi.ac.in


   Abstract
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 Abstract
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 materials and methods
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Background: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) is a ligand-dependent transcription factor involved in various disease processes including inflammation and carcinogenesis. We investigated the association of Pro12Ala PPAR{gamma} polymorphism and Helicobacter pylori infection with gastric cancer and peptic ulcer disease (PUD).

Patients and methods: In total, 348 adult patients [62 gastric adenocarcinoma, 45 PUD and 241 nonulcer dyspepsia (NUD)] who underwent an upper gastrointestinal endoscopy were enrolled. PPAR{gamma} polymorphism was analyzed by PCR-based restriction fragment length polymorphism. H. pylori infection was diagnosed by rapid urease test, culture, histopathology and PCR.

Results: PPAR{gamma} G carrier had significant association with gastric adenocarcinoma [P = 0.023, odds ratio (OR) = 2.136, 95% CI = 1.112–4.104] and PUD (P = 0.028, OR = 2.165, 95% CI = 1.008–4.306) when compared with NUD. Combination of G carrier and H. pylori infection further increased the risk of gastric adenocarcinoma (OR = 3.054, 95% CI = 1.195–7.807) and PUD (OR = 11.161, 95% CI = 3.495–35.644). PPAR{gamma} polymorphism did not increase the risk of gastric adenocarcinoma and PUD in H. pylori-negative subjects.

Conclusions: The study suggests that Pro12Ala PPAR{gamma} polymorphism is associated with gastric adenocarcinoma and PUD, and is a potential marker for genetic susceptibility to these two diseases in the presence of H. pylori infection.

gastric adenocarcinoma, Helicobacter pylori infection, peptic ulcer disease, PPAR{gamma} polymorphism


   introduction
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 introduction
materials and methods
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 discussion
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Helicobacter pylori has been classified as a major cause of peptic ulcer disease (PUD) and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma [1–3]. On a global scale, gastric cancer is the second commonest cancer in the world. There is a substantial international variation in gastric cancer incidence with the highest rates reported from China, Japan and other Eastern Asian countries. Epidemiological studies have proved that H. pylori infection is considered as risk factor for gastric cancer and World Health Organization International Agency for Research on Cancer has classified this bacterium as a definite carcinogen [2]. However, while the majority of the infected individuals develop no significant clinical disease, others develop two kinds of divergent clinical outcomes—PUD and gastric cancer [4]. The reasons for developing these two extreme phenotypes remain poorly understood and are not explained by bacterial virulence factors alone [4, 5]. This highlights the need to explore potential candidate genes of the host involved in the H. pylori-associated gastric carcinogenesis.

Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) is a member in the nuclear receptor superfamily that regulates adipocyte differentiation, fatty acid uptake and storage [6]. Human PPAR{gamma} expression was first described in hematopoietic cells [7] and later also in spleen, liver, testis, skeletal muscle and brain [8]. PPAR{gamma} exists as two isoforms, {gamma}1 and {gamma}2, generated by alternative promoters and differential splicing of at least three different transcripts from the PPAR{gamma} gene on chromosome 3p25. PPAR{gamma}2 is the most important isoform in adipose tissue, where it is almost exclusively expressed [8, 9]. Several polymorphisms in PPAR{gamma}2 have been identified so far. Only a few, however, seemed to have a functional effect on the transcription. Recently, Yen et al. [10] reported a common C > G polymorphism located in codon 12 of exon B of the PPAR{gamma} gene, which codes the aminoterminal polypeptide that defines the PPAR{gamma}2 isoform, results in a Pro12Ala substitution. With the strong effects of PPAR{gamma} on the growth of cancer cells, the role of Pro12Ala polymorphism has been recently studied in cancer [6]. Individuals with the Ala12 allele were found to have a reduced risk of colorectal cancer [11, 12]. A similar observation, however, could not be established in prostate cancer [13], endometrial cancer [14], sporadic colorectal adenoma [15] and gastric cancer [16, 17]. Little information is available regarding interactions of PPAR{gamma} polymorphism and risk for gastric cancer. The aim of the present study was to determine the role of Pro12Ala PPAR{gamma} polymorphism and H. pylori infection in patients with gastric adenocarcinoma and PUD.


   materials and methods
Top
 Abstract
 introduction
 materials and methods
results
 discussion
 funding
 References
 
study population
We enrolled 348 adult patients [62 gastric adenocarcinoma, 45 PUD and 241 nonulcer dyspepsia (NUD)] who underwent upper gastrointestinal endoscopy at two tertiary referral centers in northern India between September 2002 and May 2007. The diagnosis of gastroduodenal diseases was on the basis of clinical, endoscopical and histopathological examinations. Patients with NUD were considered as controls in our study. The ethics committee of the institute granted approval for the study and the consent was obtained from all the patients. Subjects who had received antimicrobial therapy, H2 receptor blockers, proton pump inhibitors and nonsteroidal anti-inflammatory drugs in the preceeding 30 days before endoscopy or anti-H. pylori treatment in the past were excluded from this study.

detection of H. pylori infection
During each endoscopy, antral biopsies were obtained and subjected to the following tests: rapid urease test, culture, histopathology and H. pylori-specific ureA PCR following the standard protocol as described earlier [18].

Pro12Ala PPAR{gamma} polymorphism
Genomic DNA was isolated from gastric tissues using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as per the manufacturer's instruction. The analysis of Pro12Ala PPAR{gamma} polymorphism was determined by PCR-based restriction fragment length polymorphism (PCR-RFLP) as previously described [17]. The sequences of PCR primers were as follows: forward 5'-GCC AAT TCA AGC CCA GTC-3' and reverse 5'-GAT ATG TTT GCA GAC AGT GTA TCA GTG AAG GAA TCG CTT TCC G -3' (Metabion, Martinsried, Deutschland). PCR conditions were as follows: an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 51°C for 40 s and extension at 72°C for 40 s. The final extension was continued at 72°C for 10 min and cooling to 4°C. Template-free water was used as negative control. After amplification, the purified PCR products were subjected to restriction digestion by Bst UI restriction endonuclease (Fermentas, Vilnius, Lithuania) for 8 h at 60°C. The DNA fragments were then separated on 2% agarose gel electrophoresis. Fragment sizes of 227 and 43 bp indicated the presence of a homozygous GG genotype (Ala12Ala), a single 270-bp fragment indicated the presence of wild-type homozygous CC genotype (Pro12Pro) and three fragments of 270, 227 and 43 bp indicated the presence of heterozygous CG genotype (Pro12Ala) (Figure 1). All the experiments were repeated twice for the confirmation of RFLP results.


Figure 1
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  		Figure 1. PCR-based restriction fragment length polymorphism analysis of Pro12Ala peroxisome proliferator-activated receptor gamma polymorphism; lane 1: 100 bp DNA ladder, lane 2: PCR product (undigested), lane 3: negative control, lanes 4–6: homozygous GG genotype, lanes 7–9: heterozygous CG genotype, lanes 10–12: homozygous CC genotype.

 
statistical analysis
The data analysis was carried out by SPSS software (version 12.0, SPSS, Chicago, IL). The association of H. pylori status in relation to gastroduodenal diseases was carried out by the chi-square test. Multivariate logistic regression analyses were used to identify the independent risk factors for gastric adenocarcinoma and PUD. Gender and age were included in regression analysis, and assessment for interaction was considered in the model. A two-sided P value <0.05 was considered significant.


   results
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 materials and methods
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discussion
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patient characteristics
A total of 348 patients (mean age 46.78 ± 15.96 years; 216 males) were enrolled in the study and their distributions were as follows: gastric adenocarcinoma 62 (mean age 56.60 ± 15.42 years; 47 males), PUD 45 (mean age 49.47 ± 17.22 years; 31 males) and NUD 241 (mean age 43.75 ± 14.76 years; 138 males) (Table 1).


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  		Table 1. Demography of the study populations and Helicobacter pylori infection

 
detection of H. pylori infection
Prevalence of H. pylori infection in our study population was 58.6%. H. pylori infection was significantly higher in patients with PUD than with gastric adenocarcinoma (80% versus 56.5%, P = 0.01) and NUD (80% versus 55.2%, P = 0.002) (Table 1).

Pro12Ala PPAR{gamma} polymorphism
Genotypic distributions of patients with gastric adenocarcinoma, PUD and NUD were in Hardy–Weinberg equilibrium. The potential association of Pro12Ala PPAR{gamma} polymorphism in patients with gastroduodenal diseases is shown in Table 2. Presence of G carrier was almost similar in patients with gastric adenocarcinoma and PUD. The frequency of G carrier was significantly higher in patients with gastric adenocarcinoma [37.1% versus 22.8%, P = 0.023, odds ratio (OR) = 2.14, 95% CI = 1.11–4.10] and PUD (37.8% versus 22.8%, P = 0.028, OR = 2.17, 95% CI = 1.09–4.31) when compared with NUD.


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  		Table 2. Allelic distribution of Pro12Ala peroxisome proliferator-activated receptor gamma (PPAR{gamma}) polymorphism in gastric adenocarcinoma, peptic ulcer disease (PUD) and nonulcer dyspepsia (NUD)

 
interaction between H. pylori infection and Pro12Ala PPAR{gamma} polymorphism
We also examined the potential interaction between H. pylori infection and Pro12Ala PPAR{gamma} polymorphism for the development of gastric adenocarcinoma and PUD in our population. Presence of G carrier with H. pylori infection significantly increased the risk for the development of gastric adenocarcinoma (P = 0.020; OR = 3.05, 95% CI = 1.21–7.81) and PUD (P < 0.001; OR = 11.16, 95% CI = 3.49–35.64). Interestingly, the risk was not increased in H. pylori-negative individuals with gastric cancer (P = 0.682; OR = 1.23, 95% CI = 0.46–3.32) (Table 3) and PUD (P = 0.944; OR = 1.06, 95% CI = 0.19–5.86) (Table 4).


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  		Table 3. Helicobacter pylori (HP) infection and Pro12Ala peroxisome proliferator-activated receptor gamma (PPAR{gamma}) polymorphism as risk for gastric adenocarcinoma

 

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  		Table 4. Helicobacter pylori (HP) infection and Pro12Ala peroxisome proliferator-activated receptor gamma (PPAR{gamma}) polymorphism as risk for peptic ulcer disease (PUD)

 

   discussion
Top
 Abstract
 introduction
 materials and methods
 results
 discussion
funding
 References
 
We investigated the potential association of Pro12Ala PPAR{gamma} polymorphism and H. pylori infection with gastric adenocarcinoma and PUD. We report that Pro12Ala PPAR{gamma} polymorphism in the presence of H. pylori infection could be a marker for genetic susceptibility to gastric adenocarcinoma and PUD. PPARs represent a family of nuclear receptors that are closely related to thyroid or retinoid receptors. Three PPAR subtypes (PPAR{alpha}, PPAR{delta} and PPAR{gamma}) have been identified so far [19]. PPAR{gamma}, however, is the most extensively studied of the three PPAR subtypes to date. Upon ligand binding, PPAR activates the transcription of many PPAR-responsive genes involved in the regulation of adipocyte differentiation, the enhancement of target tissues to insulin, inflammation, carcinogenesis and cell cycle control [19]. PPAR{gamma} was expressed in both cancer and normal gastric epithelium; activation of PPAR{gamma} inhibits the growth and induces apoptosis of gastric cancer cells [20]. Lu et al. [21] showed that treatment with the PPAR{gamma} ligand troglitazone substantially reduced the development of gastric cancer in mice induced by carcinogens. More interestingly, the chemopreventive effect of troglitazone was absent in PPAR{gamma} heterozygous-deficient (+/–) mice; it suggests the importance of functional PPAR{gamma} receptors in preventing gastric cancer development. Presence of the Ala12 polymorphism, which is associated with reduced PPAR{gamma} activity, may increase the risk of gastric adenocarcinoma [22, 23]. PPAR{gamma} may modulate the inflammatory response of the host cells associated with chronic H. pylori infection. Activation of the PPAR{gamma} pathway attenuates the ability of H. pylori to induce NF-kappa B-mediated apoptosis in gastric epithelial cells [24]. It may be speculated that H. pylori infection and PPAR{gamma} polymorphism further enhance the resistance to apoptosis which ultimately results in cellular proliferation [17]. In our study, G carriers (Ala12) were frequently present in gastric adenocarcinoma when compared with controls (Table 3). We found that patients with G carriers had 2.14-fold (95% CI = 1.11–4.10) increased risk of progression to gastric adenocarcinoma. Tahara et al. [16] described a 2.4-fold increased risk gastric adenocarcinoma due to Ala12 polymorphism. Liao et al. [17] reported nearly 2.5-fold increased risk of progression to gastric adenocarcinoma. It appears that the risk for the development of gastric adenocarcinoma related to Pro12Ala PPAR{gamma} polymorphism in our population is similar with the other published studies. When we analyzed the combination of G carrier and H. pylori infection in patients with gastric adenocarcinoma, the risk was further increased (P = 0.020, OR = 3.05, 95% CI = 1.21–7.81). The risk, however, was not increased in H. pylori-negative individuals (P = 0.682, OR = 1.23, 95% CI = 1.21–7.81). The present study clearly shows that G carriers in the presence of H. pylori infection are susceptible to develop gastric adenocarcinoma. So far, only one study from China had analyzed the association between H. pylori infection and Pro12Ala PPAR{gamma} polymorphism in the development of gastric adenocarcinoma and reported 12.8-fold risk [17]. The increased risk in Chinese study may be related to high prevalence of H. pylori infection in gastric adenocarcinoma (81.7%) as compared with our patients (56.5%). The reason for such discrepancy in H. pylori infection between Chinese and present studies may be because of different methods used for detection of H. pylori infection. The presence of anti-H. pylori immunoglobulin G was the diagnostic criteria in Chinese study, while in the present study H. pylori infection was diagnosed on the basis of gastric tissue, which is considered more specific.

We also analyzed the association of Pro12Ala PPAR{gamma} polymorphism and H. pylori infection with PUD. We found that patients with G carriers had increased risk for PUD (P = 0.028, OR = 2.17, 95% CI = 1.09–4.31). When we analyzed the combination of G carrier and H. pylori infection, the risk increased to 11.16-fold in H. pylori-positive individuals (P < 0.001, OR = 11.16, 95% CI = 3.49–35.64); however, the risk did not increase in H. pylori-negative individuals (P = 0.944, OR = 1.06, 95% CI = 0.19–5.86) (Table 4). There are no data available in literature to compare our observations with. To the best of our knowledge, this is the first study to show that patients with G carriers of Pro12Ala PPAR{gamma} polymorphism are susceptible to develop PUD in the presence of H. pylori infection.

In conclusion, the study suggests that Pro12Ala PPAR{gamma} polymorphism could be a potential marker for genetic susceptibility to gastric adenocarcinoma in the presence of H. pylori infection. We also report for the first time, patients with H. pylori infection and Pro12Ala PPAR{gamma} polymorphism have increased risk to develop PUD. Thus, Pro12Ala PPAR{gamma} polymorphism might be useful in predicting the risk of gastric adenocarcinoma and PUD in the presence of H. pylori infection. Further studies on different ethnic groups are needed to confirm the association of this polymorphism with the risk of gastric adenocarcinoma and PUD.


   funding
Top
 Abstract
 introduction
 materials and methods
 results
 discussion
 funding
References
 
Council of Science and Technology, Government of Uttar Pradesh, India (CST/SERPD/D-3402); Indian Council of Medical Research, New Delhi, India (80/512/2004-ECD-I to A.S.).

Received for publication January 4, 2008. Revision received February 3, 2008. Accepted for publication February 4, 2008.


   References
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 Abstract
 introduction
 materials and methods
 results
 discussion
 funding
 References
 
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9. Farmer S. Transcriptional control of adipogenesis: interplay between C/EBPs and PPAR{gamma} in regulating adipocyte gene expression. In: Progress in Obesity Research—Medeiros-Neto G, ed. (2003) Paris: John Libbey Eurotext. 15–18.

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