Polymorphism at position T-129C in the promoter region of ABCB1 gene in peptic ulcer patients: a case control study

Background Peptic ulcer disease is one of the most common diseases worldwide. P-glycoprotein encoded by ABCB1 gene is located on the surface of gastric tract cells. Changes in its protein function, as a consequence of polymorphism like T-129C (rs3213619), may have impact the development of some diseases. The aim of this study was to evaluate the potential role of T-129C of ABCB1 gene in peptic ulcer development. To the best of our knowledge, this is the first this kind of research in peptic ulcer diseases. Methods Patients, having attended gastroduodenoscopy because of dyspepsia symptoms and diagnosed at that time as peptic ulcer patients, were enrolled in the study. In all investigated cases, gastric mucosa specimens were taken from the antrum of the stomach. The control group consisted of healthy blood donors from local blood donation center. Overall DNA from 204 patients with peptic ulcer disease and from 94 healthy individuals was tested. The RFLP PCR technique was used for genotype analysis. Results All 204 DNA samples form investigated group and 94 DNA samples form healthy individuals were successfully analyzed. All genotypes for polymorphism in position T-129C of the ABCB1 gene in both groups were in agreement with Hardy-Weinberg equation. Statistical analysis showed no significant differences in frequency of T-129C genotypes between peptic ulcer patients and healthy individuals (p=0.3977). Also, there were no statistically significant differences in the frequency of occurrence of T-129C genotypes between patients infected and uninfected with H. pylori infection (p=0.1743). Conclusion Investigated polymorphism at position T-129C of ABCB1 gene is not associated with H. pylori infection nor with the risk of peptic ulcer disease development.


Introduction
The increasing number of investigators indicated that genetic factors regulating transport 3 of xenobiotics and inflammation factors production play a major role in the pathogenic process of H. pylori infection and, as a consequence, in peptic ulcer disease (PUD) and gastric cancer (GC) development. PUD is a common disease worldwide with a lifetime prevalence in the adult population around 5% to 10% over the past decade. Strong association of the disease with a Helicobacter pylori infection is widely recognized, but non-steroidal anti-inflammatory drugs or low-dose aspirin usage are also increasingly important causes of PUD. Eradication of this bacterium improves PUD recovery and is a prophylaxis to reduce the risk of gastric cancer. Antibiotics used in the scheme of eradication therapy are substrates for transporters belonging to ABC family [1][2].
Many polymorphisms of genes responsible in physiology for cell protection are currently being investigated to establish the risk factors of PUD development and also to check their role in the effectiveness of the therapy.
In this work, we conducted an analysis of the T-129C polymorphism of ABCB1 in the group of patients with peptic ulcer disease. ABCB1 (MDR1) encodes a drug transporter protein (P-glycoprotein; P-gp) expressed in many tissues, particularly on the surface of digestive tract cells, blood-brain and blood-testis barriers where it plays a protective role.
Polymorphisms of that gene could also lead to the reduction of the protective function of P-glycoprotein and, as a consequence, to the promotion of xenobiotic accumulation and/or inflammation [3,4]. Especially those in the promoter region of the ABCB1 gene, like single nucleotide polymorphism (SNP) in T-129C position which leads to the exchange of thymine into cytosine in 129 position of the promoter of this gene [5]. On the other hand, many groups of drugs including protein pomp inhibitors (PPIs) are substrates of P glycoprotein, which is a product of ABCB1 gene. Genetic polymorphisms of this gene alter pharmacokinetics of these substrates and may be associated with the drug resistance in the treatment of various diseases e.g. PUD. 4 The aim of this study was to determine the potential significance of SNP T-129C of ABCB1 gene (rs3213619) in the development of peptic ulcer in Polish population. According to our state of knowledge, this is the first study of this type among Polish population with a peptic ulcer disease.

Material And Methods
The presence of polymorphism in the promoter region of the ABCB1 gene in position -129 was evaluated in 204 unrelated patients (131 women, 73 men). The same cohort was investigated in one of our earlier study [6]. The mean age in the study group was 53 years of age. Patients were enrolled in the study, having attended the Department of Surgery, District Hospital, Łęczyca, Poland, for gastroduodenoscopy because of dyspepsia symptoms and diagnosed at that time as peptic ulcer patients. In all investigated cases, gastric mucosa specimen were taken from the antrum of the stomach. The control group consisted of 94 blood samples collected from healthy blood donors, from the local blood donation center (56 women and 38 men, mean age 33 years of age), geographically and ethnically matched to the patients. Data concerning exposure to carcinogens in patients and controls were not available. Patients who were treated with non-steroidal antiinflammatory drugs were excluded. The investigation was carried out in accordance with the principles of the Declaration of Helsinki and was approved by the Ethical Committee of the Medical University of Lodz (RNN/195/13/KE). All subjects included in the study gave their informed consent.

Rapid urease test
To diagnose a Helicobacter pylori infection, a rapid urease test was used (Institute of Food and Nutrition, Poland). H. pylori synthesizes the enzyme urease, which catalyzes the conversion of urea to ammonium ions and carbon dioxide, which leads to a change in the color of the rapid urease test medium from negative (yellow) to positive (red). This 5 infection was detected in half of the examined patients. Based on rapid urease test results, the severity of bacterial colonization was graded as: +, ++ and +++. In this study group mild H. pylori intensity (+) was observed in 54, moderate (++) in 26 and severe (+++) in 22 cases.

DNA isolation
DNA was isolated according to the "Genomic DNA Prep Plus" protocol (A&A Biotechnology, Poland) from material biopsy specimen of gastric membrane mucosa. The purity and concentration of DNA samples were estimated nanospectrophotometrically. The samples were stored at −20°C until analysis.

PCR-RFLP
PCR reaction for both the investigated and the control group were performed according to Firstly, frequencies of genotypes for studied SNP between peptic ulcer patients and healthy individuals were compared. No statistically significant differences were found (p=0.3977). The prevalence of TT genotype dominated in peptic ulcer patients and in healthy individuals cohort (97.55% and 95.74%, respectively). All results are shown in Table 1.
Secondly, the peptic ulcer patients were divided into patients under and equal 55 years old (median age of the group) and patients over 55 years old (median age of the group), and frequencies of SNP T-129C genotypes were compared. However, no statistical significance was found (p=0.5572).

Discussion
Glycoprotein P, encoded by ABCB1 is found on the surface of the digestive tract cellswhere it probably plays a protective function by eliminating xenobiotics from the cells to the extracellular environment [3,4]. ABCB1 gene is highly polymorphic. Since now over 50 SNPs of this gene has been described. It has been shown that several of these may be linked with the functioning of P-glycoprotein and are increasingly looking for a correlation between the occurrence of polymorphisms and predisposition to various diseases [3].
The distribution of specific genotypes occurrence frequency for the studied polymorphism obtained in this work was similar to that in the following populations: Polish, French, German, Italian, and Iranian [7,8]. However, it was different than in Japanese population where the presence of the CC genotype was demonstrated [9].
The aim of this study was to assess the potential impact of polymorphism in position T- that T-129C polymorphism T allele is associated with increased P-gp activity and thus faster removal of toxic xenobiotics and protection function of this protein [5].

Conclusions
In Written informed consent was obtained from the patients prior to their participation in the research.

Not applicable
Availability of data and material: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.