Helicobacter pylori cagA gene Polymorphism in Patients with Gastroduodenal Diseases

Helicobacter pylori is a genetically diverse bacterial pathogen and its CagA gene is a major virulence factor that plays an important role in gastroduodenal pathologies. The biological function of cagA depends on tyrosine phosphorylation within the EPIYA (Glutamate-Proline-Isoleucine-TyrosineAlanine) motifs at the C-terminal region of the protein. This region may undergo polymorphism to give different types of EPIYA motifs. EPIYA motif diversity may provide a useful tool for prediction of H. pylori pathogenic activity and accurate determination of number and type of cagA EPIYA motifs could identify the virulent H. pylori. The aim of this study was to detect H. pylori cagA gene and its polymorphism in endoscopic gastroduodenal biopsy specimen from patients with gastroduodenal diseases in Bangladesh. This cross sectional study was carried out in the Department of Microbiology & Immunology, Bangabandhu Sheikh Mujib Medical University and Center for Advanced Research in Sciences, University of Dhaka during the period from March 2014 to February 2015. Gastric biopsies were collected from 78 patients with gastritis, duodenal ulcer, gastric ulcer and gastric carcinoma. H. pylori was identified by rapid urease test and ureC gene PCR. Presence of cagA gene and number and pattern of cagA EPIYA motif were determined by PCR. DNA sequencing was carried out to confirm the PCR detection method of cagA EPIYA motif and to analyse their peptide sequence. Among 31(39.7%) H. pylori positive cases, 19 (61.3%) were cagA gene positive in 11(55%) gastritis, 4(66.7%) duodenal ulcer, 2(66.7%) gastric ulcer and 2(100%) gastric carcinoma. A significant association was found between cagA gene and duodenal ulcer (p=˂0.05). EPIYA motif of all H. pylori cagA positive cases showed Western type cagA EPIYA ABC. No East Asian EPIYA ABD motif was found. Majority of gastroduodenal cases (57.9%) had 3 copies of EPIYA (ABC type), 26.3% had 4 copies (ABCC type) while remaining 10.5% had AC and 5.2% AB type EPIYA motif. EPIYA ABC was found in 75% of duodenal ulcer followed by 54.5% of gastritis and 50% of both gastric ulcer and gastric carcinoma patients. EPIYA ABCC motif was found in 50% of gastric ulcer and gastric carcinoma patients. Most of the EPIYA motif was EPIYA ABC and some were ABCC which has the risk of developing gastric carcinoma.


Introduction
Helicobacter pylori is a spiral shaped microaerophilic bacterium that colonizes gastric mucosa of more than half of the world's population and is associated with development of complications such as peptic ulcer disease, gastric carcinoma and mucosa associated lymphoid tissue lymphoma. 1The association between H. pylori and gastric carcinoma led the World Health Organization to classify it as a class-I carcinogen in 1994.Bangladesh is a developing country and epidemiological studies shown 92% adult population were seropositive for H. pylori. 2 Helicobacter pylori strains can be divided into two major types based on their ability to produce a 120-145kDa immune-dominant protein called cytotoxin associated gene A (CagA) antigen. 3H. pylori strains possessing the CagA gene were linked with an increased risk of developing gastric cancer and peptic ulcer.The risk of developing gastric cancer in H. pylori infected CagA-positive subjects is six fold higher than that in CagA-negative subjects. 4More than 90% of isolated strains from East Asia including Korea, Japan, and China are known to harbor cagA, while 50%-60% of isolated strains from Western countries are positive for it. 5e 3′-end region of cagA where the tyrosine phosphorylation sites are located are highly polymorphic. 6Phosphorylation occurs on specific tyrosine residues within repeating penta amino acid Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, present at the C-terminus of the protein. 7Four different CagA EPIYA motifs, EPIYA-A, -B, -C, and -D have been defined based on the amino acid sequences surrounding the EPIYA residue. 8agA proteins nearly always possess an EPIYA-A and an EPIYA-B, followed by various number of EPIYA-C repeats in Western-type or EPIYA-D motifs in East Asian type strains. 6,9It has been suggested that the considerable variation in number of repeating EPIYA-C or -D motifs determines the biological activity of cagA. 7gA strains possessing multiple number EPIYA C segments predisposes to precancerous lesions and gastric cancer. 7,10 estern type EPIYA ABC pattern present in 100% H. pylori strains isolated from patients with various gastroduodenal diseases in India. 11The phylogenetic analysis of the 5ˊ end of the cagA gene indicates that Bangladeshi isolates are more closely related to H. pylori isolates from India and are different from isolates of East Asia. 12 large number of populations in Bangladesh are seropositive for H. pylori so, it is important to know the number and pattern of cagA EPIYA motifs for identifying the H. pylori infected patients who tend to develop severe gastroduodenal diseases.Therefore, EPIYA motif diversity may provide a useful tool for prediction of H. pylori pathogenic activity and accurate determination of number and type of cagA EPIYA motifs could identify the virulent H. pylori causing severe Gastroduodenal diseases in Bangladesh.So, this study was designed to detect H. pylori cagA gene polymorphism in gastric biopsy specimen from patients with gastroduodenal diseases.

Materials and Methods
Patients with dyspeptic symptoms who underwent endoscopic examination in the Department of Gastroenterology of Bangabandhu Sheikh Mujib Medical University (BSMMU) and Dhaka Medical College Hospital had endoscopic findings of any inflammation, ulcer or growth in gastroduodenal mucosa were enrolled in the study.Six biopsy specimens were collected from each patient: three from the gastric antrum and three from the gastric body. 13Each specimen from antrum and body was used for rapid urease test, PCR for ureC gene and histopathological examination.All procedures were performed in the Department of Microbiology and Immunology, BSMMU, Dhaka during the period of March, 2014 to February, 2015.Patients with history of previous gastric surgery, H. pylori eradication therapy or treatment with antibiotics, bismuth containing compounds, H2-receptor blockers or proton pump inhibitors within four weeks prior to enrolment were excluded from the study.Based on the endoscopic and histopatho-logical assessments, samples were divided into four groups of gastritis, duodenal ulcer, gastric ulcer and gastric carcinoma.Institutional Review Board, BSMMU and Ethical Review Committee of Dhaka Medical College approved the study protocol and informed consent was obtained from each patient prior to endoscopy and biopsy specimen collection.DNA extraction from gastric tissues: DNA from gastric tissues was extracted by using the QIAamp (QIAGEN) DNA Mini Kit according to the manufacturer's instruction.
After checking concentration of DNA, six samples were selected for sequencing.Purified products were sequenced using a BigDye Terminator Cycle Sequencing Kit in an ABI 3130 Genetic Analyzer (Applied Biosystems, USA).The sequences obtained were aligned using the CAP3 Sequence Assembly Program.After alignment, nucleotide sequences were transformed into amino acid sequences using the Blastx program and compared to previously published CagA gene sequence of strains H. pylori 26695 (AE000511) deposited into the GenBank. 15atistical Analysis: After collection all data were checked, edited and analyzed by using computer based SPSS (Statistical package of social science) software.p value was calculated using Chi-square test to find the significant relationship.p value less than 0.05 was statistically significant.

EPIYA-C EPIYA-C EPIYA-A EPIYA-B
Nucleotide sequencing of the cagA 3ˊ variable regions was performed for 6 selected cases including 2 from gastritis, 1 from gastric ulcer, 2 from duodenal ulcer and 1 from gastric carcinoma patients.Sequence analyses confirmed that three types of EPIYA motifs were observed: EPIYA-A for EPIYAKVNKKK (A/T/V/S) GQ; EPIYA-B for EPIY (A/T) (Q/K)VAKKVNAKI; and EPIYA-C for EPIYATIDDLGGPFPL (figure 2).

Discussion
CagA gene is considered to be a major virulence factor associated with gastric carcinoma.As large numbers of populations in Bangladesh are seropositive for H. pylori so, the present study was done to detect the number and pattern of H. pylori cagA EPIYA motifs using PCR based typing and sequencing analysis to identify the H. pylori infected patients who are at risk to develop gastric carcinoma.
In this study, out of 31 H. pylori positive cases, 19(61.3%)were cagA gene positive.This finding is consistent with the findings of previous studies showing cagA positivity rate 61% in china, 65.9% in Brazil and 68.4% in Bangladesh. 12,15,16 the present study cagA gene was positive in 66.7% cases in each of duodenal ulcer and gastric ulcer patients compared to 55% cases of gastritis.CagA gene was significantly associated with duodenal ulcer cases (p<0.05).8][19] For this difference in the cagA status, one possibility is the large genomic variations in the H. pylori genomes that amplifies the cagA gene from H. pylori isolated in one country failed to detect cagA in isolates from another country.
Western type cagA EPIYA-C motif was found in all 19 cagA positive H. pylori cases but there was no East Asian cagA EPIYA-D motif.Similar findings were reported from India and Colombia. 11,20.23Among 19 cagA positive cases majority (11, 57.9%) were EPIYA ABC motif followed by 5(26.3%)EPIYA ABCC, 2(10.5%)AC and 1(5.2%)AB motif.These results are comparable with the results of previous study done in Colombia. 24The multiple numbers of EPIYA motifs, especially EPIYA-C, is thought to be related to the development of gastroduodenal diseases.CagA positive H. pylori strains with multiple EPIYA-C motifs are reported to be associated with a higher gastric cancer risk than strains with only one EPIYA-C motif. 8,25In the present study more than one EPIYA-C (ABCC) was found in 26.3% of cagA positive cases which is comparable to the findings reported in South Africa and Colombia. 24,26Geographic variations in the frequency of H. pylori strains with one or two EPIYA-C repeats may be explained by different acidic conditions in the gastric mucosa, which could be related to differences among populations in the frequencies of cytokine gene polymorphisms that attenuate gastric acid secretions. 27esent study showed that the variation in the EPIYA motifs in CagA protein was not directly associated with the outcome of the disease caused by H. pylori and that there is no relation between the number of EPIYA-C motifs and the gastroduodenal diseases (p>0.05).5] These differences might be due to different study designs, sample size, populations and geographical diversity of H. pylori markers of pathogenicity.

Conclusion
This study shows the pattern of cagA EPIYA motif in gastric biopsy specimen from patients with gastroduodenal diseases.Possibly this is the first study in Bangladesh that shows Western type cagA EPIYA motif was found in all 19 cagA positive H. pylori cases and there is no East Asian EPIYA motif.This finding demand further study to be conducted with more clinical specimens and phylogenetic analysis of the H. pylori genome to determine the ancestry relationship among the overall international isolates.The multiple numbers of EPIYA motifs, especially EPIYA-C, is thought to be related to the development of severe gastroduodenal diseases and associated with higher gastric cancer risk and was found in 26.3% of cagA positive cases in the present study which also demand more studies to be conducted further to estimate the actual risk of severe gastroduodenal diseases.

Figure 1 :
Figure 1: Amplification of cagA EPIYA motif by PCR from H. pylori strains.(1) ABC motifs and (2) ABCC motifs.M: 100-bp DNA marker.No East Asian type of EPIYA-D (EPIYATIDFDEANQAG) was found.So the present study confirmed that PCR methods correctly classified the EPIYA motif types (i.e.strain-14, 23, 61, 62, 72 possessed ABC type, and strain-77 possessed ABCC type by PCR).The data are identical to data by sequencing.

Figure 2 :
Figure 2: Alignment of amino acid sequences among cagA strain (showing the EPIYA motifs) from 6 H. pylori strains including the H. pylori reference strain 26695.

Table I :
H. pylori cagA gene among H. pylori positive cases with different gastroduodenal diseases (n=31) Distribution of cagA EPIYA motif in H. pylori cagA positive cases and their relation to gastroduodenal diseases are shown in table II.

Table II :
Distribution of cagA EPIYA motif in H. pylori cagA positive cases in relation to gastroduodenal diseases (n=19)