What is the Relationship Between Helicobacter Pylori CagA Genotypes and Gastrointestinal Diseases in the Iranian Population? A Systematic Review and Meta-Analysis

Background: Helicobacter pylori (H. pylori) is one of the most well-known risk factors for getting the gastric cancer disease. In recent studies, the relationship between its virulence factors, specially CagA (cytotoxin ‐ associated gene A) toxin and development into the gastrointestinal diseases is taken into consideration. According to review of literature, despite the presence of four motifs A, B, C, and D in CagA toxin, two motifs C and D are more associated with gastrointestinal complications in patients who are infected by H. pylori. Methods: In the present study, we researched about theses ambiguities using a comprehensive meta-analysis study. In this study, we assessed the information of 1762 Iranian patients for potential relationship between all genotypes of cagA gene and gastrointestinal diseases. Results: According to statistical analysis, the abundance of cagA genotypes AB, ABC, ABCC, ABCCC, and ABD in Iranian population is 5.52%, 80.18%, 22.81%, 2.76%, and 0% respectively. In addition, it was determined that there is a signicant relationship between cagA genotypes ABCC and ABCCC on the one hand and cagA genotype ABCCC on the other hand with susceptibility to chronic gastritis and gastric cancer respectively. Conclusions: Overall, it can be concluded that the higher number of EPIYA-C copy numbers lead to the higher risk of gastric cancer. According to our results, it seems that the presence of EPIYA-ABCCC motif in strains of H. pylori should be considered as an appropriate marker in preventing the gastric cancer among the Iranian population.

increased risk of gastric cancer (33)(34). The main goal of this meta-analysis was evaluation of abundance EPIYA motifs, and potential association of prevalent genotypes with gastritis, peptic ulcer, and gastric cancer in Iranian population.

Methods
In the present systematic review and meta-analysis, all stages of research including literature search, selection criteria, quality assessment, data extraction, and statistical analysis was done according preferred items in the systematic review and meta-analysis guidelines (35).

Search strategy
To receive all reports about Iranian population associated EPIYA motifs, conducted studies till August 2020 were collected from PubMed, Scopus, Google Scholar, Magiarn, IranMedex, and ISC databases. Studies were research without language limitation and also we used some keywords such as Iran, cagA gene, EPIYA, and Helicobacter pylori.

Selection criteria
Identi cation of eligible articles (titles, abstracts, and full text) was done by two authors, separately. According to standards protocols, the inclusion criteria included Iranian patients, collected clinical specimens, original articles (cross-sectional, case-control, and cohort), EPIYA motifs (AB, ABC, ABCC, ABCCC, and ABD), and reliable material and methods. In addition, excluded studies included the articles with insu cient information, letter to editors, review articles, case reports, congress abstracts, repetitive samples, and duplicate articles.

Quality assessment and data extraction
The quality of studies was evaluated by Newcastle-Ottawa quality assessment scale criteria, and articles which received score ≥ 5 were included in present meta-analysis (data not shown). Then, the process of data extraction from eligible studies was done by two authors, separately (Table 1). Extracted information included titles such as rst author, publication year, city, numbers of patients, distribution of age and gender in each study, frequency of EPIYA motifs in each study, and diagnostic methods.

Results
In this study, from all 92 articles, 11 articles met the inclusion criteria ( Fig. 1).
Among the included articles, ten was in English language, and 1 was Persian. The studies had been conducted from various cities such as Tehran, Ardebil, Sari, Kerman, Shiraz, and Ahvaz. In the present study, we evaluated the comprehensive information of 1762 patients.
Approximately 51.2% of patients were men, and 48.8% were women, and also, mean age of them was 49.2 ± 5. Frequency of cagA gene in isolated strains from different areas of Iran was assessed around 71.34%. Based on statistical analysis, it was found that there is a signi cant relationship between cagA gene and susceptibility to gastrointestinal complications, especially gastric cancer (ORs: 2.77 with 95% CIs) and peptic ulcer (ORs: 1.05 with 95% CIs) diseases (Fig. 2).
In all include studies, EPIYA motifs was identi ed by PCR and DNA sequencing techniques. Based on studies, so far no reports has been reported of EPIYA motif D and cagA genotype ABD from Iran. However, in present study, the most prevalent EPIYA motifs, A, B, and C, and cagA genotypes, ABC and ABCC, were assessed. Geographical distribution of CagA genotypes in Iran has been noted in Fig. 3.
According to statistical analysis, abundance of cagA genotypes AB, ABC, ABCC, ABCCC, and ABD in Iranian population is 5.52%, 80.18%, 22.81%, 2.76%, and 0% respectively. In addition, it was determined that there is a signi cant relationship between cagA genotypes ABCC and ABCCC on the one hand and cagA genotype ABCCC on the other hand, with susceptibility to chronic gastritis and gastric cancer respectively (Table 2). Publication bias was not seen in our study (data not shown).

Discussion
The cagA gene is one of the most important virulence factors in H. pylori genome, which locates in the end of I region in PAIs. The GC content this gene is 35%, which is less compared to other bacterial genes (40%) of bacterial genome. This gene can be transferred between bacterial strains through horizontal transmission (48)(49). The cagA gene encodes a 128-145 kDa protein in different strains, and its frequency has reported between 40-97% (50-51). According to review of literature, H. pylori strains which carry cagA gene are more virulent (more pathogen). Frequency of cagA positive strains in East Asia (regions with high incidence of gastric cancer) is very high (51)(52). For example, frequency of this gene in South-East Asian countries such South Korea (97%), Japan (95%), China (90%) is much more compared to Western countries (16,53). Also, Iran is one of the regions of the world with high prevalence of gastric cancer.
Based on GLOBOCAN the incidence of gastric cancer in Iran has been estimated about 62.3 cases per 100000 population. Based on our statistical analysis, frequency of cagA gene in Iranian population was evaluated about 71.34%, which con rmed previous ndings. In addition, the presence of cagA has a meaningful relationship with gastrointestinal diseases such as peptic ulcer, gastric atrophy, and gastric cancer (54)(55). In our project, also, abundance of cagA positive strains in peptic ulcer and gastric cancer patients was very high, and we also saw a meaningful relationship between the presence of cagA gene and gastric cancer (ORs: 2.277; p-value: 0.00). In recent, molecular studies have shown that upon translocation of CagA toxin into the cytoplasm of gastric epithelial cells, it is phosphorylated by Src kinases family in its Tyrosine residues of EPIYA motifs (56)(57). Phosphorylated CagA reacts with about 20 various proteins in host cell, and depending on host epigenetic situations, alternative signaling pathway leads to intracellular events such as to loss polarity and junction, increase of motility due to alternation in cytoskeletal rearrangement, stimulation of cell proliferation, DNA damage and aberrant cell survival, stimulation of pro-in ammatory response via stimulating of NF-κB signaling pathway and induction of hummingbird phenotype, and nally gastric cancer (58)(59)(60)(61). Nowadays, based on difference in surrounding amino acids sequences (32-40 residues), the EPIYA motif is subdivided to four classes A, B, C, and D (62). Each of these motifs effect on different signaling pathways. Even, in recent, it has found that nucleotide sequences and their number of repeats are unique in each EPIYA motif, and this situation in turn effects on binding a nity with host proteins, and has determinative role in nal outcomes of H. pylori infection (63)(64)(65). In addition H. pylori, in recent researches it has been demonstrated that EPIYA motifs in other human pathogens such as enteropathogenic Escherichia coli (Tir), Haemophilus ducreyi (LspA), and Anaplasma phagocytophilum (AnkA) play as PEIYA effectors which can affect signaling pathway in host cell (57,66). In recent years, evaluation of EPIYA motifs has attracted a lot of attention. For example, despite of high colonization rate with H. pylori in Africa, Latin America and some East Asian countries such Thailand and Malaysia, there is a decreased incidence of gastric cancer in those countries, which considering differences in EPIYA motifs, this paradox was justi ed (34,(67)(68)(69). It seems that EPIYA motifs solving the puzzle of being carcinogenic of cagA gene, and also, these days the EPIYA motif can be used as a tool for epidemiologic studies and also monitoring of circulating of H. pylori strains worldwide (14). position) compared to EPIYA-D (73-74). However, according to literature, the presence of the higher number of EPI-X repeats is associated with the greater tendency of binding to SHP-2 (75). According to this, in a study on Mexico population, the risk of gastric cancer due to strains containing two and more than two repeats in EPIYA-C was 32 and 51 fold respectively (76).

Studies in Brazil and
Columbia showed that the possibility of gastric cancer due to strains with 2, 2 or 3, and 3 repeats of EPIYA-C was 5.9, 3.8, and 12 fold respectively (77)(78). Also, EPIYA-A and B bind C-terminal Src kinase (Csk) and p85 (subunit of PI3K) complex respectively, but these motifs have been less studied and their effects remain unknown (66). In general, cagA genotypes recognized from around the world include AB, ABC, ABCC, ABCCC, ABCCCC, and ABD. In studies from Iran, so far there is no any document based on detection of cagA genotypes ABCCCC and ABD, which is arising from genetic differences between isolated strains of Iranian patients and East Asian's strains (37)(38)(39)(40)(41)(42)(43)(44)(45)(46). Nevertheless, according to studies, in Iran, cagA genotypes ABCC and ABCCC are more prevalent in peptic ulcer and gastric cancer. However, based on our statistical analysis, only cagA genotype ABCCC had a meaningful relationship with gastric cancer. Like our results, studies in South America, North America, and European showed a signi cant relationship between infection by multiple EPIYA-C strains and gastric cancer in the patients (71,75). Recently, Gomez et al. (2020) found a meaningful relationship between EPIYA-ABCC and ABCCC with gastric cancer which con rms our study (75). Furthermore, according our results, it seems that infection by strains possessing EPIYA-ABC motif has a preventive role gainst gastritis, peptic ulcer, and gastric cancer. Regarding less a nity of EPIYA-C motif to binding to SHP-2, hence, it concluded that the presence of one copy of EPIYA-C has less effect in induction of gastric cancer, especially that EPIYA-D is less be able to induce interleukin 8 (IL-8) compared to EPIYA-C (66,79). Our country, Iran, locates in Middle East, and recent studies have demonstrated that gastric cancer is the most prevalent cancer in Iran. Based on recent studies, the prevalence of gastric cancer in Iran has been estimated about 13.7 per 100000 population. In addition, the recent studies have shown that north provinces, in particular, northeast provinces are dedicated the most gastric cancer patients to themselves, and this is while based on epidemiologic studies these areas have the most abundance of infection by H. pylori (80)(81)12). According to this meta-analysis it was demonstrated that infected patients to H. pylori are 2.26 fold more exposed to gastric cancer, which has conformity with received results from Iranian population (82). In developing countries such Iran the age of infection with H. pylori is low, and based on recent studies, about 80% of children in the rst ten years of their lives are affected to this infection. Vic versa, in developed countries infection with H. pylori elevate with increasing age. Therefore, the age is accounted as a determinative factor in increasing of the numbers of gastric cancer patients in developing countries, especially in Iran (82)(83). Also, various studies have demonstrated that cagA positive strains of H. pylori are more virulent compared to the cagA negative ones, and hence, these strains are directly related to peptic ulcer and gastric cancer (71). In the present study, frequency of cagA gene in patient with peptic ulcer and gastric cancer was reported about 71%. Based on our statistical analysis, infection with H. pylori has a meaningful relationship with affecting to gastric cancer in Iranian population. According to Ghotaslou et al. (2018) study, the infection with strains possessing cagA and vacA s1m1 genes causes the increased risk to gastric cancer in Iranian population, which con rms our declarations (84). However, although the rate of gastric cancer in Iran is like East Asian countries such Japan, Korea, and China, but EPIYA motifs pattern in these countries is different of Iran (34,37,85). While in East Asian countries, the cagA2a (EPIYA-ABD) genotype is common, but so far in Iran no report has been issued about detection of EPIYA-D, and this is due to geographical differences of circulating strains (34). In addition, in other regions such as Africa, Latin America, or even East Asian countries e.g. Thailand and Malaysia, despite high colonization rate of H. pylori but the frequency of gastric cancer is low; one of the most probable reasons for this difference is diversity in H. pylori strains. According to estimates, it was determined that circulating H. pylori strains in Iran have ve different genetic patterns, which three patterns are like to identi ed patterns from Arabia, Turkey, and Uzbekistan, while two other patterns speci cally are belong to Bandar Abbas and Yazd (86). Lati -Navid et al. (2010), showed that Iranian strains fall in a same clade with European H. pylori (hpEurope) strains (Fig. 4).
According to phylogenetic analysis, Iranian patients' strains fall into a same clade with isolated strains of England, Spain, Finland, Turkey, and Italy (86). The hpEurope strains are formed from combination of both Ancestral Europe1 (AE1) and Ancestral Eroupe2 (AE2) population. AE1 is more related to Northern Europe, while AE2 is related to isolated strains from Southern Europe regions (87). Available information show that strains of Central Asia are more AE1, whereas North East Africa strains originate from AE2 (12,86). It seems that circulating strains among Iranian patients originate from hpEurope strains, and have transferred to Iran from some groups such as Arabs in the 7-8th centuries, Uzbeks ght in 16th century, and Ottoman Empire during the 20th century (86). Existence of cagA2a genotype (EPIYA-ABC motifs) in Iran's studies con rm this hypothesis (37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). However, why the number of gastric cancer cases in Iran is as high as cases of Japan, South Korea, and China? It seems that the presence of EPIYA-C motif, especially in strains which have more than one repeat of EPIYA-C can be reinforcement of binding to SHP-2 and is accounted a risk factor for gastric cancer (75,88). In our study, the presence of EPIYA-ABCCC was signi cantly related with gastric cancer in Iranian population. Moreover, life style, ethnicity, and etc. cause to raise the prevalence of gastric cancer in Iran (81). Finally, it should be noted that our study had several limitations such: high degree of heterogeneity in the analyzed studies, limitation in number of studies, Lack of solidarity between cagA genotypes and other virulence factors with gastrointestinal diseases development. In many of studies it has determined that the simultaneous presence of some alleles of cagA and vacA genes cause increase the risk of gastric cancer in different populations of the world (84). Therefore, further studies will be essential to elucidate the dominant role of cagA genotypes for assessment of susceptibility into gastrointestinal diseases.

Conclusions
In the present study, we declared the high prevalence of cagA gene in gastrointestinal diseases of Iranian population. We demonstrated that this gene signi cantly is related to susceptibility of patients to gastric cancer. Notwithstanding EPIYA-ABD motif is related to increase the risk of gastric cancer in East Asian population, so far no evidence has been found based on identi cation of EPIYA-ABD in Iranian population, and regarding of high prevalence of gastric cancer in Iran, we are faced with a paradox. This question has also been observed in the Thailand and Malaysian population. Sahara et al. (2012) in their study demonstrated that the frequency of EPIYA-D motif is low in these countries, and this reason justi es the decrease of frequency of gastric cancer in Thai and Malaysian population (34). We observed that there was a signi cant relationship between EPIYA-ABCCC and gastric cancer in Iranian population. Overall, it can be concluded that the higher number of EPIYA-C copy numbers leads to the higher risk of gastric cancer (55,88). Therefore, according to our results, it seems that the presence of EPIYA-ABCCC strains of H. pylori should be considered as an appropriate marker in prevention of gastric cancer in Iranian population.

Mucosa-associated lymphoid tissue (MALT) lymphoma
Nonsteroidal anti-in ammatory drugs (NSAIDs) Comprehensive meta-analysis (CMA) Odds Ratio (ORs) Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information les

Competing interests
There is no any con ict of interest among the all authors.

Funding
We have not received any funding for this research. Schematic illustration of search strategy process in current meta-analysis.  of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.