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Section 1. Biology
Dalimova Dilbar Akbarovna, Ph D., Doctor of Philosophy, Center for Advanced Technologies of the Republic of Uzbekistan, Tashkent E-mail. dilbar.dalimova@gmail.com Abdurakhimov Abrorjon Akramovich, Researcher, Institute of Biophysicsand Biochemistry at the National University of Uzbekistan, Tashkent E-mail: Abror1978@mail.ru
Turdikulova Shakhlo Utkurovna, Doctor of biological science, Center for Advanced Technologies of the Republic of Uzbekistan, Tashkent E-mail: shahlo.ut@gmail.com
DISTRIBUTION OF HELICOBACTER PYLORI GENOTYPES IN TWO AREAS OF UZBEKISTAN WITH DIFFERENT GASTRIC CANCER RISK
Abstract. Genotyping of H.pylori from the region of Aral Sea, a zone of ecological disaster with a high prevalence of gastric cancer, was compared with Tashkent - region with low gastric cancer rate. PCR analysis revealed that 66,1% samples from the Aral Sea and 84.2% samples from Tashkent was CagA positive. In Tashkent, we found the prevalence of VacAsl m2 alleles and VacAsl ml alleles from Khorezm region. In most samples from Khorezm had mixed genotype of IceA1/IceA2 (80%), whereas in Tashkent mostly IceAl allele was distributed. The predominant genotypes in high-risk of gastric cancer area (Aral Sea) were cagA+/vacAs1m1/iceA1/iceA2 and in low-risk Tashkent, the district was cagA+/vacAs1m2/iceA1, which correspond to the less aggressive strains. Comparison of H.pylori virulence genes distributed in high- and low-risk area of gastric cancer in Uzbekistan showed the differences between genotypes, more aggressive form of H.pylori was revealed in the Aral Sea region.
Keywords: Helicobacter pylori, the gene of virulence, CagA, VacA, IceA, gastric cancer, genotypes.
Background ated diseases. This may be related to a complex of environ-
Helicobacterpylori is a gram-negative pathogen that colo- mental factors, host characteristics, and bacterial virulence
nizes the human stomach. H. pylori colonization results in a determinants. Several virulence-associated factors ofH.pylori
persistent, lifelong infection if left untreated [1]. The infection were associated with clinical outcomes of the infection [3].
of H.pylori is associated with duodenal ulcer, gastric ulcers, ad- Previous studies in Uzbekistan showed a significant relation-
enocarcinoma of the distal stomach and MALT- lymphoma [2]. ship between gastric diseases including gastric cancer or pre-
Each year, at least 7 million cases of these diseases oc- cancerous lesions and H. pylori infection [4]. Consequently, it
cur worldwide, resulting in hundreds of thousands of deaths. is important to know the genetic diversity of H. pylori strains
754000 people around the world died from gastric cancer in Uzbekistan, a region with a high frequency of H. pylori-
in 2015 and 70% of those makes to poor countries. In 2016 induced gastric disease.
alone, 900000 people were diagnosed with gastric cancer. By Aral Sea region of Uzbekistan become a zone of ecologi-
2035, almost 15 million cancer-related deaths per year are cal disaster and known as a high-risk area of gastric cancer.
forecast to occur. More than half of the world population is The case of gastric cancer in this region is three times more
infected with H. pylori, but not all individuals develop associ- than in Tashkent - low cancer risk area. But whether the high
incidence of gastric cancer in the high-risk area has a relationship with specific H. pylori genotype is unclear. The aim of this study was to compare the distribution of Hpylori genotypes in the two areas with different cancer risk and furthermore explore its geographic characteristics.
Methods
For this study, gastric biopsies were collected from 147 individuals including 109 cases from Aral Sea region (58 men and 51 women, 25-62 years, mean age: 42.5 years) and 38 cases from Tashkent (20 men and 18 women, 21-59 years, mean age: 44.33 years). Their biopsies were obtained during endoscopy with informed consent.
DNA extraction from the biopsy and PCR analysis
Isolation of DNA was obtained using the kits of Ampli Prim RIBO-PREP reagent kit (Inter Lab Service, Russia). Genotyping was performed for CagA, VacA h IceA genes, and subtypes of these genes. Genotypes were determined by polymerase chain reaction [5; 6; 7].
Statistical analyses
Data were analyzed using SPSS for windows version 11.0. The x2 test or Fisher's exact test was used to assess the relationships between different areas. P < 0.05 was considered statistically significant.
Results and Discussion
A total of147 H.pylori clinical isolates from two geographic areas in Uzbekistan were obtained. The high-risk group comprised 109isolates from Khorezm (Aral Sea region). The low-risk group comprised 38 strains from Tashkent.
Detection of the cagA gene in two geographical areas
CagA positive genotypes were found in 109 cases from total analyzed 147, that corresponds to 70%, and geographical distribution in Uzbekistan regions was: 66.1% samples from Khorezm was CagA positive, in Tashkent region, 84.2% of samples have CagA gene. CagA gene is considered as a marker for the presence of Cag PAI, which is associated with the most virulent H.pylori strains [8]. High prevalence of cagA-positive H.pylori is 90-95 % in Asian countries and 50-60% in western countries [9]. Bravo et al., also reported that a higher frequency of cagA is in patients from high risk areas of gastric cancer than in those from low risk areas of gastric cancer [10]. Some persons showed that cagA positive strains are associated with higher gastric cancer risk [11]. But in our study, there was no significant difference in cagA status between the two areas of Uzbekistan. Study of association of CagA genotype with the different type of diseases showed that in the case of gastric ulcer percent of CagA positive strains (87%) was higher compared with gastric cancer - 60.5%.
Detection of the vacA gene in two areas
The vacuolating cytotoxin (VacA) induces the formation of intracellular vacuoles in epithelial cell lines. Besides its di-
rect cell-damaging effect in vitro, VacA also plays a major role in inducing cytoskeletal changes, apoptosis, suppression of epithelial proliferation, and migration [12; 13; 14; 15]. VacA also causes cell death by mitochondrial damage, via signaling pathways that are not fully defined [16]. VacA gene is present in all H. pylori strains and the three most extensively studied regions of heterogeneity correspond to the signal "s" region, the intermediate "i" region, and the middle "m" region. Type s1/i1/m1 forms of VacA are more active in assays of cell- vacuolating activity than are forms of VacA designated s2, i2, m2 [17; 18]. The s-region, which encodes the signal peptide, coexists as either s1 or s2 allelic types. The m-region (middle) occurs as m1 or m2 allelic types. Production of VacA is related to the mosaic structure of vacA [19]. In human stomach, strains with vacA m1 allele are associated with severe epithelial damage compared to those with m2 allele.
The presence of VacA gene was found in all samples, and distribution ofVacA subtypes was s1 variant was found in 112 (76.2%) cases, s 2 in 26 (17.7%) cases. From this 84 (77.1%) samples from Khorezm region have s1 allele, and 16 (14.7%) have s2 allele of VacA gene. In Tashkent this distribution was different - s1 genotype was determined in 73.7% cases, little lower than in Khorezm and in 26.3% was found s2 variant. In 9 cases all from Khorezm "s" allele wasn't typed, probably because high mutation rates of this gene and used primers have not specificity in these cases.
In 31 cases (20.2%) we found m1 allele ofVacA gene, and 82 (55.8%) samples have an m2 subtype of this gene. Among geographic area percent amount of this alleles was different and consist of 49.5% for the Khorezm region and it was much higher in Tashkent - 73.7%. In addition, we did not find vacA m genotype in 33 isolates. This is currently unexplained and may be due to the existence of additional vacA. The frequency of predominant VacA alleles found in our study was close to revealed in Thailand and China [20] but different from allele distribution in isolates from East Asia.
Studies of association of VacA genotypes with clinical outcomes showed the prevalence of s1 and m1 alleles in strains isolated from patients with gastric cancer, and in the cases of gastric ulcer was found s1 and m2 genotypes.
The results of our study demonstrate that vacAs1 is very common among H.pylori strains in Uzbekistan (76.2%), and the most frequent H. pylori vacA genotypes in Uzbekistan are s1/m2 (41.5%) and s1/m1/m2 (26%). In different studies, it was shown that patients infected with vacA s1m1 strains have a higher risk of carcinogenesis [21]. Type s1-m1 forms of VacA promote the death of gastric epithelial cells in vitro [22; 13; 23]. In our study, distribution of the vacA s1m1 genotype was found in the high risk areas, this may be a factor
contributing to the higher gastric cancer incidence in the Aral Sea region.
Since the H. pylori genome contains only one copy of the vacA gene, detection of multiple genotypes is indicative of the presence of several strains in one sample [24; 25]. We found a very high proportion of mixed genotypes (36%) with respect to the vacA gene in clinical isolates. In addition, a high incidence (up to 30%) of multiple H. pylori strains in single biopsy specimens was noted.
The risk of coinfection or superinfection is known to be higher in countries where the general level of infection is higher. In Uzbekistan, 90% of the adult population is infected with H. pylori and, in some areas, the incidence reaches 100%. However, it remains unclear whether mixed infection with several strains of H. pylori increases the risk of severe clinical manifestations, such as gastric ulcer or cancer. The presence of multiple-strain colonization should be considered when planning therapeutic strategies, as well as when studying the pathogenesis of H. pylori infection.
Detection of IceA gene alleles
The iceA gene has two main allelic variants, iceA1 and iceA2. IceA gene could be genotyped as iceA1 and iceA2 with specific primers. IceA1 is upregulated upon contact of H. pylori with the gastric epithelium and has also been suggested as a marker for peptic ulcer disease [20; 25]. In our
study IceA2 genotype was found in 9 cases, IceA1 genotype in 28 cases (19%) and mainly in 65.3% cases was found IceA1/A2 genotype, however in Tashkent region percent of mixed IceA1/A2 variant was much lower than in Khorezm and consist of 23.7% and 65.3% correspondingly.
Conclusion
In our study, the predominant genotypes in high-risk of gastric cancer area (Aral Sea) were cagA+/vacAs1m1/ iceA1/iceA2 and in low-risk Tashkent district was cagA+/ vacAs1m2/iceA1, which correspond to the less aggressive strains. Combined analysis of vacA, cagA, and iceA genotypes may permit identification of high-risk patients infected with more pathogenic H. pylori strains. Eventually, patients infected with such strains could be selected for prophylactic anti-H.py-lori treatment to prevent associated gastric diseases later in life. Further studies are required to determine the epidemiological and clinical importance ofH.pylori virulence-associated genotypes in different geographic areas in Uzbekistan.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
This work was supported by NATO Reintegration grant № 220207 and Science and technology development coordination committee under the Cabinet of Ministers Republic of Uzbekistan Grant № FA-F4-T106.
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