Association Between HLA-DQ Genotypes and Haplotypes vs Helicobacter pylori Infection in an Indonesian Population

More than 50% of the world’s population is infected with Helicobacter pylori (H. pylori) in the pylorus of the stomach (Brown, 2000). In particular, H. pylori infection is more prevalent in developing countries, varying with the geographic area, age, socioeconomic status, race, and ethnicity. The precise transmission route is unknown, but most individuals may become infected in childhood (Klein et al., 1991). H. pylori is an important gastrointestinal pathogen associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer (Asaka et al., 1994; Honda et al., 1998; Peek et al., 2000 ). Although the majority of H. pylori-infected patients develop asymptomatic gastritis, gastric or duodenal ulcers, only a small proportion of them suffer from gastric cancer. The different outcomes of H. pylori infection seem to be due to bacterial genotypes and strain diversities, duration of infection, or factors involving the host and environment. Host individual differences in immune response also have appeared to play a key role in the establishment and


Introduction
More than 50% of the world's population is infected with Helicobacter pylori (H. pylori) in the pylorus of the stomach (Brown, 2000).In particular, H. pylori infection is more prevalent in developing countries, varying with the geographic area, age, socioeconomic status, race, and ethnicity.The precise transmission route is unknown, but most individuals may become infected in childhood (Klein et al., 1991).H. pylori is an important gastrointestinal pathogen associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer (Asaka et al., 1994;Honda et al., 1998;Peek et al., 2000 ).Although the majority of H. pylori-infected patients develop asymptomatic gastritis, gastric or duodenal ulcers, only a small proportion of them suffer from gastric cancer.The different outcomes of H. pylori infection seem to be due to bacterial genotypes and strain diversities, duration of infection, or factors involving the host and environment.Host individual differences in immune response also have appeared to play a key role in the establishment and
In Asia, infection rates of H. pylori are high in Japan (Tokudome et al., 2005), Korea (Lee et al., 2008), China (Brown et al., 2002) and Vietnam (Nguyen et al., 2006) and the incidence rates of gastric cancer are generally high in the areas with a higher prevalence of H. pylori infection.In Indonesia, on the other hand, not only urea breath testing (UBT) but also IgG antibodies to H. pylori were found to be low in men and women, significantly lower than the 62% and 57%, respectively, in Japan.For example gastric cancer incidence in Yogyakarta and Semarang, Indonesia appeared exceedingly low, only approximately 1% of that in Japan (Tokudome et al., 2005;Tokudome et al., 2005 ).
Human leukocyte antigen (HLA) class II molecules are α-β heterodimeric membrane glycoproteins that are expressed on the surface of antigen-presenting cells such as macrophages, dendritic cells and B lymphocytes (Kaufman et al., 1984).Only helper T cells can recognize peptides derived from extracellular antigens which are associated with HLA class II molecules.The interaction

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of T cell receptors, peptides and HLA class II molecules determines T cell activation and an immune response to various antigens (Sette et al., 1987;Peace et al., 1991;Topalian et al., 1994).HLA polymorphism is responsible for variation in the immune response of different individuals to various antigens, and contributes to the susceptibility or resistance to infections and autoimmune diseases (Amar et al., 1984;Payami et al., 1986;Todd et al., 1987;1988;Hill et al., 1991).Previous studies have shown that adhesion of H. pylori to gastric epithelial cells transferred with HLA genes leads to different degrees of apoptosis (Fan et al., 1998).The HLA-D region accounts for over 50% of the heritability in hosts (Azuma et al., 1995), and it was also reported that there is an association of HLA-DQA1 and DQB1 genotypes with gastric disease (Rotter et al. 1984;Etoglu et al., 1992;Ohmori et al., 1997).The present study was conducted in order to investigate the association of HLA-DQA1 and DQB1 genetic diversity in individuals infected with H. pylori in an Indonesian population.

Study Population
Since 2004, international collaborative epidemiologic studies on host and environmental factors for stomach cancer have been conducted in several Southeast Asian countries, including Thailand, Vietnam and Indonesia.In March 2007, we conducted an epidemiologic study on 107 male and 187 female participants from the general population in the city of Mataram, Lombok Island, Indonesia.Their age ranged from 6 to 74 years with a mean age of 34.0 (±14.4)(±SD).Informed consent was obtained from all subjects for the examination of H. pylori infection and the analysis of host factors, including HLA-DQ antigen genotypes.The protocol was approved by the Ethics Committee of the Nagoya City University Graduate School of Medical Sciences.

Statistical Analyses
Differences in the distribution by age according to prevalence of H. pylori infection were examined by t-test.Differences in the distribution by sex and genotype were assessed with Chi-square test.Hardy-Weinberg equilibrium was examined for HLA-DQA1 and DQB1 allele polymorphisms.Multi-comparisons for HLA-DQA1 and DQB1 alleles and haplotypes were made according to the Bonferroni method.Associations of the HLA genotypes and haplotypes with H. pylori infection were examined by odds ratio (OR) and 95% CI (confidence interval) using unconditional logistic regression analysis.Statistical significance was determined as p<0.05.All the statistical analyses were performed using SAS software package (version 9.1).

Discussion
We investigated associations between host HLA-DQ variation and H. pylori prevalence in an Indonesian population with an H. pylori infection rate of 11.2% in people residing in Mataram, Lombok Island.Individuals carrying DQB1*0401 genotypes were noted to be at a significantly greater risk for developing H. pylori infection, compared to those with DQB1*0301 genotypes.Haplotypes of DQA1 or DQB1 were not related to H. pylori infection.
Human major histocompatibility complex (MHC) class II molecules, such as HLA-DP, HLA-DQ, and HLA-DR play a key role in immune reactions to pathogens, including bacteria and toxins (Liewelyn et al., 2004).The HLA-D region accounts for over 50% of the heritability in hosts (Azuma et al., 1995) and appears responsible for variation in the immune response of different individuals to various exogenous antigens, suggesting that there are varieties in the host's response to the same organism, and individuals with various HLA types differ in their immune response.Allele-specific antigenic peptides to T-cells may contribute to the differences between HLA-DQ genotypes and susceptibility or resistance to H. pylori infection (Herrera-Goepfere et al., 2006).Possible associations between HLA-DQ genotypes and H. pylori infection have been reported in China (Wang et al., 2004;Huang et al., 2005).Distributions of HLA-DQA1 and DQB1 genotypes detected in the Indonesian people in the present study may, in part, explain the low prevalence of H. pylori infection there.
In addition to HLA-DQ genotypes, other host factors, not only oncogenes or suppressor genes (Yasui et al., 2006) but also genetic polymorphisms, associated with bacterial infection and inflammatory cytokines, seem of interest because they are known to be related to H. pylori infection and H. pylori-associated lesions/diseases, including gastritis, gastric/duodenal ulcer and gastric cancer (Veldhuyzen et al., 1994).We are now analyzing genetic polymorphisms, including CD14, IL1-beta, IL4, IL8, TNF-alpha, and PTPN11 (Hamajima et al., 2006), and comparing the prevalence of alleles of the genetic polymorphisms between H. pylori infected vs. noninfected subjects in the people of Mataram, Lombok island, Indonesia.
Because bacterial infection is established via interactions among host factors, bacterial factors and environmental/vehicle factors, H. pylori bacterium genotypes and strains are important.There are genotypes, including cytotoxin-associated genes A (cagA) and vacuolating cytotoxin A (vacA) genotypes in respective H. pylori strains, including East-Asian type, South/Central Asian type, African type and European type (Yamaoka et al., 2002).There are various genotypes in different H. pylori strains in diverse ethnic groups, including Southeast Asian countries.We also detected East-Asian type in H. pylori bacteria isolates from patients with gastric ulcer in Mataram, Lombok island (unpublished).Thus, associations between H. pylori bacterium genotypes and strains vs. H.pylori prevalence in the Indonesian people should be further studied.
H. pylori may be transmitted via the fecal-oral or oral-oral infection route in early childhood (Klein et al. ,1991;Kusters et al., 2006 ), environmental/lifestyle factors, including sanitary conditions, water sources, living conditions, family size, and socioeconomic status, appeared critical.It seemed that sanitary/environmental conditions remain basic in Mataram, Lombok island, Indonesia.Some people still use the fingers when eating (Cairncross et al., 1997), consume well, pond or river water (Cairncross et al., 1997;Mckeown et al., 1999 ) and use privy/latrine type toilet (Feachem et al., 1983).Prevalence of hepatitis A, for instance, a typical waterborne infectious disease, is very high at no less than 90%,  1250 and the infection rates did not differ between the two areas (Brown et al., 1985;Hoang et al., 2008) Effects of pre-or co-infection of other organisms, including dengue, should be taken into account because the altered immune system may modify sensitivity or resistance to infection by H. pylori (Nurgalieva et al., 2005).Dietary factors cannot be overlooked.Investigations on consumption of fresh fruit and vegetables, isoflavone-rich soybean products (Ko et al., 2010), such as tempe (fermented soybean curd) and vegetable oils, in particular, are now in progress.The present approaches are ecological studies using international groups/areas as units.Naturally, the level of evidence is low (Da Cunha et al., 2007).There may exist ecological fallacies when drawing individual-based inferences on the basis of group-level data.The studied may not represent the Indonesian people as a whole because the subjects were not, strictly speaking, randomly sampled from the general population of Mataram, Lombok island, Indonesia.In addition, the number of study subjects did not seem to be sufficient when genomic alleles are rare, in particular, or the statistical power may not be sufficient to detect the supposed differences.
In conclusion, HLA-DQ genotypes may play an important role in H. pylori infection, and low H. pylori infection rates in Indonesian people may be, in part, explained by the diversities in the distribution of HLA-DQ genotypes along with the differences in bacterial genotypes and strains, sanitary or environmental conditions and dietary factors.Further studies are warranted to confirm the present observations.