Study On The Correlation Between HLA-DRB1Gene Polymorphism And Helicobacter Pylori Infection And Gastric Disease

Background:Helicobacter pylori (H. pylori) is a microaerophilic, spiral, gram-negative bacillus thatcolonizing in the human gastric mucosa. More than50%of the word population has beeninfected by this bacterial, and the infection rate is much higher in China and otherdeveloping coutries as compared with that in the developed coutries. Human is the onlynature host of H. pylori. The infection of the pathgon usually turns to chronic infection andpersists for the life of the host. Despite the high infection rate, only10to20percent ofthose who infected exhibit clinical symptoms. H. pylori infection is the cause of chronicgastritis (CG), gastric ulcer (GU), duodenal ulcer and gastric mucosa-associated lymphoidtissue lymphoma and is closely related to the development of gastric cancer (GC), thus ithas been classified as class one carcinogen by the World Health Organization.The development of gastritis and gastrointestinal diseases are not only associated withthe infection of H. pylori, some other factors, such as host genetics and environmentdiversity should also be taking into consideration. Host genetic factors that contribute togastritis and other gastrointestinal diseases are mainly immune associated genetic factors,especially the major histocompatibility complex (MHC) systerm that encoded humanleukocyte antigen (HLA), which playing key role in the immune response.HLA was mainly divided to type Ⅰa ndⅡ molecules, which mediate the CD8~+andCD4~+T lymphocyte response, respectively. It has been believed that HLA class-IImolecules were associated with H. pylori infection and gastric diseases. However, due tothe different in ethnic, regions and research methods, results were different from each other.Nowadays PCR reaction based genotyping methods are used for HLA typing, such asPCR-RFLP、PCR-SSOP、PCR-SBT、PCR-SSP、PCR-SSCP. The PCR-SBT typing methodis recognized as the most direct and accurate method for the moment. However, this method is not cost effective enough for research using.In this study, based on the principle of PCR-SBT method, we established the PCR-SBTgenotyping for HLA-DRB1by using eight specific PCR primers designed by ourselves. Weuse this method to analysis the disribution and the polymorphism of the HLA-DRB1alleleof Chongqing Han population, and to investigate the relationship between HLA-DRB1andH. pylori infection related gastric diseases. The above study provided an experimental basisfor revealing the the immunogenetics mechanism of H. pylori infection and related gastricdiseases.Aim:In this study, after the establishment of the PCR-SBT method for HLA-DRB1allelegenotype, we aimed to investigate the frequency and polymorphism of the HLA-DRB1allele and the relationship with H. pylori infection and related gastric disease among theHan population in ChongQing district.Methods:1. Periphery venous blood genomic DNA extractionUsing blood genomic DNA kit to extracte collected peripheral venous blood, and thenfrozen at-20℃, the follow up to amplified the target fragment.2. Establishment of the PCR-SBT method for HLA-DRB1allele genotypeAccording to the HLA-DRB1gene sequence in the IMGT/HLA database and thedifference between different subtypes of the second exon, we use primer5.0to design eightpairs of primers to amplify the target fragment specific to special HLA genotypes. And thenthe PCR production was sequenced. Chromas and Genetool were used to analyze thesequencing, and then the HLA-DRB1genotype we determined by blasting these sequencesin the IMGT/HLA database.3. H. pylori infection assayELISA assay for surem H. pylori specific IgG antibody was performed to screen H.pylori infected subjects.4. Statistic analysisThe allele frequencies of HLA-DRB1were compared among groups by the chi-square(χ~2) test. Odds ratio and95%confidence interval were used to evaluate the risk ofdiseases. Results:1. H. pylori infection rate is34.2%in Chongqing.2. The allele frequency of DRB1*09:01(29.1%) was the highest among these alleles,and the allele frequency of DRB1*12:01, DRB1*12:02, DRB1*14:05and DRB1*15:02were the lowest (1.26%respectively).3. No significant difference of the HLA-DRB1allele frequency was observed betweenmale and female individuals.4. The allele frequency of HLA-DRB1*04:05was negatively correlated with theinfection of H. pylori (infecte vs. uninfected P=0.035, OR=0.265,95%CI:0.076-0.922)5. The allele frequency of HLA-DRB1*15:01was negatively correlated with thedevelopment of gastric cancer and gastritis (gastric cancer vs. healthy control: P=0.039,OR=0.287,95%CI:0.091-0.905；gastritis vs. healthy control: P=0.001, OR=0.061,95%CI:0.007-0.494).Conclusions:1. The method of HLA-DRB1genotyping with PCR-SBT was successfully established.We found that the HLA-DRB1alleles were highly polymorphic in Chongqing district whichwas a convincing evidence for the study of the association between genetic factors anddisease.2. The HLA-DRB1*04:05allele is a genetic factor for protecting host from H. pyloriinfection3. The HLA-DRB1*15:01allele is a host genetic protective factor for gastric cancerand gastritis.