The Association Research Of The Rs2246945Gene Polymorphism Of A4GnT In Chinese Population With Helicobacter Pylori Gastropathy

Helicobacter pylori(Hp) is first isolated by Nobel laureate BarryJMarshall and J.Robin Warren from chronic gastritis patients in1983. It is asingle pole, multi-flagella, ends obtuse, curved spiral Gram-negative bacillus.It has a high infection rate of more than50%in human population, and theinfection rate is higher in developing countries.Hp is considered a leadingcause of chronic active gastritis, peptic ulcer, gastric mucosa-associatedlymphoid tissue lymphoma and gastric cancer.In1994,World HealthOrganization classified Hp infection as class Ⅰ carcinogen.In spite of Hp has a high infection rate, a majority of infected individualremain asymptomatic,that suggests the presence of human body defensemechanisms against Hp pathogenesis.Studies have shown that α-1,4-N-acetylglucosaminyltransferase (A4GnT) exist inmucous neck cells,cardiac gland and pyloric gland cells of gastric mucosa.A4GnT is aglycosyltransferase that mediates transfer of GlcNAc to βGal of O-glyan toform the terminal mucous α1,4-linked N-acetylglucosamine(αGlcNAc).Themucin with terminal αGlcNAc inhibits proliferation,motility and causesmorphologic change of Hp due to the inhibition of thecholesteryl-α-glucoside,because rs2246945codes the218amino acid ofA4GnT protein, The amino acid changing may affect the structure andcatalytic activity of A4GnT leading to the change of expression of A4GnTproduct α GlcNAc.Therefore, analysis the relationships between A4GnTgenes rs2246945polymorphisms and gastric disease in Chinese Hanpopulation will help us to understand the protection of gastric mucosa ofA4GnT against Hp.Objective: To study the A4GnT gene coding region SNP rs2246945allele polymorphism in Chinese Han population.to explore the relationship between rs2246945and gastric disease caused by HP infection.Methods:1Sample collection and DNA extraction: The case group was selected inEndoscopy room of The Third Hospital of Hebei Medical University, All thecase group biopsy of gastric musosa were Hp-positve measuredwith rapid urease test. and the biopsy samples DNA were extracted. The bloodsamples of healthy control were collected from health examination people.Theserum level of anti-Hp IgG was measured by ELISA kit, The anti-Hp negativeones were selected as the controls group The genomic DNA were extractedfrom whole blood.2MAMA-PCR reactions:To detect the SNP rs2246945, missamplification mutation assay PCR (MAMA-PCR) were performed. Twoupstream primers were designed with mismatched nucleotide at the3’extremity.The PCR action condition is：initial denaturation5minutes；denaturation is40seconds. Primer annealing is40seconds. Extension is40seconds.Then take7μl PCR products for2%agarose gel electrophoresis.scanning PCR product pictures by the gel image analysis system.3Sequencing reaction: Part of MAMA-PCR results were confirmed bydirect sequencing using Beckman-culter CEQ8000Genetic Analyzer4Statistical analysis: Calculated for frequencies of each genotype andallele.Use Hardy-Weinberg equilibrium to test the sample,Measurement datato x+s.comparison among groups use t-test. comparison among genefrequency and genotype frequency.Use SPSS statistical software19.0tostatistical anal-ysis all of the data.Result: three different genotypes of SNP rs2246945was tested usingchi-square test.There was significant difference in genotype distributionbetween Hp group and control group (χ2=24.833, P＜0.05).The AA genotypehas no statistical significance between Hp group and control group(χ2=2.067,P＞0.05，OR=1.514，95%CI0.859-2.670).The CC genotype has statisticalsignificance between Hp group and control group(χ2=12.706, P＜0.05). TheCC genotype of Hp group and control group was tested using odds ratio.We find CC was genotype associated with a lower risk of Hpinfection(OR=0.202,95%CI0.079-0.520).The frequency of AC genotypedistribution in the case group was48.37%,in control group was40.40%,Thereis a trend that Hp group higher than control group.But the AC genotype hasnot statistical significance between Hp group and control group(χ2=1.299, P＞0.05，OR=1.385,95%CI0.791-2.425).Conclusion:1.homozygous CC at the rs2246945locus was associated with a lowerrisk of H. Pylori infection.2.homozygous AA at the rs2246945locus was not statistical significancewith H. Pylori infection,but it has a trend that increase risk of H. Pyloriinfection.3.heterozygote AC at the rs2246945locus was not statistical significancewith H. Pylori infection,but it has a trend that increase risk of H. Pyloriinfection.