Association Between An Insertion/deletion Polymorphism Of Pepsinogen C Gene And Gastric Cancer Risk

Association between an insertion/deletion polymorphism of pepsinogen C gene and gastric cancer riskIntroductionGastric cancer is the result of cooperation between environmental factors and genetic factors. The gene polymorphism is one of the material bases of the individual suspceptibily to gastric cancer. The application of the suspceptive gene polymorphisms as molecular markers for gastric cancer prevention is the frontier of research world widely.Human pepsinogen(PG)is the inactive precursor of the gastric mucosa specific functional enzyme—pepsin C, which is considered to be a mature marker of stomach cells. PGC gene located at 6 p21.1-pter, and has nine exons and eight introns. An about 100bp insertion-deletion RFLP polymorphism was observed between exon 7 and 8 with several restriction enzyme. Our previous study reported that the frequency of PGC homozygotic allel 1 genotype(310bp/310 bp) carrier in patients of gastric cancer was significantly higher than that in other genotypes. It suggested that PGC polymorphism was associated with susceptibility to gastric cancer susceptibility. China is a high risk country of gastric cancer, and Zhuanghe county in Liaoning province, is a high risk area in northern China. Up to now, it is not distinct that whether the distribution frequency of PGC gene polymorphism in residents is correlated with the high incidence of gastric cancer in Zhuanghe,and what is the role of PGC gene polymorphism in the development of the gastric cancer(normal→superficial gastritis→atrophic gastritis→gastric cancer). Furthermore, the mechanism of the PGC gene polymorphism in genetic predisposition to gastric cancer is worth more investigation.Helicobacter pylori(H.pylori), as the main environmental factor, trigger and accelerate the gastric cancer in the pathogens of gastric cancer. Persistent gastritis induced by H.pylori is the strongest known risk factor for adenocarcinoma of the distal stomach, yet few patients with H.pylori infection develop gastric carcinoma. In addition to H.pylori constituents itself, host inherited factors also present the contribution. Therefore, the interaction of genetic susceptibity and enviromential factors is important. But it is not clear that whether the interactions of PGC gene polymorphism and H.pylori, or PGC gene polymorphism and H.pylori genotypes appear in the development of gastric cancer. And the effect of the interaction on gastric cancer still deserves further study.ObjectiveIn this present research, we implemented the population-based genetic epidemiologic case-control study in Zhuanghe, Liaoning, a high risk area of gastric cancer. The purpose is to investigate the correlation of PGC gene polymorphism and gastric cancer risk, and the mechanism. It is expected to help exploring a valuable molecular marker for gastric cancer, screening individuals in high risk conditions, and furthermore providing experimental evidences and reference for individualization prevention from gastric cancer.Materials and methodsThe subjects in present study, whose samples including serum, sludged blood and gastric mucosa, consisted of1. people from Zhuanghe, Liaoning, gastric cancer high risk area, who were screened for gastric cancer.2. patients from Zhuanghe Central Hospital for gastroscopy.3. patients from the first Affiliated Hospital of China Medical University for gastroscopy.4. patients from the second Affiliated Hospital of China Medical University for gastric operation.5. patients from Liaoning Cancer Hospital for gastric operation. Information about gender, age and other factors was obtained by means of a questionnaire administered to each subject. The study protocols were approved by the Human Ethics Review Committee of China Medical University. Written informed consent was obtained from participants in accordance with the Declaration of Helsinki and its later revision.Hematoxylin and Eosin(HE)Staining were taken part in histopathologic examination. Genomic DNA was isolated from the sludged blood or gastric mucosa by standard phenolchloroform extraction. PGC genotypes were determined by polymerase chain reaction(PCR)and confirmed by sequencing. Serum immunoglobulin(Ig)G antibodies to H pylori and serum PGC concentrations were measured by an enzyme linked immunoadsorbent assay(ELISA). H.pylori genotype,such as ureB, cagA, vacAs1, vacAm1b, vacAm2, iceA1, iceA2 were determined by PCR. PGC protein was observed by immunohistochemistry methods.Theχ~2 test or Fisher's Exact Test were used to examine the differences in the different groups. The medians of variables were compared between two groups by the Mann-Whitney U tests. The Odds ratio(ORs)and their 95％confidence interval(CIs) were calculated by unconditional logistic regression models. An interaction was assessed by crossover analysis; synergy index and attributable proportion of interaction were calculated.ResultsPaper1 The characteristic of PGC gene polymorphism in population distribution1. PGC homozygous allele 1 genotype frequencies in the high/low risk area of gastric cancerThe frequencie of PGC homozygous allele 1 genotype in high risk area(20.06％)was significantly higher than that of low risk area(12.92％, P=0.033). The OR value adjusted by age and gender was 1.961(95％CI 1.139-3.376).2. PGC homozygous allele 1 genotype frequencies in the familial history/non-familial history group The frequencie of PGC homozygous allele 1 genotype in familial history group(33.67％) was significantly higher than that of non-familial history group(14.16％, P=0.001). The OR value adjusted by age and gender was 3.638(95％CI 1.864-7.100).Paper2 The impact of PGC gene polymorphism on the gastric cancer and its precancerors risk1. PGC homozygous allele 1 genotype frequencies in the different gastric diseases groupThe frequencies of homozygous allele 1 genotype among NOR, GEU, GA and GC were 7.09％, 11.35％, 19.15％and 18.44％, respectively. The frequencies of GA and GC were significantly higher than that of NOR controls(P=0.003, P=0.004, respectively), but it was not significant in GEU(P=0.217). Logistic regression analysis adjusted by age and gender showed that subjects carrying the homozygous allele 1 genotype were at a 3.112-fold(95％CI 1.443-6.710)elevated risk for GA and 3.000-fold(95％CI 1.382-6.512)for GC.2. PGC homozygous allele 1 genotype frequencies in IM or non-IM groupThe frequencies of homozygous allele 1 genotype among IM and non-IM were 19.16％and 11.26％, respectively. The frequencie of IM was significantly higher than that of non-IM(P=0.022). Logistic regression analysis adjusted by age and gender showed that subjects carrying the homozygous allele 1 genotype were at a 1.900-fold(95％CI 1.106-3.265).Paper3 The association of the interaction between PGC gene polymorphism and H.pylori and gastric diseases risk1. the single effect of PGC gene polymorphism, H.pylori infectionThere was no significant difference in the frequencies of PGC homozygous allele 1 genotype between GEU(11.35％)and NOR controls(7.09％, P=0.217), the significant differences between GA(19.15％)or GC(18.44％) and NOR controls(P=0.003, P=0.004, respectively). The OR value adjusted by age and gender were 3.112 (95％CI 1.443-6.710)and 3.000(95％CI 1.382-6.512), respectively.Compared with the frequencie of H.pylori-IgG positive in NOR controls(28.06％), that in GEU(46.10％) was significantly higher(P=0.002), and the OR value adjusted by age and gender was 2.063 (95％CI 1.259-3.380). There were no significant differences in GA(33.33％,P=0.319)or GC(38.30％,P=0.069).2. the synergistic effect of PGC gene polymorphism and H.pylori infectionIn the GEU group: Synergy index of PGC gene polymorphism and H.pylori infection was 5.40, the OR value was 8.692(P=0.049), suggesting a super multiplicative interaction. The result showed a significant type of 4 gene-environment interaction. The attributable proportion of interaction was 72.09％.In the GA group: Synergy index of PGC gene polymorphism and H.pylori infection was 6.48, the OR value was 11.159(P=0.024), suggesting a super multiplicative interaction. The result showed a significant type of 4 gene-environment interaction. The attributable proportion of interaction was 77.00％.In the GC group: Synergy index of PGC gene polymorphism and H.pylori infection was 4.34, the OR value was 10.612(P=0.028), suggesting a super multiplicative interaction. The result showed a significant type of 4 gene-environment interaction. The attributable proportion of interaction was 69.69％.Paper4 The relationship of PGC gene polymorphism and PGC protein expression1. PGC genetpye and PGC protein expression in different gastric diseasesAlong with GS→GEU→GA→GC:The frequencies of PGC homozygous allele 1 genotype increased in sequence, and those in GA and GC were significantly higher than that in GS respectively(P=0.020, P=0.010).Compared with that in GS, the concentration of serum PGC in GEU and GC were significantly higher(P=0.000, P=0.000, respectively), While that in GA was not(P= 0.127).The positive rates of PGC expression in situ decreased in sequence(P＜0.01), and there were significant differences among groups, except for GS-GEU(P＜0.05).2. Association of PGC gene polymorphism and PGC protein expressionThere were no significant correlation between PGC gene polymorphism and serum PGC concentration(P=0.435). After adjustment of age and gender, PGC homozygous allele 1 genotype was negatively correlated with PGC protein expression in situ(r=-0.1085, P=0.023). Along with PGC homozygous allele 1 genotype→heterozygous allele 1 genotype other→genotype: the positive rate of PGC increased in sequence, and there were significant differences between PGC homozygous allele 1 genotype and other genetype(P=0.009). While there were no significant differences of the hadro-strong positive rate among three groups.In the GS group: compared with other genotype, the hadro-positive rate decreased in PGC homozygous allele 1 genotype(P=0.047).There were no significant differences in group GEU, GA, and GC.Conclusion1. The frequencie of PGC homozygous allele 1 genotype carrier in zhuanghe, high risk area, was significantly higher than that in low risk area. The frequencie of PGC homozygous allele 1 genotype cartier in familial history group was significantly higher than that in non-familial history group. PGC gene polymorphism may be the special genetic factor for the high incidence of gastric cancer in Zhuanghe.2. The frequencies of homozygous allele 1 genotype carrier in IM, GA and GC were significantly higher than that in NOR and non-IM. There were significant increased risk of atrophic gastritis and gastric cancer in PGC homogenous allele 1 carriers. PGC gene polymorphism may impact on individual susceptibility to gastric cancer and precancerous atrophic diseases.3. PGC gene polymorphism and H.pylori infection seem to present a positive interaction in the development of gastric cancer, while no significant interactions of PGC gene polymorphism and H.pylori gene hypotype. 4. PGC homozygous allele 1 genotype was significantly negative-correlated with PGC protein expression in gastric mucosa. PGC gene polymorphism may effect on PGC protein expression in situ, while not on serum PGC.