The Associations Study Of Single Nucleotide Polymorphisms Of DNA Repairgenes XRCC1and OGG1with Susceptibility To Pancreatic Cancer

Objective:1. Exploring the correlation between human X-ray repair cross complementary gene1（XRCC1） c.1471g> A single nucleotide polymorphisms and pancreatic cancer risk.2. Exploring the correlation between human X-ray repair cross complementary gene1（XRCC1） c.1686c> G of single nucleotide polymorphisms and pancreatic cancer risk.3. Exploring the correlation betweenhuman8-hydroxy guanine DNA glycosidase gene1（OGG1） c.269c> A single nucleotide polymorphism and the risk of pancreatic cancer in the association.4. Exploring the correlation between human8-hydroxy guanine DNA glycosidase gene1（OGG1） c.6271> c single nucleotide polymorphisms and pancreatic cancer risk.Materials and Methods:1. In XRCC1gene research:328han pancreatic cancer patients（case） and350nontumorous with hospitalized patients （control） were chose for experiment. And the venous blood was collected at the same period, the age, gender, smoking, drinking, family history, body mass index, diabetes factors and frequency are matched between case and control samples, And All samples were from the third affiliated hospital, third military medical university hospital （Jan.2009to Oct.2012）2. In XRCC1gene research:347han pancreatic cancer patients（case） and364nontumorous with hospitalized patients （control） were chose for experiment. And the venous blood was collected at the same period, the age, gender, smoking, drinking, family history, body mass index, diabetes factors and frequency are matched between case and control samples, All the samples were from third affiliated hospital of third military medical inpatients （Jan.2009to Oct.2012）3. Case-control study was used to analysis the genotype data. First of all, The PCR-RFLP parting technology were used to detect the genotypes of c. XRCC1gene1471G> A and c.627T> C locus single nucleotide polymorphisms. Then, the chi-square （x2） test was used to analysis the genotype and allele frequency of Hardy Weinberg equilibrium. And using chi-square （χ2） test to evaluate the factors related to clinical pancreatic cancer, And unconditioned Logistic regression analysis was used to estimate the odds ratio （OR） corresponding to95%confidence interval （CI）, line of single nucleotide polymorphisms associated with pancreatic cancer risk analysis, threshold of statistical significance is P values<0.05.4. OGG1gene c.269c> A and c.1686c> G locus polymorphic genotypes were detected by PCR-RFLP parting technology, though the case-control study. And using chi-square （χ2） test to evaluate the factors related to clinical pancreatic cancer Using chi-square （χ2） test analysis, to assess the genotype and allele frequency of Hardy Weinberg equilibrium, and unconditioned Logistic regression analysis was used to estimate the odds ratio （OR） corresponding to95%confidence interval （CI）. For all the statistical significance analysis, threshold of statistical significance is P values<0.05.Results:1. In the study, A total of678subjects were collected for detect the2SNPs of human XRCC1gene, including328patients with pancreatic cancer and350healthy controls.Between the case and control groups, gender, age, smoking status, alcohol use, body mass index, diabetes, and family history of pancreatic cancer control study showed no significant statistical difference （P>0.05）. And no Hardy-Weinberg equilibrium were found （P>0.05）.2. Two genetic variants （c.1471G> A and c.1686C>G） were found in this study.Sequence analysis indicated that the c.1471G>A genetic variant was a non-synonymous mutation and genotyped by CRS-PCR method, which caused by G to A mutations in exon13of human XRCC1gene and resulted in glutamic （Glu） to lysine （Lys） amino acid replacement （p.Glu491Lys, reference sequences GenBank IDs:NC₀00019.9, NM₀06297.2, and NP₀06288.2）. The PCR-amplified products of this genetic variant were digested with AlwNI restriction enzyme and divided into three genotypes, GG （198and19bp）, GA（217,198, and19bp）, and AA （217bp, Table2）. As for c.1686>G genetic variant, it was a synonymous mutation and detected by PCR RFLP method, which caused by C to G mutations in exon15of human XRCC1gene （p. leucine （Leu）562Leu）. The PCR amplified products of this genetic variant were digested with Mbol restriction enzyme and divided into three genotypes, CC （232bp）, CG （232,181, and51bp）, and GG （181and51bp）.3. The PC patients showed a higher frequencies of G-allele of c.1471G>A SNP and C-allele of c.1686>G genetic variants than cancer-free controls.The G-allele of c.1471G>A SNP and C-allele of c.1686>G SNP were predominant alleles in the studied subjects. The allele frequencies of c.1471G>A SNP in PC patients （G,71.49%; A,28.51%） were significantly different from cancer-free controls （G,62.00%; A,38.00%; χ2=13.7203, P=0.0002）. In addition, the genotype frequencies in PC patients were not consistent with cancer free controls, the differences being statistically significant （χ2=12.8496, P=0.0016）. As for c.1686C>G SNP, the allele frequencies of PC patients （C,72.87%; G,27.13%） were significantly different from cancer-free controls （C,65.14%; G,34.86%, χ2=9.4227, P=0.0021）. The genotype frequencies in PC patients were not consistent with cancer free controls, the differences being statistically significant （χ2=8.8232, P=0.0002）.4. The association between the risk of PC and XRCC1SNPs.As for c.1471G>A, the significantly decreased risk of PC were found in the homozygote comparison （AA vs. GG:OR=0.43,95%CI0.26-0.70, χ2=11.91, P=0.001）, heterozygote comparison （GA vs. GG:OR=0.72,95%CI0.52-1.00, χ2=4.01, P=0.045）, dominant model （AA/GA vs. GG:OR=0.63,95%CI0.47-0.86, χ2=8.65, P=0.003）, recessive model （AA vs. GA/GG:OR=0.50,95%CI0.31-0.79, χ2=8.82, P=0.003） and allele contrast （A vs. G:OR=0.65,95%CI0.52-0.82, χ2=13.71, P<0.001）. Similarly, as for c.1686C>G, the significantly decreased risk of PC were detected in the homozygote comparison （GG vs. CC:OR=0.48,95%CI0.29-0.81, χ2=7.98, P=0.005）, dominant model （GG/GC vs. CC:OR=0.69,95%CI0.51-0.93, χ2=5.99, P=0.014）, recessive model （GG vs. GC/CC:OR=0.55,95%CI0.34-0.89, χ2=5.98, P=0.014）, and allele contrast （G vs. C:OR=0.70,95%CI0.55-0.88, x2=9.42, P=0.002）.5. In the study, A total of711subjects were collected for detect the2SNPs of human OGG1gene, including347patients with pancreatic cancer and347healthy controls.The chi squared （x2） test was performed to assess the Hardy-Weinberg equilibrium in genotype and allele frequencies and the differences of characteristics such as gender, age, smoking status, alcohol consumption, body mass index, diabetes mellitus, and family history of PC between cases and controls. The odds ratios （ORs） with their95%confidence intervals （CIs） for PC risk in relation to selected SNPs were estimated using an unconditional logistic regression analysis. A P value less than0.05was regarded as statistically significant.6. Two single nucleotide polymorphism loci mutation of human OGG1gene were found association with the risk of PC. The results of sequence analysis revealed that the c.269C>A SNP was a non-synonymous mutation and genotyped by CRS-PCR method, which caused by C to A mutations in exon2of human OGG1gene and resulted in proline （Pro） to Gln amino acid replacement （p.Pro90Gln, reference sequences GenBank ID nos. NG₀12106.1, NM₀02542.5, and NP₀02533.1）. The PCR products of this SNP were digested with HpaⅡ restriction enzyme and divided into three genotypes:CC （192and18bp）, CA （210,192, and18bp）, and AA （210bp）. As for c.627T>C SNP, sequence analysis indicated that it was a synonymous mutation and genotyped by PCR-RFLP method, which caused by T to C mutations in exon4of human OGG1gene （p.Ser209Ser）. The PCR products of this SNP were digested with HhaⅠ restriction enzyme and divided into three genotypes:TT （243bp）, TC （243,164, and79bp）, and CC （164and79bp）.7. Comparing Pancreatic cancer group with control group, The PC patients showed a higher frequencies of G-allele of human OGG1gene loci cDNA263alleles A> G mutation and G-allele of Human OGG1gene cDNA627C> G than cancer-free controls.The C allele of C.267>A SNP and T allele of c.627T>C SNP were predominant alleles in the studied populations. As for c.269C>A SNP, the allele frequencies of PC patients （C,72.77%; A,27.23%） were significantly different from healthy controls （C,64.70%; A,35.30%; χ2=10.7453, P=0.0010）. Besides, the genotype frequencies of PC patients were not consistent with healthy controls, the differences being statistically significant （χ2=l0.7380, P=0.0047）. As for c.627T>C SNP, the allele frequencies of PC patients （T,72.91%; C,27.09%） were significantly different from healthy controls （T,66.07%; C,33.93%, χ2=7.8271, P=0.0051）. The genotype frequencies of PC patients were not consistent with healthy controls, the differences being statistically significant （χ2=7.3475,P=0.0254）. 8. As the analysis results showed:SNPs of human cDNA263OGGl gene have remarkable correlations with pancreatic cancer risk.For c.269C>A SNP, the significantly decreased risk of PC was found in the homozygote comparison （AA versus （vs.） CC, OR=0.44,95%CI0.27-0.73, Z=3.18, P=0.001）, dominant model （AA/CA vs. CC, OR=0.69,95%CI0.52-0.93, Z=2.42, P=0.015）, recessive model （AA vs. CA/CC, OR=0.49,95%CI0.31-0.80, Z=2.88, P=0.004）, and allele contrast （A vs. C, OR=0.69,95%CI0.55-0.86, Z=3.27, P=0.001）. As for c.627T>C SNP, the significantly decreased risk of PC was detected in the homozygote comparison （CC vs. TT, OR=0.57,95%CI0.35-0.94, Z=2.19, P=0.028）, heterozygote comparison （TC vs. TT, OR=0.71,95%CI0.52-0.97, Z=2.13, P=0.033）, dominant model （CC/TC vs. TT, OR=0.68,95%CI0.50-0.91, Z=2.59, P=0.010）, and allele contrast （C vs. T, OR=0.72,95%CI0.58-0.91, Z=2.79, P=0.005）.Conclusion:1. The allele A of c.269C>A and allele C of c.627T>C might be associated with a protection from PC （for c.269>A, A versus （vs.） C, OR=0.69,95%CI0.55-0.86, P <0.001; for c.627T>C, C vs. T, OR=0.72,95%CI0.58-0.91, P=0.005）.2. Results from this study indicate that the c.269>A and c.627T>C SNPs of OGG1gene are associated with PC susceptibility of Han nationality in southwest China.3. The A allele of c.1471G>A and G allele of c.1686C>G genetic variants could contribute to decrease the risk of PC （for c.1471G>A:A vs G, OR=0.65,95%CI0.52-0.82,30=13.71, P<0.001, for c.1686>G:G vs C, OR=0.70,95%CI0.55-0.88, X2=9.42,P=0.002）.4. Our studies indicate that the c.1471G>A and c.1686>G polymorphisms of XRCC1gene are associated with PC risk of Han nationality in southwest China.