The Research Of The Relation Between Bladder Cancer Recurrence And Multiple Loci

Bladder cancer is a common malignant tumor with a high morbidity and recurrence rate. Until now, there are a small number of genome-wide association studies (GWAS) screening reports of bladder cancer risk related genes. But there is no report of screening of bladder recurrence-related genes with this method. This issue, which contains2parts as "the verification of the genes which might be associated with the risk of bladder cancer" and "the screening of genes which might be associated with bladder cancer recurrence", verified a part of the loci which have been reported as related genes with the risk of bladder cancer were also associated with bladder cancer among Chinese population. It has also screened some loci which might be associated with the recurrence of bladder cancer, proving the feasibility of GWAS to screen the candidate genes associated with the recurrence of bladder cancer.Part I The verification of the genes which might be associated with the risk of bladder cancerObjective:To verify whether the genes which have been reported as related SNPs with the risk of bladder cancer were also associated with bladder cancer among Chinese population by the molecular genetics method with genome-wide association studies (GWAS) as the core means. To discuss the feasibility of explore the differences between different groups of people among Chinese population with the methods above.Materials and Methods:The patients who were diagnosed as urothelial carcinoma by pathology were recruited from2011in Huashan Hospital of Fudan University with a number of164. A5ml venous blood was collected from each patient after they signed informed consent. The control group with a number of1008tumor free people was also recruited, and a5ml venous blood was also collected from each people.16SNPs which have been reported might be associated with the risk of bladder cancer were selected as the focus of the study. The5ml venous blood of the patients with bladder cancer and the healthy control volunteers was placed into the ethylenediamine tetraacetic acid dipotassium (EDTA) anticoagulant tube, mixed immediately, centrifuged, removed the serum and obtained the blood cells. The total RNA was extracted from peripheral blood samples. The cDNA was synthesized by reverse transcription of total RNA. Then the cRNA was synthesized, purified, labeled and fragmentized. The hybridization of gene chips was performed and the scanning of the chips was finished at last. The clinic characters of the patients were summarized, including name, sex, age, smoking history and the pathological diagnosis, etc. The characters of the healthy control volunteers were also summarized. The genotype distributions for all SNPs were tested for Hardy-Weinberg equilibrium (HWE) according to the Fisher’s exact test. The allelic odds ratio (OR) and95%confidence interval (CI) were estimated using a logistic regression model, adjusting for age and smoking status (yes/no). All analyses were conducted using PLINK software. P-values were two-tailed. An alpha of0.05was used to claim statistical significance.Result:The clinic characters, including sex, age, and smoking history of the2groups showed no significant difference.2SNPs were found with the significant differences between the patient group and control group of the distribution of allele. They are rs798766(C>T, p=0.00163), which is in the5th intron of gene tacc-3on4p16, and rs9642880(G>T, p=0.04587), which is at the upstream of myc gene on8q24. A potential SNP which might also be associated with bladder cancer was also found which is rs2294008(C>T) in the1st extron of the gene psca.Conclusion:rs798766, rs9642880and rs2294008showed varying degrees of differences of the distribution between the patient group and control group, which pointed these SNPs could be genetic diagnostic marker of bladder cancer. This part of the issue also proved that it is feasible to explore the differences of allele distribution in some SNPs between different groups among Chinese population by the theory of GWAS. Part Ⅱ The screening of genes/loci which might be associated with bladder cancer recurrenceObjective:To screen the candidate SNPs which might be associated with bladder cancer recurrence among Chinese population by the molecular genetics method with genome-wide association studies (GWAS) as the core means based on the results of part Ⅰ. To discuss the feasibility of screening the genes/loci associated with bladder cancer recurrence among Chinese population with the methods above.Materials and Methods:The164patients with bladder cancer were recruited and classified into recurrence group (n=40) and non-recurrence group (n=124). The5ml venous blood of the patients with bladder cancer and the healthy control volunteers was placed into the ethylenediamine tetraacetic acid dipotassium (EDTA) anticoagulant tube, mixed immediately, centrifuged, removed the serum and obtained the blood cells. The total RNA was extracted from peripheral blood samples. The cDNA was synthesized by reverse transcription of total RNA. Then the cRNA was synthesized, purified, labeled and fragmentized. The hybridization of gene chips was performed and the scanning of the chips was finished at last. The16SNPs which have been reported might be associated with the risk of bladder cancer were also selected as the focus of the study. The detail clinic characters of the patients were summarized, including name, sex, age, smoking history, the pathological diagnosis, the number and size of the tumors, the circumstances of recurrence, etc. The genotype distributions for all SNPs were tested for Hardy-Weinberg equilibrium (HWE) according to the χ2test and Fisher’s exact test. The allelic odds ratio (OR) and95%confidence interval (CI) were estimated using a logistic regression model, adjusting for age and smoking status (yes/no). All analyses were conducted using STATA12.0and PLINK software. P-values were two-tailed. An alpha of0.05was used to claim statistical significance.Results:The clinic characters, including sex, age, smoking history and the stage/grade of the tumors of the2groups showed no significant difference.2SNPs were found with the significant differences between the patient group and control group of the distribution of genotype whose specific genotypes showed protective function to decrease the recurrence of bladder cancer. They are rs1495741(GG genotype, p=0.013), which is on8p22, and rs13181(GT genotype, p=0.024), which is on19q13.1SNP was found with the significant differences between the patient group and control group of the distribution of allele, which is rs8102137(T>C, p=0.03) in the gene ccne-1on19q13.Conclusions:rs1495741,rs13181, rs8102137showed significant differences in the distribution of genotype or allele, which pointed these SNPs could be genetic diagnostic marker of bladder cancer recurrence. This part of the issue also proved that it is feasible to explore the differences of allele distribution in some SNPs between different groups among Chinese population by the theory of GWAS and the core technical of gene chip.