Identification Of Sequence Polymorphisms In The Mitochondrial Cytochrome C Oxidase Genes As Risk Factors For Sporadic And Familial Breast Cancer

Objective: Currently,the breast carcinoma has been the most common malignant tumor in female.The breast carcinoma that occupied 25% of all malignant tumors in female which made it ranked the second incidence and the fifth cancer-related death worldwide.Aetiology of breast cancer including dietary patterns,obesity,occupational exposure,stress levels,smoking,alcohol consumption and heavy metals.Furthermore,family history of breast carcinoma also contribute to the carcinogenesis.But the true mechanism of this cancer remains unknown.Single nucleotide polymorphisms(SNPs)accumulated in the mitochondrial DNA(mtDNA)links to the tumor formation.Our previous researches discovered that SNPs in the mitochondrial displacement loop(D-loop)raised the risk of breast carcinoma.In this study,cancer risk-associated SNPs in the(cytochrome c oxidase)COX genes of mt DNA were assessed in sporadic breast carcinoma(SBC)and familial breast carcinoma(FBC)patients.Methods:1 Tissue specimens and DNA extraction.All the peripheral blood samples were collected at breast cancer center of the Fourth Hospital of Hebei Medical University(China)including 161 samples of sporadic breast carcinoma patients(SBC),and controls(C),67 samples of familial breast carcinoma patients(FBC),and 41 samples of relatives(R)of familial breast carcinoma from January 2008 to October 2015.All the breast cancer patients were confirmed by pathology.mtDNA extraction were carried out by using the TIANamo Genomic DNAki(TIANGEN Beijing China).2 Sequencing the mt DNA CO??CO??CO? genes.The primer pairs used for amplify the CO?(5904~7445 base),CO?(7586~8269 base)and CO?(9207~9990 base)genes were listed in Table 1.PCR was performed with the PCR Green Master Mix(Thermo,USA)and PCR production was purified prior to sequencing.Cycle sequencing was carried out with the Dye Terminator Cycle Sequencing Ready Reaction Kit(Applied Biosystems,Foster City,CA,USA)and separated using the ABI PRISM Genetic Analyzer 3100(Applied Biosystems).3 Statistical analysis.The ?~2 test was used to analyze statistical difference between different groups,such as the presence or absence of an individual SNP between sporadic breast cancer patients and healthy controls.P value under 0.05 was considered statistically significant.As to SNPs frequency,Kruskal-Wallis H test was used to compare the differences when there were four groups(P value of < 0.0126 was considered statistically significant.)and assisted by nemenyi grammar to do the comparison among groups.All of the statistical analysis was done with the SPSS version21 software package(SPSS Company,Chicago,IL).Results:In this study,We first analyzed mtDNA CO??CO??CO? genes sequences in 30 sporadic breast carcinoma patients,controls,familial breast carcinoma patients,and healthy relatives of familial breast carcinoma.When the P value was near to 0.05 or under it,we then expanded specimen volume.The volume of samples was expanded by using No.4 and No.6 primers.1 Between the sporadic breast carcinoma and the controls,clinical diagnosis staging,lymphatic metastasis,Menopausal status,HER-2 expression and ER/PR expression showed no statistical difference between this two groups.2 In the target genes,CO?(5904~7445 base)?CO?(7586~8269 base)?CO?(9207~9990 base),a total 60 SNPs(30 in CO?,5 in CO?,25 in CO?)were identified in the 3010 bp mt DNA coding region from breast cancer patient,controls and healthy relatives of familial breast cancer.Kruskal-Wallis H test assisted by nemenyi grammar was used to evaluate the association between the sum of COI,CO?,CO? genes SNPs per breast cancer patient and healthy relative of familial breast cancer versus the “no-cancer” control.We compared SNPs frequency in two different groups.Overall higher SNPs frequency was identified in sporadic breast cancer,relatives and familial breast cancer patients compared to healthy controls,but there was no difference among sporadic breast cancer,familial breast cancer patients and healthy relatives in the target genes 7100bp-7700 bp and 9300bp-10000 bp.3 Using ?~2 test,three of sixty SNPs(7196C/A,9545A/G,9548G/A)were identified as cancer risk-associated SNPs for sporadic breast cancer by comparing sporadic breast cancer patients and controls(Table4).Cancer risk-associated SNPs(9548G/A)for sporadic breast cancer were also identified as cancer risk-associated SNPs for familial ones.No SNPs were detected significantly different between healthy relatives and controls.Conclusion:1 In the target genes CO?(5904~7445 base)?CO?(7586~8269 base)?CO?(9207~9990 base),three of sixty SNPs(7196C/A,9545A/G,9548G/A)were identified as cancer risk-associated SNPs for sporadic breast cancer by comparing sporadic breast cancer patients and controls.2 Cancer risk-associated SNPs(9548G/A)for sporadic breast cancer were also identified as cancer risk-associated SNPs for familial ones3 higher SNPs frequency was identified in sporadic breast cancer,relatives and familial breast cancer patients compared to healthy controls in the target genes 7100bp-7700 bp and 9300bp-10000bp.