Single Nucleotide Polymorphisms In The Mitochondrial Displacement Loop And Age-at-onset Of Familial Breast Cancer

Objective: Breast cancer is the leading cause of cancer in women, lots ofrisk factors, including family history, have been implicated in the developmentof breast cancer. Mitochondria play an important role in modulating oxidativestress, many studies have showed that mtDNA variations may contribute tocarcinogenesis. The mutation rate of mtDNA is much higher than that ofnuclear DNA due to its lack of histone protection, limited repair capacity andproximity to electron transport chain dysfunction. Single nucleotidepolymorphism is a variation that occurs when a single nucleotide sequence inthe genome differs, which is widely distributed in the entire human genome. Itcontributes to the genetic variations among individuals in term of genesequence. In cancer patients, single nucleotide polymorphisms have beenshown to accumulate extensively in the mitochondrial D-Loop region, and theassociation of these polymorphisms with cancer risk and disease outcomehave been exhaustively studied. We sequenced a region of the majority of theD-Loop in the DNA from the blood of breast cancer patients and the controlsfrom Hebei province to identify cancer risk-associated D-Loop SNPs, andinvestigate the association between germline SNPs of the D-Loop and age-at-onset in familial breast cancer patients. The analysis of genetic polymorphismsin the D-Loop may help to predict cancer risk for familial breast cancer,thereby helping to detect and refine therapeutic decisions earlier.Methods:1Sample collection: Blood samples were collected from sporadic breastcancer patients, familial breast cancer patients and their relatives, and healthycontrols. The relatives were selected from the female first-degree relatives ofthe familial patients, who had a cancer-free history and had no other knowndiseases that could be associated with mitochondrial defects. All the patients received treatment at the Breast Center of the Fourth Hospital of HebeiMedical University between2008and2013. All of the patients werediagnosed histopathologically. All procedures were supervised and approvedat the hospital by the Human Tissue Research Committee, informed consentwas obtained from all participants before enrollment.2DNA extraction, amplification and sequencing: The genomic DNA wasextracted using the Wizard Genomic DNA Purification Kit and stored at-20°C.PCR was performed with a PCR Master Mix Kit (Promega Corporation)and purified prior to sequencing. Primer pairs amplifying a982base pair (bp)product of D-Loop region were designed as follows: forward primer5′-CCCCATGCTTACAAGCAAGT-3′(nucleotides16190-16209) and reverseprimer5′-GCTTTGAGGAGGTAAGCTAC-3′(nucleotides602-583). Cyclesequencing was carried out with the BigDye Terminator v3.1CycleSequencing Kit (Life Technologies, Carlsbad, CA, USA), and the productswere then separated on an ABI PRISM3100Genetic Analyzer (LifeTechnologies). Polymorphisms were confirmed by repeated analyses fromboth strands.3Statistical analysis: χ2test (Pearson Chi-Square test) was used toanalysis the difference of clinical characteristics between familial breastcancer patients and sporadic breast cancer patients, and the distributionfrequency of SNP numbers among each group. The univariate analysis wasperformed by Kaplan-Meier method and Log-Rank method. All of thestatistical analysis was carried out with the SPSS19.0software. A P-value of<0.05was considered statistically significant.Results:1Ninety-two sporadic breast cancer patients,60familial breast cancerpatients and41relatives, and93healthy controls were enrolled in our study.The distribution of pathological types was significantly different betweensporadic and familial breast cancer patients (p=0.020), while clinicalcharacteristics of age, TNM classification, lymphatic metastases, menopausalstate, Her-2expression and ER/PR expression showed no statistical difference between the two groups.2SNPs were detected in101sites within the982bp mitochondria D-loopregion from the four groups. The distribution frequency of SNP numbers wascompared among the breast cancer patients, relatives and controls. The SNPswere more highly concentrated in sporadic and familial breast cancer patientsthan in the controls, with even the healthy relatives of familial breast cancersshowing higher SNP numbers than the controls.3Fifty-nine SNPs with a minor allele frequency higher than5%in eithergroup were used for cancer risk analysis by the χ2test subsequently. Thedistribution frequency of315C/Cinsert,524C/del,16247A/del,16248C/del,16249T/C,16257C/A,16258A/del,16259C/del,16262C/del,16268C/del,16279C/del,16280A/del,16297T/C and16300A/del were significantlydifferent between sporadic breast cancer patients and the controls. All of thecancer risk-associated SNPs for sporadic breast cancer were also identified ascancer risk-associated SNPs for familial ones.4Nucleotide positions at positions310,16319and16362were identifiedas cancer risk-associated SNPs specific for familial breast cancer bycomparing the familial breast patients and controls.5As a cancer risk-associated SNP for both familial breast cancer andsporadic breast cancer, the distribution frequency for the minor allele atposition16247was higher in familial breast cancer than in sporadic breastcancer.6The allele489T/C was identified as a specific risk-associated SNP forthe families of breast cancer patients by comparing familial breast cancerpatients with their relatives.7The age-at-onset of the patients with the C genotype was significantlylower than that of patients with the T genotype at the site16311(p=0.022).Conclusions:1Familial breast cancer patients were more susceptible to non invasiveductal carcinoma, including some highly invasive ones, such as medullarycancer， which might contribute to the more malignant of familial breast cancer.2The D-loop region of mtDNA is the polymorphisms hotspot. Themajority of the mtDNA polymorphisms observed in breast cancer patientswere the transition or deletion of the single base, most of which were locatedin the hypervariable segment I (HV1) and segment II (HV2) region.3Nucleotide positions at310,16319and16362were identified as cancerrisk-associated SNPs specific for familial breast cancer. The transition ofposition16319might accelerate the cancer-associated risks of the familialbreast cancer members, and the transition of positions at310and16362mightdecrease it.4The distribution frequency for the minor allele at16247was higher infamilial breast cancer than in sporadic ones. Which may suggest that agenealogy has high transition of this position would have more chance to getbreast cancer.5489T/C was identified as a specific risk-associated SNP for the familiesof breast cancer patients. Which means that a family member，who has Tgenotype at this position, would be more susceptible to breast cancer.6The SNP sites of nucleotides16311T/C were identified for theirassociation with age-at-onset of familial breast cancer.