The Microsatelliteinstability At Position 8272 In Mitochondrial DNA And Age-at-onset Of Breast Cancer

Objective: Breast cancer is one of the most common malignant tumor in women, it is also one of the important cause of tumor which leading to the death in women, multiple genes and lots of factors involved, including obvious family history. Many studies have showed that the mutation of mitochondrial DNA is much common due to its lack of histone protection and effective repair system and proximity to electron transport chain dysfunction. Microsatelliteinstability is comparison between tumor tissue and normal tissue, the copy number variation of allele is that the change of microsatellite variable number of tandem repeats. In cancer patients, microsatelliteinstability have been shown in mt DNA, such as the 9-bp deletion at position 8272 of mt DNA in esophagus cancer and renal carcinoma.We sequenced and analyzed a region of MSI 8272 of mt DNA in peripheral blood of familial breast cancer patients, first-degree health female relatives and sporadic female breast cancer patients, and investigate the relationship of morbidity risk and age-at-onset between mt DNA microsatelliteinstability and breast cancer. The analysis may help to realize the breast cancer, thereby helping to provide new way of genetic diagnosis and treatment in familial breast cancer.Methods:1 Sample collectionThe collected sample are all female with a cancer-free history. Blood samples were collected from sporadic breast cancer patients, familial breast cancer patients and their relatives, and healthy controls. The relatives were selected from the female first-degree relatives of the familial patients, who had a cancer-free history and had no other known diseases that could be associated with mitochondrial defects. All the patients received treatment at the Breast Center of the Fourth Hospital of Hebei Medical University between 2008 and 2013. All of the patients were diagnosed histopathologically. All procedures were supervised and approved at the hospital by the Human Tissue Research Committee, informed consent was obtained from all participants before enrollment.2 Experimental ProcessThe genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit. PCR was performed with a PCR Master Mix Kit(Promega Corporation) and purified prior to sequencing. Primer pairs amplifying a 8272 base pair(bp) product of mt DNA region were designed as follows: forward primer 5′-CCCCATGCTTACAAGCAAGT-3′ and reverse primer 5′-GCTTTGAGGAGGTAAGCTAC-3′. The PCR product was sent to sequencing company.3 χ2 test(Pearson Chi-Square test) was used to analysis the differences of clinical types between sporadic breast cancer patients and familial breast cancer patients, meanwhile analysis mt MSI distribution frequency of each group.Statistical analysis:All of the statistical analysis was carried out with the SPSS 19.0 software. P<0.05 was considered statistically significant. 0.05<P<0.1 may be considered statistically significant.Results:1 Thirteen Polymorphism variant locuses were showed in the gene sequencing of 8154-8380 mt MSI DNA,aberration rate was about 5.7%.High frequency was defined as more than 5% MSI sample cases in each samples.In 192 experimental samples,there were 19 ones occured the 9-bp deletion at position 8272,about 9.9% of the total sample.2 χ2 test(Pearson Chi-Square test) was used to analysis MSI distribution frequency of 8272 site of 9-pb delection in the four experimental groups.P value was greater than 0.05 was defined as breast cancer had no association with the 9-bp deletion at position 8272.3 Kaplan-Meier was used to draw the curve of the age of onset.Log-Rank was used to analysis if the position 8272 MSI had relationship with the age of onset of sporadic breast cancer or familial breast cancer:the age of onset of familial breast cancer had no association with the 9-bp deletion at position 8272(P=0.876),The 9-bp deletion at position 8272 of mt DNA may be associated with the age-at-onset of sporadic breast cancer(P=0.090).Conclusions:1 The 9-bp deletion at position 8272 is a high frequency site of the 8154-8380 gene fragments in the mitochondrial gene.2 The 9-bp deletion at position 8272 of mt DNA was irrelevan to familial breast cancer and sporadic breast cancer.3 The 9-bp deletion at position 8272 of mt DNA was irrelevan to the age-at-onset of familial breast cancer, it may be associated with the age-at-onset of sporadic breast cancer.