Genetic Polymorphism Research Of Hot-spot Mutation Regions Of BRCA1/2 And VDR Gene And Their Relationships With Kazakh’s Breast Cancers In Xinjiang

Objective: Nowadays, breast cancer is the most common malignant tumor in female population all over the world. Malignant tumor is the outcome of combined action of many genes, and the scholars in the whole world committed to the research of breast cancer susceptibility genes, in the hope of using genetic screening in high-risk groups not only for early intervention but also to reduce morbidity and mortality of breast cancer.BRCA1 and BRCA2 are the primary breast cancer susceptibility genes. The research confirmed that BRCA1 and VDR gene with common targets, thus inhibiting the growth of breast cancer cells. China’s breast cancer susceptibility gene mutation spectrum obviously inadequate or even blank, especially in the areas inhabited by minority nationalities in Xinjiang, breast cancer susceptibility gene research mainly revolves around Han, Uygur.As the third largest ethnic groups in Xinjiang- Kazakh was few related in gene research reports, so our study is in order to preliminarily discuss the polymorphism distributions of BRCA1/2 and VDR hot spot mutations area/loci of Kazakh breast cancer. At the same time, we also analyzed the correlation of three genes, protein expressions, the relationships between genes or protein expressions and clinical pathological features, and also to complement the national breast cancer susceptibility gene mutation spectrum, ultimately for early prevention, early treatment and prognosis judgement of the Kazakh breast cancer.Methods:(1) We detected hot mutation fields of BRCA1 and BRCA2 between 164 Kazakh breast cancer patients and 127 Kazakh healthy adult women in Xinjiang, and then compared the SNPs which were detected in our research with human genome database to make sure whether the presence of newly discovered SNP loci by polymerase chain reaction and direct sequencing method. We explored the susceptibility SNP loci of Kazakhnational breast cancers, analyzed the correlations between susceptibility SNPs and breast cancer, and discussed the clinical pathology characteristic in case-control study.(2) We detected five hot-spot mutation locus of VDR gene between 164 Kazakh breast cancer patients and 127 Kazakh healthy adult women by polymerase chain reaction- restriction fragment length polymorphism methods. We looked for the SNP locus associated with the Kazakh breast cancer, analyzed its clinical pathological features.(3) 57 Kazakh breast cancer patients were collected to detect and compare the protein expression of BRCA1,BRCA2 and VDR between carcinoma tissues and normal breast tissues adjacent to the lesions using immunohistochemical method. The connection between the protein expression and gene polymorphisms was explored. The correlations protein expressions of three genes in carcinoma tissues and their clinical pathological features were analyzed.Results:(1) 18 SNP loci were detected in the hot-spot mutation regions of BRCA1 and BRCA2, including 7 SNPs in exon 10 of BRCA1 gene, 1 SNP in intrion 18, 3 SNPs in exon 10 of BRCA2 gene and 7 SNPs in exon 11, which were neither new SNPs nor pathogenic mutations. Only 3 SNPs’ allele frequency distributions were different in the case and control groups(P<0.05). The risk of breast cancer were significantly increased while carrying mutation genotypes of these 3 SNPs(P<0.05), including rs1799950 in BRCA1 and rs1801499 and rs1799944 in BRCA2, which were closely related with clinical pathological factors. We observed a statistically significant interaction for rs1799950 polymorphism and ER or PR status in a case-only model(P<0.05). Comparing AG genotype carriers with noncarriers, we found a statistically significantly increased OR of 4.422 for ER-negative tumors, and an OR of 3.888 for PR-positive tumors(P<0.05). No statistically significant interaction was observed between rs1799950 polymorphism and Her-2 status(P > 0.05). Comparing AG genotype carriers with noncarriers, we found a statistically significantly increased OR of 4.397 for Ki-67 high expression tumors, an OR of 4.163 for invasive carcinoma tumors(P<0.05), an OR of 6.496 for TNM Ⅱ stage(P<0.05), and ORs of 3.782 and 4.401 for histological grade levels 2 and 3 period(P<0.05). Comparing TC genotype carriers of rs1801499 and AG genotype carriers of rs1799944 with noncarriers, we found a statistically significantly increased OR of 3.017 for ER-positive tumors, and an OR of 2.915 for PR-positive tumors(P<0.05). We found a statistically significantly increased OR of 2.123 for Her-2-positive tumors, an OR of 2.373 for Ki-67 high expression tumors, an OR of 3.375 for node-negative tumors(P<0.05),an OR of 4.163 for invasive carcinoma tumors(P<0.05), ORs of 2.526 and 3.207 for TNMⅠand Ⅲ stage(P<0.05), and ORs of 3.170 and 2.342 for histological grade levels 1 and3 period(P<0.05).(2) The allele frequency distributions of 5 hot-spot SNPs in VDR were compared between case and control groups, which contained Fok I(rs2228570), Bsm I(rs1544410), Tru9 Ⅰ(Mse I)(rs757343), Apa I(rs7975232), Taq I(rs731236), and statistically significant differences were found(P<0.05). The risk of breast cancer were significantly reduced while carrying mutation genotypes of these 2 SNPs(P<0.05),including Fok I(rs2228570) and Apa I(rs7975232) in VDR, and Apa I(rs7975232) was closely related with clinical pathological factors. Comparing TT genotype carriers with noncarriers, we found a statistically significantly reduced OR of 0.639 for ER-negative tumors, an OR of 0.604 for Her-2-positive tumor, an OR of 0.523 for TNMⅢstatus and an OR of 0.540 for lymph node-negative tumors(P<0.05). No statistically significant interaction was observed between GT and TT genotypes and pathological type or histological grade(P > 0.05). Comparing GT genotype carriers with noncarriers, we found a statistically significantly reduced OR of 0.475 for TNMⅡstatus(P<0.05).(3) We found the protein expressions of BRCA1, BRCA2 and VDR present in different parts with various degrees between breast carcinoma and adjacent tissues of 57 Kazakh breast cancer patients. BRCA1 proteins were mainly located in cytoplasm and nuclei, BRCA2 proteins were mainly distributed in the cytoplasm and cell membrane, the VDR protein mainly distributed in the cytoplasm and cell membrane. BRCA1 and VDR expressed negatively or lowly in normal breast tissue, while more significantly higher expressing in malignant tumor(P<0.05). And BRCA2 expression in the breast tissue was close to normal breast tissue(P>0.05). BRCA1/2 protein expression are positively correlated with PR and Ki-67(P<0.05). The protein expression of BRCA1 respectively positively correlated with protein expression of BRCA2 and VDR(P < 0.05). Conclusion:(1) We found susceptibility/protective SNPs of BRCA1/2 and VDR gene hot-spot mutation regions/sites in Kazakh breast cancers in Xinjiang, which were different from mutations of other ethnic groups. The results not only complemented the Kazakh breast cancer susceptibility gene mutation spectrum, but also helped screening for the Kazakh population at high risk of breast cancer.(2) The susceptibility/protective SNPs of BRCA1/2 and VDR gene were closely related to the different clinical pathological status, which provided important reference basis for judging the clinical treatment and prognosis of Kazakh breast cancer patients.(3) BRCA1 and VDR may jointly participated in the process of breast cancer. We can detect BRCA1 and VDR protein expression level by immunohistochemical method to judge the prognosis of breast cancer and to guide treatment. Vitamin D may be applied to the targeted therapy of breast cancer in the future, and provide a certain theoretical basisfor prevention in high-risk group. The function of common mechanism need further verification.