Molecular Characterization of Breast Cancer in Sudanese Women Using PvuII, Xbal, and Hinfl Single Nucleotide Polymorphisms of ESR1 Gene

Abstract

Abstract Background Breast cancer (BC) is the most frequently diagnosed and leading cause of cancer deaths among females world wide. It has been identified as the most common malignancy among Sudanese Women, accounting for 34% of all female cancers. The prolonged exposure to estrogen is well established risk factor for breast cancer. The effect of estrogen hormone on target tissues is mediated by its alpha receptor, through the binding of the hormone to the receptor Which in turn promote the proliferation and differentiation of mammary tissues. The estrogen receptor alpha is coded by ESRI gene which is genetically polymorphic. Aim The aim of this study was to evaluate the association between (Pvull, Xbal, and Hinfl) single nucleotide polymorphisms of the estrogen receptor alpha gene and BC risk, types, estrogen receptor status, and other risk factors such as mammographic density, family history, age, parity, and age at menarche, in Sudanese BC Women. Materials and Methods In an analytical case-control study, the three single nucleotide polymorphisms were genotyped in 139 BC cases and 139 age-matched breast cancer-free controls. A specimen of 3 ml of peripheral blood was drawn from each subject, from which the leukocyte DNA Was extracted using a modified salting out method, the purity and quantity of the extracted DNA was then assessed spectrophotometerically. The genotyping of the three SNPs was carried out by PCR-RFLP method using Pvull, Xbal, and

Hinfl restriction enzymes, the products were then separated electrophoretically. The estrogen receptor statuses for 65 cases were determined on a freshly obtained breast tissues that were fixed in formalin and embedded in paraffin according to standard histological procedures, the sections prepared are then stained using immunoperoxidase staining method. The mammographic densities of cases and controls were determined by assessing the mammogram of every subject by a radiologist and mammographam specialist independently. Data obtained were analyzed using SPSS, to calculate the frequency distribution for age, marital status, parity status, menopausal status, and age at menarche. The associations were calculated using chi square test. The unadjusted and adjusted logistic regression were used to calculate the odds ratio (Ors) and 95% confidence intervals (CI) for assessing the association between a genotypic and allelic frequencies and BC. Hardy-Weinberg equilibrium online calculator was used to determine the deviation of genotypic frequencies from Hardy-Weinberg equilibrium. Results The results of BC cases showed a young mean age 46.5il0.4SD years, high frequencies of married women 97.1%, and parity 89.9%. The family history of breast or other type of cancers in Sudanese women, proved to be the strongest risk factor as compared to all factors such as age, mammographic density, age at menarche, parity, marital status, and menopausal status previously believed to be associated with BC, as those have family history of the disease showed an 11 folds increase in the risk of developing BC (OR: 11.8, 95%CI: 4-34.2, P-value .000) as compared to those have no family history. Breast cancer among Sudanese women also showed a higher frequency of the aggressive invasive ductal carcinoma (70.5%), with higher percentage of receptor positive status (63%). The results of minor allele frequency showed an association between the G allele of the Xbal SNP and the risk of BC (P-value = 0.014). Of all the three studied SNPs, rs9340799 (X12411) was the only one to show statistically significant association with the risk of BC (P-value I 0.03), where the calculated odds ratios (OR) and 95% confidence intervals (95%CI) using unadjusted logistic regression of the genotypic variants of this SNP showed that Xx genotype had (OR:l.6, 95%CI : 0.85-3.04), and xx genotype (OR= 2.6, 95%CI:l.23-5.46), while using the adjusted logistic regression, the Xx genotype showed (OR= 1.02, 95%CI: 0.75-1.4) (P-value = 0.000), and xx genotype showed (OR= 1.65, 95%CI: 0.95-2.87) (P-value = 0.01). The effect of xx genotype was evidently seen in parous women (OR=2.3, 95%CI:l.l-5.01) (P-value = 0.03). The association of genotypic variants of different SNPs with estrogen receptor status, showed a statistically significant association between Xx variant of (Xbal) with positive receptor status (OR: 5.3, 95%CI: 1.8-15.8) (P-value = 0.002). The other two SNPs (Pvull and Hinfl) showed statistically significant association only with mammographic density (P- value =0.000 and 0.01 respectively). Conclusion In conclusion, the findings of this study provide a persuasive indication of strong association between the homozygous recessive and heterozygous genotypes Xbal SNP and risk of BC in Sudanese women, and no clear association between the genotypic variants of the other two SNPs, namely, Pvull and Hinfl with the risk of developing the disease.