br In this study the polymorphic
In this study, the polymorphic variation in EPZ31686 that take part in the formation of catechol estrogens (CYP1A1 and CYP1B1) and their conjugation (SULT1A1) were analyzed to determine the association of functional polymorphisms in these genes to the risk of breast malig-nancy.
2. Materials and methods
The present study consisted of 200 histologically confirmed breast
Genotype and allele frequencies of studied SNP's in steroid metabolizing pathway in breast cancer patients and controls.
Genes regulating steroid metabolizing pathway: activation of procarcinogens/estrogens
A χ2 test was performed to evaluate the association between SNP and breast cancer cases. The genotypes were verified to comply with the HW; OR and 95% CI were calculated to assess the relative risk; p < 0.05 was considered to be statistically significant.
Fig. 1. An LD map showing the pairwise LD between the variants (SNPs) in the gene CYP1A1 (rs4646903, rs1048943) in the cases and normal controls. Measures of pairwise linkage disequilibrium (LD) for the CYP1A1 gene poly-morphism is presented. The distance between these two marks is 1.34 kb and pairwise LD values (D′ = 0.12 and r2 = 0.001) indicating these variants were not in LD and failed to LD blocks (Fig. 1). Haplotype analysis was not in-formative.
cancer patients from Southern India. The patients were from the Department of Oncology of Sri Ramachandra Institute of Higher Education and Research, Chennai. Relevant clinical and pathological data were gathered from all the patients. Pathological grading of the tumors was obtained by the histopathological examination. The in-clusion criteria for the healthy volunteers include no previous diag-nosis of any benign breast diseases; no history of mastectomy, oo-phorectomy, or hysterectomy; no family history of ovarian, breast, endometrial, and prostate cancer; and no mental or physical dis-ability. The patients and controls were similar in ethnicity and nu-tritional habits, and they were age matched. The criteria for breast cancer patients were no previous treatment for cancer and con-firmation of breast malignancy with histological diagnosis. Informed consent was mandatory for both groups. Ethical clearance was ob-tained from the institutional ethical committee for use of samples from control and breast cancer–affected patients. Informed consent was obtained in written format from all the subjects enrolled for the study.
2.2. Molecular analysis by Taqman allelic discrimination assay
Peripheral blood lymphocyte samples of about 3 mL were obtained
Stratified analysis of the genotypes of the steroid metabolizing pathway with clinical characteristics like menopausal status.
Genotype Controls No (%) Patients No (%) OR 95% CI p value
Carcinogen/estrogen metabolizing pathway
CYP1A1 (rs4646903) genotype among breast cancer patients and control stratified by age at diagnosis
CYP1A1 (rs1048943) genotype among breast cancer patients and control stratified by age at diagnosis
CYP1B1 rs1056836 genotype among breast cancer patients and control stratified by age at diagnosis
SULT1A1 rs9282861 genotype among breast cancer patients and control stratified by age at diagnosis
by veinpuncture and collected into K2-EDTA vacutainers. DNA isolated from peripheral blood samples using salting-out method was analyzed for selected SNPs by Taqman SNP methodology with real-time poly-merase chain reaction technology (Taqman SNP Genotyping Assay, Life Technologies Applied Biosystems, Carlsbad, USA). The DNA isolated was amplified using Taq Gold Polymerase of Taqman PCR master mix in ABI machine ABI Quant studio 6 flex using sequence-specific primers. The reaction volume was set to 5 μL, consisting of 2.50 μL of (2×) Taqman genotyping master mix, 0.25 μL of (20×) Taqman genotyping assay mix, and 2.25 μL of the genomic DNA of 10 ng concentration obtained on diluting the DNA with distilled water. Thermal cycle re-action condition is programmed for initial denaturation at 95 °C for 10 min followed by further denaturation at 95 °C for 15 s for 40 cycles and then annealing and extension at 60 °C for 1 min in 384 wells in ABI Quant studio 6 flex machine. Fluorescent signals were obtained for the amplification of each allele by the Taqman probes. After the amplifi-cation by PCR, an end-plate read was performed on the ABI machine. The fluorescent measurement taken during the plate read was used by the sequence detection system and fluorescence the (Rn) values are plotted with the signals obtained from each well. Alleles that are pre-sent in each sample are indicated by the fluorescence signal. Each fluorescent detector will be a perfect match for the allele 1 (wild type) or the allele 2 (mutant).