RESUMO
Breast cancer is currently the most common type of cancer in females. The majority of the hereditary forms of breast cancer are caused by mutations in the BRCA1 and BRCA2 genes, whose main function is the DNA repair of double-strand breaks. Genetic testing of females with a family history of breast cancer is recommended to determine their hereditary predisposition for this type of cancer. The variants with no clear clinical significance may represent a diagnostic challenge when performing targeted resequencing. In this study, DNA samples were obtained from 24 breast cancer patients (mean age, 35±10 years) with a positive family history and from 71 age-matched healthy controls. Informed consent was obtained from all the subjects. Sequence-targeted BRCA1 and BRCA2 libraries were prepared using the TruSeq Custom Amplicon method and sequenced on the Illumina MiSeq system. A wide range of variants were identified in the BRCA1 and BRCA2 genes. Two pathological/presumably pathological variants were detected in the breast cancer patient group: a mutation in BRCA2 at the chromosomal (chr) position chr13:32890665, which affected the first position of the 5' splice region following exon 2; and a mutation in BRCA1 at chr17:41219635, causing an in-frame triple nucleotide deletion of valine 1688 (8.3%). In the patient and control groups, 7 likely polymorphic variants and 13 common variants were detected in the BRCA1 and BRCA2 genes. To the best of our knowledge, this study was the first to identify 3 common polymorphisms in BRCA2, characteristic solely of the Bulgarian population, including chr13:32973737, T/-, a single-nucleotide polymorphism (SNP) within the 3'-UTR of exon 27; chr13:32973280, A/-, a mononucleotide deletion within the 5'-UTR of exon 27; and chr13:32973924, T/-, a mononucleotide deletion downstream of the gene sequence. To the best of our knowledge, this study was the first to apply next-generation sequencing of the BRCA1 and BRCA2 genes in a Bulgarian population, prompting further investigation for local founder mutations and variants characteristic for this particular region.
RESUMO
PURPOSE: Though p53, BRCA1, ATM, PIK3CA, and HER2 genes are shown to be involved in various aspects of breast carcinogenesis, their functional relationship and clinical value are still disputable. We investigated the genetic status or expression profile of these genes to further elucidate their clinical significance. METHODS: PCR-SSCP-Sequencing of p53, BRCA1, ATM, and PIK3CA was performed in 145 Bulgarian patients with sporadic breast cancer. Expression profiles of HER2 were determined by ICH and CISH. Relationship between mutations and clinicopathological characteristics was evaluated by Chi-squared and Fisher's exact tests. Multivariate Cox proportional hazard test and Kaplan-Meier analysis were used to evaluate differences in overall survival between groups. RESULTS: The frequency of p53 (22.07%), BRCA1 (0.69%), ATM (7.59%), and PIK3CA (31.25%) alterations and HER2 (21.21%) overexpression was estimated. Mutated p53 was associated with tumor size (P = 0.033) and grade of malignancy (P = 0.001), ATM--with grade of malignancy (P = 0.032), and PIK3CA--with PR-positive tumors (P = 0.047). HER2 overexpression correlated with age of diagnosis (P = 0.009), tumor size (P = 0.0004), and ER expression (P = 0.011). Univariate survival analysis showed that mutated p53 is an indicator for worse outcome (P = 0.041). Combination of two genetic abnormalities did not correlate with more aggressive carcinogenesis and worse overall survival. CONCLUSIONS: Our data indicated that p53, BRCA1, ATM, PIK3CA, and HER2 alterations specifically correlate with clinicopathological characteristics of Bulgarian patients with breast cancer. Of these genes, only mutated p53 showed significant, though not independent, negative effect on overall survival.