The Role of HER2/Neu and BRCA1 Genes in the Diagnosis of Breast Cancer among Sudanese Women

Background: Knowledge of HER2/Neu and BRCA1 Genes might be helpful for development of strategies for decreasing the burden of risk of breast cancer. Therefore, the aim of this study to detect the role of HER2/Neu and BRCA1 Genes expression in diagnosis of breast cancer in Sudanese women. Methodology: A total of 100 tissue samples obtained from patients with breast cancer in addition to 50 tissue samples obtained from patients with benign breast lesions, were detected the expression of HER2/Neu and BRCA1 Genes by Polymerase Chain Reaction (PCR). Results: The prevalence of HER2/Neu and BRCA1 Genes, among cases was 6%, and 10% respectively. Conclusion: HER2/Neu and BRCA1 Genes have a considerable contribution to etiology of breast cancer in Sudan that requires further consideration.


Introduction
Breast cancer is the most common cancer in women worldwide, leading to about 300,000 deaths each year. It is considered a biologically heterogeneous disease that is influenced by complex and still incompletely understood interactions between multiple genetic and environmental risk factors. These interactions may play an important role in the significant geographical differences in breast cancer incidence [1]. The incidence in developed countries is higher than in developing countries, as well as in urban and rural areas [2]. In Sudan, it was reported that about 70% of the women diagnosed with breast cancer were younger than 50 years old and invasive ductal carcinoma was the most common type (71.5%) [3]. The BRCA1 tumor suppressor gene account for approximately 45% of families with a significantly high breast cancer incidence and the HER2/Neu oncogene is in proximity on the long arm of chromosome 17 (17q11-21) [4] [5]. Absence of BRCA1 or functional overexpression of the HER2/Neu gene may be characteristic of the somatic phenotype of breast cancer in premenopausal women, characterized by a poor prognosis, such as a high tumor grade, negative hormone receptors, and a high proliferation rate [6].

Materials and Methods
In this study 100 women with breast lesions were retrospectively investigated for the presence of HER2/Neu and BRCA-1 expression by Polymerase Chain Reaction (PCR). Of the 100, Formalin fixed paraffin embedded tissue samples, 50 tissue samples were obtained from women with breast cancer and the remaining 50 tissue samples were from women with benign breast lesions.

DNA extraction
DNA was extracted according to the steps described in DNA extraction kit purchased from Sacace biotechnologies-Casera-Italy. The pellet obtained from previous steps was treated with 300 μl of Reagent 2 (lysis buffer) in addition 100 μl of sample, vortexed, incubated at 65˚C for 5 min and centrifuged at (12,000 -16,000 g) for 10 min at 25˚C and transfer the supernatant into new tube (sterile 1.5 ml Eppendorf tube) for DNA extraction. Vortexed vigorously sorbent and added 20 μl to each tube, Vortexed for 5 -7 sec and incubated all tubes for 3 min at room temperature, then this step was repeated. Then all tubes were centrifugated for 30 sec at 5000 g and used a micropipette with a plugged aerosol barrier tip, carefully removed and discarded supernatant from each tube without disturbing the pellet. Tips were changed between the tubes. 500 μl of Washing Solution was added to each tube. Vortexed vigorously and centrifuged for 30 sec at 10,000 g. Supernatant was removed and discarded from each tube.
This step was repeated and incubated all tubes with open cap for 5 -10 min at 65˚C. The pellet was resuspended in 100 μl of DNA-eluent. Incubate for 5 min at 65˚C and vortex periodically. The tubes were centrifuged for 1 min at 12,000 ×g.
The supernatant was containing DNA ready for amplification stored at −20˚C until used.

PCR amplification of HER2/Neu and BRCA1 Genes
The PCR was carried out in a total reaction volume of 40 µl containing between 20 µl mix-1 (contained in PCR tubes), 10 µl of mix-2 and 10 µl of extracted DNA (sample). The PCR program was described in Table 1.  Figure 1.

Discussion
Brest cancer is the most common cancer in women in both Sudan and worldwide [7]. It is considered the leading cause of female related mortality in Sudan [8]. The incidence rates are rising all over the world increasing the disease burden and necessity to increase awareness of such devastating disease. The amplification and/or overexpression of the HER2/Neu and BRCA1 oncogenes have been proposed as an important prognostic marker in breast cancer1, in our study, HER/neu protein was expressed in 3 (6%) of breast cancers and not expressed in benign breast lesion. They are involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double-strand breaks [12]. If BRCA1 or BRCA2 itself is damaged by a BRCA mutation, damaged DNA is not repaired properly, and this increases the risk for breast cancer [12].
In this study we found that BRCA1 protein was expressed in 10% of breast cancer, this study is supported by study of Dokyung et al. [13]. who reported that BRCA protein was expressed in 24.7% of breast cancers, In the present study we also found the expression of BRCA1protein in breast cancer is higher than in normal breast lesion, this finding is inconsistent with the study of [14] who reported that a high uniform expression of BRCA1 was observed in normal breast tissue while absent or reduced expression was found only in malignant tissues.

Conclusion
In conclusion, the present results suggest there is a weak expression of HER/Neu and BRCA1 proteins in breast cancers among Sudanese women and breast cancers are the most prevalent cancers in Sudanese women. HER/Neu and BRCA1 Genes mutation is not clearly involved in breast cancers among Sudanese women.