Identification of Mutations in Gene BRCA1/2 in Breast Cancer Cases from Balochistan, Pakistan

Asma Yousafzai1,*, Muhammad Luqman2, Nisar Ahmed1, Muneeza Arbab1, Muhammad Murad1, Naheed Sajjad3, Sara Naudhani1, Shakeela Daud1, Abdul Hameed Baloch4, Khushnaseeb5, Sana Zahoor6, Zahid Rauf7, Muhammad Mohsin Javed6 and Jamil Ahmad1

1Department of Biotechnology, Balochistan University of Information Technology, Engineering and Management Sciences, Quetta

2Department of Environmental Sciences, Faculty of Bio-Sciences, UVAS, Lahore

3Department of Biotechnology, Sardar Bahadur Khan Women University, Quetta

4Department of Animal Breeding and Genetics, Lasbela University of Agriculture, Water and Marine Sciences, Uthal

5Center for Nuclear Medicine and Radiotherapy, Quetta

6Department of Biotechnology, Virtual University of Pakistan

7Faculty of Information and Communication Technology, Balochistan University of Information Technology, Engineering and Management Sciences, Quetta

ABSTRACT

BRCA1/2 genes are highly susceptible genes for both breast and ovarian cancer and account for 15-20% of all the hereditary breast cancer cases. Present study was aimed at identifying mutation in BRCA1/2 in breast cancer patients in Balochistan. Blood samples of 100 subjects including 50 breast cancer cases and 50 normal subjects were used to determine genetic variants in BRCA1 and BRCA2. Nine variants were identified in BRCA1 and four were identified in BRCA2. In case of BRCA1, six missense substitutions (p.Asp343Tyr, p.Gly393Asp, p.Ser561Phe, p.Ser616Phe, p.Pro871Leu and p.Ser1613Gly) two frameshift (p.Ser423fs and p.Gly1770fs) and nonsense mutation (p.Glu1250X) was identified. In case of gene BRCA2, all the four variants were missense substitutions (p.Glu58Lys, p.Asp99Asn, p.Asp104Asn and p.Pro999Gln). The data reported here will be valuable addition for the future genetic screening of the breast cancer patients of Balochistan.

Article Information

Received 03 October 2018

Revised 20 December 2018

Accepted 27 January 2019

Available online 30 May 2019

Authors’ Contributions

AY executed the experimental work. NA and SN provided help in Wet Lab. SD helped in molecular techniques. ML, K, NS, AH, SZ, ZR and MMJ helped in sampling. MM, MA and JA helped in data analysis. JA supervised the work.

Key words

Breast cancer, Missense mutation, Nonsense mutation, Heterozygous, Gene BRCA1/2.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.4.sc2

* Corresponding author: [email protected]

0030-9923/2019/0004-1579 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Breast cancer is the most frequently diagnosed and second leading cause of death accounting for about 25% of all cancer cases among women worldwide (Ferlay et al., 2012). 5-10% of the breast cancer cases are considered to be hereditary, out of which 15-25% cases are caused by mutations in BRCA1 and BRCA2 genes. BRCA1/2 gene mutations are most frequently diagnosed in both breast and ovarian hereditary cancers (Narod, 2012; Baloch et al., 2014; Esposito et al., 2016).

The human gene BRCA1 is located on the chromosome 17q2.1, from base pairs 41,196,312 to 41,277,500; whereas gene BRCA2, cytogenetic location is 13q13.1, ranging from base pairs 32,315,480 to 32,399,672 on chromosome 13. Germ line alterations of the BRCA1 (MIM113705) or BRCA2 (MIM600185) genes result in vulnerability to breast and ovarian tumor. Many germ line alterations in both genes have been reported. Most of these mutations are small deletions or insertions that result in frame shift and trimmed protein (Ford et al., 1998).

The protein encoded by BRCA1 is a tumor suppressor nuclear phosphoprotein that plays a critical role with the interaction of the other proteins forming a protein complex in the nucleus of different cells to repair DNA damage (Cao et al., 2003; Deng, 2006; Buckley and Mullan, 2012; Dine and Deng, 2013). The BRCA1 protein contains 1863 amino acids and is divided into domains including ring domain at NH2 terminus, central DNA binding domain and BRCT domain at COOH terminus. Missense and truncating mutations in BRCA1 are most frequently been found in a wide range of breast cancer cases with family history (Dine and Deng, 2013).

An increased incidence of other malignances, such as prostate, pancreatic and colorectal cancer is also observed among BRCA1/2 mutation carriers. The proportion of described mutations in BRCA1 relative to BRCA2 varies between different populations. With the exception of a strong BRCA2 founder effect in Iceland, however, BRCA1 mutations are more regularly reported. In the majority (>80%) of families with breast cancer and ovarian cancer, the disease has been linked to the BRCA1 gene.

Present study was performed on 50 breast cancer cases and 50 normal subjects. The prime objective of the current study was to analyze germline mutations in BRCA1/2 gene in the patients of breast cancer and to compare it with normal population. Current study revealed 13 different genetic variants including missense, nonsense and frameshift mutations in gene BRCA1/2 in breast cancer cases from Balochistan.

Materials and methods

Breast cancer cases (n=50) and normal subjects (n=50), all females, belonging to different ethnic groups, were enrolled in current study. Informed consent forms were signed from the volunteers of both breast cancer patients and normal subjects. History of the disease including the family history of breast or other cancers, onset and date of diagnosis was enquired from the patients. Clinical history including histopathology was obtained from the patient files enrolled for chemotherapy and/or radiotherapy in CENAR (Center for Nuclear Medicine and Radiotherapy). Venous blood (5 ml) was collected in 15 ml tubes containing EDTA as an anticoagulant agent.

DNA was extracted from the blood samples through an inorganic method. Primers were designed for all the coding exons of BRCA1/2 genes using the Prime3 software. BRCA1/2 exons were amplified through PCR by using the primers designed for BRCA1/2 and visualized through gel documentation system after gel electrophoresis. All the amplified products were sequenced on 3100 ABI prism DNA sequencer using the Big Dye Terminator Cycle Sequencing Kit. The results of DNA sequences were analyzed for the genetic variants by comparing the sequences of the exons with the normal sequences of BRCA1/2 genes (ENST00000357654.7) through ENSEMBLE genome browser.

Results and discussion

All the coding exons and intron-exon boundaries of BRCA1/2 gene were analyzed. From among 50 breast cancer patients, nine genetic variants in BRCA1 and four in BRCA2 were recorded. In the case of BRCA1, six missense substitutions (p.Asp343Tyr, p.Gly393Asp, p.Ser561Phe, p.Ser616Phe, p.Pro871Leu and p.Ser1613Gly) two frameshift (p.Ser423fs and p.Gly1770fs) and one nonsense mutation (p.Glu1250X) was identified (Table I). In the case of gene BRCA2, all the four variants were missense substitutions (p.Glu58Lys, p.Asp99Asn, p.Asp104Asn and p.Pro999Gln) (Table II). Sequence data of all exons of BRCA1/2 were compared with normal sequences of BRCA1/2 cDNA sequences and genomic data from public databases.

Table I.- List of genetic variants identified in gene BRCA1.

Table II.- List of genetic variants identified in gene BRCA2.

Among the Asian states, Pakistan is considered to have the highest incidences of breast cancer (Liede et al., 2002). BRCA1 and BRCA2 are the most susceptible breast cancer genes. Proteins encoded by both genes play an important role to protect the genome. BRCA1 encodes a pleiotropic protein in response to DNA damage that functions to activate cell cycle checkpoint and DNA repair (Gudmundsdottir and Ashworth, 2006; Roy et al., 2012; Almeer et al., 2018).

The identified missense mutations was studied by bioinformatics software Polyphen-2 that showed that most of the missense mutations were benign (Fig. 1); while the other nonsense mutation and frameshift mutations may have been the case of breast cancer in the patients (Fig. 2). Studies suggest that BRCA1 and BRCA2 are most predisposing genes to hereditary breast and ovarian cancers. Germline mutations in both BRCA1 and BRCA2 genes may increase the risk of developing breast cancer up to about 80% throughout the life time of an individual (Roy et al., 2012; Narod, 2012). In Pakistan, consanguineous marriages are very common. In current study, parents of 7 patients were reported with consanguinity. Hashmi (1997) and Hussain and Bittles (1998) reported in their study that Pakistan has the highest rate of consanguinity globally with 60-70% consanguineous marriages, which may play major role in spreading the hereditary cancers. Liede et al. (2002) reported five BRCA1 mutations (2080insA, 3889delAG, 4184del4, 4284delAG, and IVS14-1A→G) and one BRCA2 mutation (3337C→T) in multiple case subjects in Pakistani population. Rashid et al. (2006) identified thirty deleterious germ-line mutations in the 176 families (17.0%), including 23 in BRCA1 and 7 in BRCA2. Four mutations, 185delAG, 185insA, S1503X and R1835X, were recurrent; these accounted for 52% of all identified BRCA1 mutations.

The data reported here will be valuable addition for the future genetic screening of the breast cancer patients either with a family history or not and for the other family members of the patients who are at risk.

Conclusion

Current study revealed 13 different genetic variants including missense, nonsense and frameshift mutations in gene BRCA1/2 in breast cancer cases from Balochistan.

Statement of conflict of interest

The authors declare no conflict of interest.

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