Germ-Line Mutanome Profiling of The Breast Cancers in Pakistani Population

Statement of the Problem: Cancers are complex disorders. Consanguineous populations, by virtue of reduced genetic diversity, provide a unique model for the investigations of the underlying genetic component(s). A prototype example in the breast cancers is the identification of BRCA1/2 gene defects in Ashkenazi Jews. In Pakistan, the age-standardized rate (ASR) of breast cancer incidence in females is among the highest in Asia, whereas the morality rate is one of the highest in the world. With the consanguinity rate of 56.4% and inbreeding coefficient (F) of 0.0331, it is extremely important to investigate the role of inherited mutations in breast cancers in Pakistani population. Methodology and Theoretical Orientation: BROCA analysis for breast cancers consists of twenty-seven (27) established and candidate breast cancer genes involved in molecular carcinogenesis. A pilot hospital-based cohort study was designed. Eighty-five breast cancer patients and three controls with no medical history of any cancer, participated in the study. The BROCA investigations were carried out by a genomic capture, massively parallel next generation sequencing assay on Illumina HiSeq2000 assay with 100bp read lengths. Copy number variations were determined by partially-mapped read algorithm. Once the mutation was identified, it was validated by Sanger sequencing. After informed consent, the mutations were screened in the familial samples. Findings: The analysis revealed germ-line mutations in 12% of the patients. These mutations were restricted to three genes (BRCA 1, BRCA 2, and TP53). The identified mutations consist of both novel and previously reported alterations and result in protein truncation. No mutations were identified in the remaining twenty-four (24) genes. Mutation screening in the familial samples identified carriers in four out of five families. Conclusions and Significance: The study provides framework for the development of preventive and treatment strategies against breast cancers in Pakistani population. The present pilot study is the first-systematic attempt to investigate the role of all known breast cancer genes in Pakistani population. We report the investigation of germline mutations in twenty-seven breast cancer candidate genes in a cohort of breast cancer patients from Pakistan. The study has significant implications in delineating the molecular pathology of inherited breast cancers. Methods Ethics Statement The project is in accordance with the Declaration of Helsinki 2013 [4]. Informed consent was obtained from the participants. The project was approved by Ethical Review Committees of collaborating institutions: Atomic Energy Medical Centre (AEMC), Jinnah Postgraduate Medical Centre (JPMC), and Dr. Panjwani Center for Citation: Sadia Ajaz, Sani-e-Zehra Zaidi, Saleema Mehboob Ali, Aisha Siddiqa, Muhammad Ali Memon (2018) Germ-Line Mutanome Profiling of The Breast Cancers in Pakistani Population. Arch Mol Med & Gen 1:106. Arch Mol Med & Gen , 2018 Volume 1(1): 46-50 Molecular Medicine and Drug Research (PCMD), International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi, Pakistan. Sampling The pilot study, conducted from July 2016 to June 2017, included blood samples from ninety-four (94) breast cancer patients and two (02) healthy volunteers. 8-10ml of blood samples were transferred to the ACD-coated vacutainers (BectonDickinson®, Franklin Lakes, NJ, USA). In case of healthy volunteers, 5ml blood samples were collected. The samples were either processed immediately or stored at 4°C until DNA isolation. After quality and quantity control, eightyfive (85) samples were analysed by next generation sequencing (NGS). Cases and Family History Instrumentation Patients volunteered the information about the family history of breast cancer, or any other cancer, number of pregnancies, number of children, the age of menarche and age of menopause. Details on Bloom Richardson grading system and tumor node metastasis (TNM) staging as recommended by the Union International Contre le Cancer (UICC) were obtained from the hospital medical records with the patient’s consent. DNA Extraction, Quantification, and Quality Control Standard Phenol-Chloroform method was used for the extraction of DNA from the WBCs of patient’s blood sample [5]. Isolated DNA was quantified on UV spectrophotometer (Beckman Coulter® DU 530). The acceptable range of 260/280 ratios was set as 1.8-1.99. Quality control was carried out on 0.7% agarose gel and visualized under UV light using Azure C-300® imaging system. None of the extracted DNA samples showed the shredded or streaked pattern on the agarose gel (Figure 1). Genomic Capture, Massively Parallel Next Generation Sequencing Assay (BROCA Analysis) After DNA extraction, quantity and quality control, germline DNA was sequenced using breast and ovarian cancer gene panel (BROCA). This assay sequences all the exonic and flanking intronic sequences of the selected genes. The analysis is able to capture all types of mutations including large genomic re-arrangements in known breast cancer genes [6-8]. The breast cancer gene panel assay investigates following 27 genes: BRCA1, BRCA2, TP53, ATR, BARD1, BRIP1, FAM175A, FANCM, GEN1, MRE11A, NBN, RAD51B, RAD51C, RAD51D, RECQL, RINT1, SLX4, BAP1, PALB2, PTEN, STK11, XRCC2, ATM, CHEK1, CHEK2, CDH1, and CTNNA1. Exon Amplification and Sanger Sequencing The exons in BRCA1, BRCA2, and TP53 genes carrying mutations as identified by N.G. S. were amplified. The amplicons were purified by an isopropanol-based method. 15μl of the purified amplified products along with the primers were used for sequencing. Sanger sequencing of samples was performed commercially. Bioinformatic Analysis ExAC and NHGRI databases were used as reference for the identification of novel mutation(s). Results and Discussion 10 samples from 85 patients (approx.12%) had a germ-line mutation. These were confined to BRCA1, BRCA2, and TP53 genes.