Association of BRCA1 185 del AG with early age onset of breast cancer patients in selected cohort from Pakistani population

Background & Objective: Large spectrum of pathogenic BRCA mutations is known as a major cause of hereditary breast ovarian cancer in human all over the world. The objective of present study was to find out the association of mutations185-del-AG and 185Ins.A at BRCA1 exon-2 with age of onset and family history of gynecological cancer among the selected cohort of breast cancer patients in Pakistani population and to provide guidelines for treatment strategies. Methods: For the present study 115 subjects were recruited from different hospitals of Punjab, Pakistan, during May, 2017 to February, 2018. The inclusion criteria were age ≥30, without any previous BRCA testing and willingness to participate in present study. Subjects were interviewed for various demographic factors. Out of 115 subjects, 46 were selected on the basis of findings of previous studies and approximately 3 ml of blood was collected in EDTA coated vials for analysis of BRCA1 exon-2. Column based DNA extraction was performed by using commercial kit and exon specific primers were used to amplify BRCA1 exon 2 and PCR products were sent for sequencing to Eurofins Genomics. Sequences were analyzed through the BLAST program at National Center for Biotechnology Information (NCBI) and Bio Edit software. Accession numbers were obtained on submission of sequences in GenBank. Results: BRCA1-185-del AG mutation was found in one of the breast cancer patient who was 33 years of age at diagnosis. None of the samples revealed positive results for BRCA1-185 Ins. A. Conclusion: BRCA1-185 Del AG mutation has association with early age onset of breast cancer. The direct sequencing is very useful approach for BRCA analysis and exon specific selected cohort from Pakistani population.


INTRODUCTION
The DNA repair associated breast cancer gene1 (BRCA1, BIC -Gene ID: 672, position: 43044295 to 43125483bp) in human being, is located on 'q' arm of chromosome 17(17q21.3), spread over 81 kb of genome, with 24 exonic regions and produces 7.8kb of mRNA which translate into type 1 breast cancer susceptibility protein. 1 BRCA1 translation products contain numerous important motifs which have strong interactions with other proteins involved in the processes; like progression of cell cycle, responses to DNA damages, maintenance of genomic integrity, ubiquitination and apoptosis. 2 BRCA1 gene products are very important transcriptional regulators having vital role in repair of double strand DNA breaks mediated by homologous recombination. 3 Germline mutations, in BRCA1 are also associated with male breast, uterine, cervical, pancreas and prostate cancers beside breast/ovarian carcinomas. 4 It has been estimated that in human female, cumulative frequency for mutations in this gene imposes a risk of 87% of breast and 50% of ovarian cancer. 5,6 The BRCA databases reported over 6000 variants among which around 1800 have been classified as likely pathogenic or pathogenic in nature. 7 The frame shift mutations named;185-del AG (c.68_69delAG-Allele ID 32701) and185 ins.A (c.66 dup A-Allele ID: 46247) are well established pathogenic variant at exon 2 of BRCA1 gene. The first one occurs by deletion of AG as a consequence of which, stop codon appears after few base pairs that results in significant truncation of protein. 8 The 185-del AG mutation was first time described as founder mutation in Ashkenazi Jews population. 9 The rapid rise in the incidence of carcinomas of gynecological origin in Pakistan is imposing challenge to expand investigations on BRCA mutations to provide guidelines to determine treatment strategies. Since according to previous studies, one of the commonly found BRCA1 aberrations in Pakistani population is 185delAG 1. 10 Thus, the present study was aimed to investigate incidence of this mutation along with another wellestablished BRCA1variant at exon 2 and relationship of these with age of onset and family history in Pakistani population. The latest development in research explored the importance of BRCA1/2 mutations for determining treatment regimes. In the recent years, Food and Drugs Administration has approved PARP (Poly adenine dinucleotide phosphate polymerase) inhibitors such as olaparib to treat certain tumors having BRCA1 mutation. 11,12 Due to administrations of such recommendations in clinical practice the trend of BRCA testing is increasing.

METHODS
Present study was carried out at Centre of Agriculture Biochemistry and Biotechnology in the University of Agriculture, Faisalabad, Pakistan. Research was approved by institutional Biosafety/ Bioethical committee through the Office of Research, Innovation and commercialization (IBC, ORIC, UAF, Pakistan). A total of 115 subjects (85 cancer patients, 30 non-cancerous) were selected from different hospitals of Punjab, Pakistan. The subjects were interviewed for personal health and reproductive features such as age at menarche, menopause and first child birth, total number of children parental consanguinity and ethnicity. Beside that family history of cancer of gynecological origin or any other kind of cancer was also recorded. Regarding this, positive family history was defined for present study as having at least one first degree or two second degree relatives with gynecological cancer, and negative family history as none of first or second degree relative with such cancer (excluding themselves in case of patients). Demographic features were recorded on SPSS version 4. Age patterns and some other features were compared with previous studies done on Pakistani population. Inclusion criteria: The inclusion criteria were ≥30 age at collection of data/blood sample (except for a few cancer patients who presented with the disease before this age), no BRCA test before this study and willingness to participate in research by informed consent. Subjects were never forced to provide any kind of information. Selection of samples: Blood samples of 46 out of 115 female subjects selected on the basis of age at sampling/onset of disease and family history, for sequence analysis of BRCA1exon-2. Subjects were classified in the following four groups. The database for these two mutations with record of related explored features has been given in Appendix-I. DNA extraction and amplification: The blood samples (3 ml) were collected in EDTA vials with the help of medical professionals with care to avoid any discomfort or contamination. Extraction of DNA was done by using thermo scientific kit (K0781) by standard procedure according to instructions given by manufacturer. Following sequences of primer were selected as used by Singh et al. 2015. 13 Forward primer 5'GAAGTTGTCATTTTATAAACCTTT3' Reverse primer 5'GTCTTTTCTTCCCTAGTATGT3' For PCR standard reaction mixture was prepared in 25ul volume for amplification of required DNA fragments, at primer annealing temperature of 51°C. The PCR products along with 250 base pairs ladder were run on 1.5% agarose in 0.5 X TBE to reveal amplified DNA fragment. DNA Sequencing: PCR products were sent for sequencing to Eurofins Genomics. FASTA files

DISCUSSION
The well-known BRCA1 -185 Del AG pathogenic variant is one of the founder effect mutations whose incidence is variable among populations of different geographical and ethnic backgrounds. 14 This variant also recognized in Pakistani population as one of the most common BRCA1 alterations identified so far. 10 In the present study design, the concept of deliberate selection of cohort for exon specific BRCA variants analysis for Pakistani population has been introduced so that most suspected individuals for different mutations could be screened. By using this approach, BRCA1-185 Del AG mutation was detected in 1/46 including all groups and 1/16 of most suspected group-I. None of the Sample was found to be positive for 185 Ins. A.
The mean age of total of 115 subjects enrolled for present study was 46.55±11.01years while for the breast cancer patients mean age at diagnosis was 45±9.86 years. According to previous findings the 45 to 49 years range of age has significance in prognosis of breast cancer. 15 To analyze variations at BRCA1 exon 2, out of 115 subjects 46 were selected by narrowing the range up to five year from upper extreme (mean age 39.95±8.8).The victim of 185 del AG was aged 33 at diagnosis this observation is in concordance with previous finding on this mutation among patients of cancer of gynecological origin with Pakistan ethnicity (Appendix-I). Regarding family history, patient in present study reported to have negative family history for any kind of cancer as Liede et al., 2002 16 also reported the victims with this mutation had negative family history for such cancers. Previous studies on Pakistani population also tried to find relation with ethnicity as Rashid 18 reported this mutation in patient with Punjabi ethnicity and currently it was also observed in patient with Punjabi ethnicity. In the two prior studies on Pakistani population that included exon-2 in investigation none of the patients found to be positive for 185 delAG. 19,20 The BRCA1-185 Ins-A mutation was also found in two of the previous studies that explored mutation in four ovarian cancer patients with Punjabi ethnicity. 16,17 None of our selected samples revealed positive result for this mutation. The present study design has drawback of not including ovarian cancer patients for analysis of BRCA1 at exon 2.
During the earlier studies, it has been revealed that a lot of heterogeneity existed in survival data and responses to cancer therapies between noncarriers and carriers and among carriers of different pathogenic variants of BRCA1. Recently during an experimental trial it has been found that 185 del AG mutation exhibited sensitivity to platinum drugs and PARP (Poly ADP polymerase) inhibitors because of loss of ring domain in BRCA1 protein. 21 This feature of treatment sensitivity to double stranded breaks (DSB) inducing agents may serve as marker to prevent ineffective therapies and to develop some promising and unconventional therapeutic methods. Thus from current study it is concluded that BRCA1 185 Del AG has association with early age onset of breast cancer and exon specific selection of cohort for direct sequencing of different mutational analysis is a useful approach to be applied on BRCA analysis. Moreover it is suggested that BRCA analysis should also be meant for determination of treatment strategies and breast cancer Pakistani patients diagnosed with age less than forty should be screened for BRCA1-185 Del AG as a part of routine diagnostic marker before treatment.