Printer Friendly

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

Byline: Durr-e-Samin, Muhammad Saif-ur-Rehman, Muhammad Shahnawaz-ul-Rehman and Muhammad Sajjad Khan

ABSTRACT

Background and 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 [greater than or equal to]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.

KEYWORDS: Breast Cancer, Pakistani population, BRCA1, Exon-2,185-Del-AG, 185 Ins. A.

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 produces7.8kb 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 well-established 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 [greater than or equal to]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.

1. Breast cancer patients with < 40 years of age at diagnosis (N= 16)

2. Breast cancer patients with [greater than or equal to]40 < 60 years of age at diagnosis (N=16)

3. Non-Cancer subjects with [greater than or equal to]30 and <40 years of age (N=7)

4. Non-Cancer subject with [greater than or equal to]40 < 60 years of age (N=7)

Regarding family history 80% (11/14) of non-cancerous subjects and 50% (16/32) of cancer patients were selected with positive family history according to definition for this study design. Selection criteria was formulated by careful examination of data from previous studies about analysis of BRCA1 mutations at exon-2. 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 For PCR standard reaction mixture was prepared in 25ul volume for amplification of required DNA fragments, at primer annealing temperature of 51AdegC. 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 were analyzed by bioinformatics tools such as BLASTn, BLASTp. Analysis of chromatograms was done by using Bio Edit software. The wild type and mutant sequences were compared with Homo sapiens Taxon 9606, reference sequence at NCBI data base and submitted to GenBank by using BankIt browsing.

Appendix-I: Data set of mutations at exon-2 in Pakistani Population.

###Liede et###Rashid et al.###Malik et al.###Moatter et al.###Aziz et al.###Present Study

###al.(2002)16###(2006)17###(2008)19###(2011)20###(2016)18

No. of

Patients1

###1(OC)###3(OC)###0###0###0###0

(Ethnicity)

###(Punjabi)###(Punjabi)###-###-###-###-

Age at

###40###41,<50, 57###-###-###-###-

diagnosis

(years)

No. of

Patients2

###1(BC)###2(BC)###0###0###1

(Ethnicity)###1(Punjabi)

###(Punjabi)###(Pathan)###-###-###(Punjabi)

Age at###35

###47###39, 40###-###-###33

diagnosis

(years)

###Direct DNA###Direct

###SSCP, PTT,###SSCP assay###Allele

###sequencing###sequencing

Methods###DHPLC and###SSCP###and DNA###specific

###PTT###of PCR

###DNA sequencing###sequencing###PCR

###(For ex-11)###products.

###2, 7, 8, 10, 11, 15,

Exons###2,11,12,15,20###2,3,13###2,5,6,16,20,22###2,15###2

###17, 20, 24

###341(BC)

No. of

###120(OC)###176###120###53###120###46

Subjects

###200(Control)

Family history###Negative

###Positive family

of patient/###family history

###history for all###Negative###-###Positive###Negative

proband with###for both

###five mutations

mutation###mutations

###Unilateral,###Case/control

###sporadic breast###Patients with###Population###with exon

Type of cohort

###Case/Control###Familial###cancer patients###moderate###based case###specific

studied

###with negative###family history###control###selection of

###family history###cohort

Table-I: Age at diagnosis of Breast Cancer patient.

Class Boundaries###Frequency###Percentage

(Years of age)

60###6###7.05%

Total###85###100%

RESULTS

Demographic data analysis: Age at sampling/ onset of cancer and family history were taken as criteria for analysis of BRCA1-exon 2 mutations in Pakistan ethnicity. The mean age at diagnosis of Breast cancer was 45+-9.86 years (Ranged 26 to 65 years) with class boundaries shown in Table-I. Maximum number of Patients (63/85-74%) fall in age [greater than or equal to] 50 years. Regarding family history 73.91% (85/115) subjects provided required information among which 32.94% (28/85) from the patients while 53.33% (16/30) from non-cancerous subjects reported that they have positive family history according to definition of this study plan. None of other recorded feature produced any significant results.

DNA extraction and BRCA1-exon-2 polymerization: DNA was successfully extracted, visualized on 1.5%agarose gel. PCR products resulted in synthesis of fragment of 258 nucleotide bases of Human BRCA1-exon 2 with flanking intron on both 5' and 3'sides (Fig.1).

Sequencing of DNA samples: PCR products sent for sequencing returned in FASTA files formats and chromatograms of the BRCA1 exon 2 coding sequence of 99 base pairs, along with intronic flanking regions on both 3' and 5' ends. Sequenced fragments produced significant alignments on BLASTn and BLASTp and were verified from GenBank by obtaining following accession numbers: MH046834, MH046835, MH046836, and MH046837.

BRCA1-185-del AG (c.68_69delAG) was found in the group-I (patients with age A BRCA1 Mutations in Breast Cancer Patients from Lahore, Pakistan. Asian Pac J Cancer Prev. 2016;17(4):1725-1727. doi: 10.7314/APJCP.2016.17.4.1725

19. Malik FA, Ashraf S, Kayani MA, Jiang WG, Mir A, Ansar M, et al. Contribution of BRCA1 germline mutation in patients with sporadic breast cancer. Int Semin Surg Oncol. 2008;5(1):1-5. doi: 10.1186/1477-7800-5-21

20. Moatter T, Aban M, Khan S, Azam I, Pervez S. BRCA1 status in Pakistani breast cancer patients with moderate family history. J Coll Physicians Surg Pak. 2011;21(11):680-684.

21. Drost R, Dhillon KK, Van Der Gulden H, Van Der Heijden I, Brandsma I, Cruz C, et al. BRCA1 185delAG tumors may acquire therapy resistance through expression of RING-less BRCA1. J Clin Invest. 2016;126(8):2903-2918.
COPYRIGHT 2018 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2018 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Publication:Pakistan Journal of Medical Sciences
Geographic Code:9PAKI
Date:Oct 31, 2018
Words:2077
Previous Article:Vitamin D levels in patients with ankylosing spondylitis: Is it related to disease activity?
Next Article:Accuracy of echocardiography in diagnosing total anomalous pulmonary venous return.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters