Abstract
Background:
Breast cancer (BC) is a leading cause of death among women. Pathogenic variations (PVs) in BRCA1 and BRCA2 genes increase the risk of hereditary and Triple-negative breast cancer (TNBC). Despite its great importance, NGS-based genetic testing of BRCA1 and BRCA2 in Bangladesh is out of reach of most patients because of its high cost.
Objective:
To overcome this challenge, this study explored whole genome databases (cBioPortal, gnomAD, dbSNP) to investigate whether sequencing selected exons in BRCA1 and BRCA2 genes may provide meaningful clinical interpretation of BC risk.
Methods:
Exons 4 and 16 of BRCA1 and exons 18, 23 and 25 of BRCA2 were selected for targeted analysis. According to the aforementioned databases, 10 and 21 mutations are pathogenic in the targeted regions of BRCA1 and BRCA2 respectively. To evaluate the presence or absence of these germline pathogenic variations (PV) in Bangladeshi BC patients, we collected blood samples from 13 subjects, among whom, 9 were diagnosed with BC, and 4 without cancerous tumors but with family history and breast lump.
Results:
Analysis of 13 patient samples revealed no PVs in the targeted regions. Within the BRCA1 gene, 5 of the 10 sites, and within the BRCA2 gene, 10 of the 21 sites associated with pathogenic variations were successfully sequenced and clearly interpretable. However, comparison with the reference sequence confirmed the absence of PVs in the targeted regions of both BRCA1 and BRCA2. In silico analysis indicated that missense mutations are the predominant type of variation in both genes.
Conclusion:
This study, although limited by sample size, demonstrates that even exons enriched for pathogenic mutations may not be sufficiently representative for detecting PVs through targeted sequencing of BRCA1 and BRCA2. Comprehensive sequencing of the full lengths of BRCA1 and BRCA2 may be necessary to ensure confident clinical interpretation in BC patients.
Introduction
Breast cancer (BC) remains a formidable health concern in the female population around the world. Bangladesh is no exception with an incidence rate of ~22.5 per 100 000 females and a prevalence rate of ~19.3 per 100 000 females.1,2 Lack of affordable screening tests, ignorance about the importance of screening and late diagnosis play a crucial role in the increasing complication and mortality rate of Bangladeshi BC patients.3-5 Although different screening and diagnostic tests are available, early detection of small tumor masses is still difficult that leads to false positive or negative results.5-11 Molecular diagnostic tests for PV screening in BRCA1 and BRCA2 genes have the potential to circumvent all of these downsides, but are still not affordable as a screening test for regular use in clinical practice. 12
BRCA1 and BRCA2 genes have a strong association with hereditary BC, where the presence of PV increases the lifetime risk of BC development 70% to 80% and 50% to 60%, respectively. 13 The mutation status of the BRCA genes is also crucial in TNBC treatment regimen, as 9% to 30% of the TNBC patients contain BRCA mutations at variable frequency across different races and ethnicities.14-18 Previous studies have shown that approximately 10% to 15% of women with BC carry germline genetic variants, most frequently in the BRCA1 and BRCA2 genes. These variants are particularly enriched in TNBC compared to other BC subtypes. Overall, BRCA1/2 PVs were identified in 15.6% of cases, and notably, 71.9% of women with BRCA1 mutations had TNBC. This highlights a strong association between BRCA1/2 genetic alterations and the TNBC subtype. 19 The TNBC patients are treated with platinum-based chemotherapy and PARP inhibitors since they are unresponsive to endocrine therapy due to reduced or no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2).20,21
Despite the crucial role of BRCA genes in (BC) screening and TNBC treatment, genetic testing of BRCA1 and BRCA2 is not widely accessible to a major portion of the Bangladeshi population due to its high cost. Most of the organizations offering this service use the Next Generation Sequencing (NGS) technology, charging around 25 000 to 35 000 taka ($220-$320). The real-time polymerase chain reaction (PCR) technology using Oncogenetics BRCA Panel Kit 22 costs ~10 000 taka ($92). This kit targets 8 BRCA1 mutations in exons 2, 4 and 10 and 1 BRCA2 mutation in exon 11. Although the frequency of these 9 mutations is considerably high in Caucasians, there is no information on their frequency in Bangladeshi BC patients. Additionally, real-time PCR detects those specific mutations, leaving out all the other mutations that might be present in those exons. As part of our study, we investigated the potential use of the documented PV on the selected exons of the BRCA1 and BRCA2 genes. Following an in-silico investigation, we established a protocol for targeted germ-line variation detection in BRCA1 and BRCA2 genes in BC patients using end-point PCR and Sanger sequencing. Since the Sanger sequencing is available in more institutions within Bangladesh due to its lower price, this protocol will be much more cost-effective compared to the current procedure. So far, 13 samples have been analyzed targeting PV on BRCA1 and BRCA2 genes. All the samples came back negative for the selected variations; however, the study can be extended further, targeting more exons and comparing the outcomes with NGS results.
Materials and Method
Data and Sample Collection
Patient data and blood samples from 13 subjects were collected for this study from the Japan Bangladesh Friendship Hospital (BFH), Bangladesh and National Institute of Cancer Research, Dhaka, Bangladesh. The protocol for this study was approved and ethical clearance was provided by the Institutional Review Board (IRB) of the Faculty of Biological Sciences, University of Dhaka (Reference: Ref. No.257lBiol. Scs.). All participants were properly informed about the procedures of the study and their verbal consents were taken before inclusion in the study. The 9 diagnosed patients were undergoing treatment. After taking their consent, blood samples were collected and kept in EDTA-coated tubes. For long-term preservation, the tubes were stored at −20°C. This study followed the case report (CARE) guideline (https://www.equator-network.org/reporting-guidelines/care/).
Genomic DNA Extraction and Quantification
Genomic DNA from blood samples was extracted using FavorPrep™ Blood Genomic DNA Extraction Mini Kit and the genomic DNA was purified by using Monarch Genomic DNA Purification Kit (New England Biolabs), 23 according to the manufacturer’s protocol. DNA concentration and purity index was evaluated by using the NanoDrop™ 2000/2000 c spectrophotometers (Catalog number: ND-2000) 24 by maintaining proper protocol.
In-Silico Study
In order to determine the target regions for this study, we explored the cBioPortal database (https://www.cbioportal.org), a depository of 26 BC studies encompassing more than 8000 samples.25-27 Since the current publicly available version of cBioPortal uses hg19/GRCh37 as a reference to the human genome, we converted the GRCh37 positions of the BRCA1 and BRCA2 cBioPortal PV to GRCh38 positions using the Liftover web tool developed by Broad Institute (https://liftover.broadinstitute.org). 28 We also went through the Human Genome Aggregation (gnomAD) database version 3.2.1 (https://gnomad.broadinstitute.org) 29 and dbSNP (https://www.ncbi.nlm.nih.gov/snp) 30 in order to explore additional PV that might not be listed in the cBioPortal. Analysis of BRCA1 and BRCA2 mutation from available online data of other studies led us to design primers for exons 4 (F4R4) and 16 (F16R16) of BRCA1 and exons 18 (F18R18), 23 (F23R23) and 25 (F25R25) of BRCA2. Primers were designed and validated using Primer3Plus version 3.3.0 (https://www.primer3plus.com/primer3plusAbout.html) and UCSC in-silico PCR tools (https://ucsc.gao-lab.org/cgi-bin/hgPcr), respectively.31,32 Sequences of the targeted regions of each primer set were retrieved from the UCSC genome browser (version: GRCh38; https://genome.ucsc.edu). The detailed information of primers is presented in Supplemental Table S1.
Optimization of Targeted PCR Amplification Protocol
PCR reagents from 3 different companies were used throughout this study.33-36 Despite minor variations in the composition of PCR reagents (Supplemental Table S2), a uniform set of optimized PCR conditions was applied using all 3 reagents. For the amplification of BRCA1 F4R4 and F16R16, as well as BRCA2 F18R18 and F25R25, the protocol entailed an initial denaturation at 94°C for 10 minutes, followed by a denaturation step at 95°C for 45 seconds, annealing at 62°C for 45 seconds, extension at 72°C for 45 seconds, and a final extension at 72°C for 1 minute. In contrast, the BRCA2 F23R23 required a distinct approach, starting with an initial denaturation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, annealing at 62°C for 45 seconds, extension at 72°C for 45 seconds and a final extension period of 72°C for 5 minutes.
PCR Product Purification and Sequencing Data Analysis
Following PCR amplification, agarose gel electrophoresis (1.5%) was performed to visualize and confirm the amplification the desired PCR product. 37 The PCR product was purified using the FavorPrep™ GEL/PCR Purification Kit and prepared to send for Sanger sequencing. The sequencing results (chromatogram files in .ab1 format) were visualized and analyzed using Chromas. 38
Results
Patient Information
Blood samples from 13 diagnosed or suspected BC patients were collected and their information was recorded. The age range of the maximum patients was 25 to 34 (38.46%), where most of the patients were female (92.31%). The familial inheritance indicated a larger portion of cases as hereditary (38.46%) and the menstrual status of maximum patients was regular (38.46%). The receptor status revealed that most of the patients showed negative status including ER (−ve; 30.76%), PR (−ve; 38.46%), and HER2 (−ve; 61.53%). The larger part of patients were suffering from diabetes (30.76%) and hypertension (38.46%). Out of 13 patients, 1 patient was male, 4 were suspected due to the presence of breast lump (s) and family history of BC and the remaining 9 patients were diagnosed with BC. All 9 of them had received either adjuvant chemotherapy or radiotherapy followed by surgical intervention. Three of those 4 individuals with breast lumps also underwent breast-conserving surgery. The demographic and Clinicopathological features of the study subject are demonstrated in Table 1.
Patient Information and the Distribution of Different Characteristics.
Abbreviations: n, no. of patients; IDC, invasive ductal carcinoma.
In-Silico Results
The analysis of mutations in gnomAD version 3.2.1, dbSNP, and cBioPortal revealed that missense mutations are predominant in both genes, with a significant number yet to be validated, while confirmed pathogenic or likely pathogenic variations tend to concentrate in particular exons rather than being distributed throughout the entire gene. For BRCA1, these exons were: 2, 4, 10, 14, 16 and 17 and for BRCA2, these exons were 10, 11, 14, 18, 21, 22, 23 and 25 (Figure 1). We selected exons 4 and 16 of BRCA1 and exons 18, 23 and 25 of BRCA2 genes as the starting point of this study (Figure 2). The mutations found in cBioPortal provided additional information on the drug sensitivity associated with the alteration, followed by the mutations, their biomarker level, and functional impact of the specific mutations (Supplemental Table S3).
Distribution of pathogenic variation across exons of BRCA1 (A) and BRCA2 (B).
BRCA1 and BRCA2 target exons and their pathogenic variations documented in breast cancer patients: (A) BRCA1 exons and the targeted mutations. There are 5 pathogenic/likely pathogenic variations targeted in exon 4 and another 5 in exon 16 and (B) BRCA2 exons and their targeted mutations. There are 5, 7 and 9 pathogenic/likely pathogenic variations in exons 18, 23 and 25, respectively.
DNA Extraction, Targeted PCR Amplification and Purification
Agarose gel electrophoresis confirmed the successful isolation of whole genomic DNA via the presence of a single DNA band (Figure 3A). Moreover, the targeted amplification of BRCA1 by F4R4 and F16R16 primers resulted in amplicons of 104 and 101 bp size and the product size of BRCA2 by F23R23, F18R18 and F25R25 primers is 172, 168 and 205 bp, respectively (Figure 3B). Successful purification of the PCR amplicon resulted in single DNA bands in 1.5% agarose gel with the product size being: 104 and 101 bp for the amplicons of BRCA1 exon 4 and 16 and 172, 205 and 168 bp for the amplicons of BRCA2 exon 23, 25 and 18 (Figure 3C). Validation of the presence of specific PCR product for BRCA1 and BRCA2 after purification (Figure 3D).
Validating the extraction of genomic DNA and PCR amplification of the desired size in 1.5% agarose gel: (A) agarose gel after genomic DNA extraction from blood samples. Genomic DNA of 5 samples is shown here in the first, D1-D5 lanes and 100 bp DNA ladder was loaded into the first lane (designated as M), (B) agarose gel after PCR amplification by the primers targeting BRCA1 exon 4 (A1) and 16 (A2) and BRCA2 exon 18 (A3), 23 (A4), and 25 (A5). Here, M denotes 100 bp DNA marker/ladder, (C) validating the presence of a specific PCR product after purification. In the first lane, 100 bp DNA ladder is denoted by M and the purified amplicons of BRCA1 exon 4 (P1) and 16 (P2) and BRCA2 exon 18 (P3), 23 (P4) and 25 (P5) are loaded from the second-sixth lanes, and (D) Agarose gel after PCR amplification by the BRCA2 F2R2 primer targeting exon 18 for patients S1 to S8.
Targeted Sequencing of the Amplicons and Status of the Pathogenic Variations
Upon examining and analyzing the chromatogram (Figure 4), it was observed that, of the 31 targeted mutation sites, 15 were located in regions that exhibited high sequencing quality, ensuring clear readability. Specifically, within the BRCA1 gene, 5 out of the 10 sites associated with PV were distinctly interpretable. Similarly, in the BRCA2 gene, 10 out of the 21 sites linked to PV were clearly readable. The results have been summarized in Table 2. As presented in Table 2, all the positions had the same nucleotide base as in the reference sequence indicating the absence of any PV in the target regions of BRCA1 and BRCA2 genes in the samples analyzed. However, the question remains about the numerous positions outside the region we targeted for this study.
Representative chromatogram image of BRCA1 and BRCA2 amplicons: (A) BRCA1 F4R4 primer (BRCA1 Exon 4), (B) BRCA1 F16R16 primer (BRCA1 Exon 16), (C) BRCA2 F23R23 primer (BRCA2 Exon 23), (D) BRCA2 F18R18 primer (BRCA2 Exon 18), and (E) BRCA2 F25R25 primer (BRCA2 Exon 25).
Summary of Observed Nucleotide Bases at the Target Regions of BRCA1 and BRCA2 from this Study.
Discussion and Future Prospect
Among the high penetrance genes, BRCA1 and BRCA2 PV increase the risk of developing BC by 11 to 12-fold in the female population.39,40 Since PV of BRCA1 and BRCA2 genes have a relatively high occurrence frequency in hereditary BC and TNBC patients, we selected these 2 genes for our study, aiming at their possible use in screening the risk of developing BC as well as in determining TNBC treatment.
Guided by the prevalence of PV documented in the databases, we focused on exons 4 and 16 of BRCA1, and exons 18, 23, and 25 of BRCA2 for primer design. Among the patients included for this study, there were 4 patients with breast lump(s) and family history since the BRCA1 and BRCA2 gene mutations are more prevalent in hereditary BC 13 and 4 TNBC patients since these genes play a crucial role in determining the treatment regime of TNBC patients and 15% to 25 % of TNBC patients have been found to have germline variations in one or both of these genes.16,41 We also included 5 IDC BC patients who were not triple-negative.
Following Sanger sequencing, we analyzed and interpreted data from regions that exhibited clear peaks in the chromatogram. A portion of the chromatogram yielded unclear peaks due to sequencing noise and the short size of the amplicons. Of the 31 targeted positions, 15 target locations were clearly readable (Table 2). Analysis of these 15 regions revealed no mutations, suggesting the possibility of a geographical variance in the mutation spectrum among Bangladeshi BC patients. This outcome is consistent with another study conducted in 52 Bangladeshi BC samples (32 BC patients, 16 undiagnosed but symptomatic patients with breast lump and 4 patients with family history but no symptoms) using exon-specific 52 primers against BRCA1, BRCA2 and ERBB2 genes, multiplex PCR and NGS technology. 42
Our study utilized basic molecular biology techniques and reagents, which are already available in a wider range of institutions in our country. Considering all expenses and potential government subsidy, the estimated cost of this screening test would be nearly 6000 to 7000 taka ($52-$62), which is one-third of the current NGS testing method available in Bangladesh. To translate this research into the clinical field, extension of this study with a larger sample size, inclusion and comparison with the results of NGS samples and larger amplicons targeting a wider region of BRCA1 and BRCA2 genes should be conducted.
Conclusion
This study explored the potential of developing a cost-effective, targeted sequencing approach for BRCA1 and BRCA2 genes to evaluate BC risk in Bangladeshi patients. By focusing on selected exons with high PV density, the approach aims to balance clinical relevance with affordability. Analysis of 13 patient samples revealed no PV in the targeted regions, consistent with previous findings in the Bangladeshi population. This suggests that these specific exons may not harbor common germline variations in this cohort. However, the small sample size limits the generalizability of the results, emphasizing the need for larger-scale studies. Expanding the analysis to include more patients and additional mutation hotspots could improve diagnostic yield and help establish a reliable, low-cost genetic screening platform for hereditary BC in Bangladesh.
Supplemental Material
sj-docx-1-cix-10.1177_11769351261445625 – Supplemental material for Exon-Specific Targeted Analysis of BRCA1 and BRCA2 Mutations in Bangladeshi Breast Cancer Patients
Supplemental material, sj-docx-1-cix-10.1177_11769351261445625 for Exon-Specific Targeted Analysis of BRCA1 and BRCA2 Mutations in Bangladeshi Breast Cancer Patients by Shuvra Dutta, S. M. Mahbubur Rashid, Shihabul Islam, SK Farid Ahmed and Mustak Ibn Ayub in Cancer Informatics
Supplemental Material
sj-pdf-1-cix-10.1177_11769351261445625 – Supplemental material for Exon-Specific Targeted Analysis of BRCA1 and BRCA2 Mutations in Bangladeshi Breast Cancer Patients
Supplemental material, sj-pdf-1-cix-10.1177_11769351261445625 for Exon-Specific Targeted Analysis of BRCA1 and BRCA2 Mutations in Bangladeshi Breast Cancer Patients by Shuvra Dutta, S. M. Mahbubur Rashid, Shihabul Islam, SK Farid Ahmed and Mustak Ibn Ayub in Cancer Informatics
Footnotes
Acknowledgements
We gratefully acknowledge the concerned staff of Japan Bangladesh Friendship Hospital for helping with data and sample collection for our current study. We also acknowledged the guardian and patients who agreed to participate in this study willingly.
List of abbreviations
Ethical Considerations
The protocol for this study was approved and ethical clearance was provided by the Institutional review board (IRB) of the Faculty of Biological Sciences, University of Dhaka (Reference: Ref. No.257lBiol. Scs.). All participants were properly informed about the procedures of the study and their verbal consents were taken before inclusion in the study.
Consent to Participate
Verbal consent under the IRB was taken from the patients.
Consent for Publication
Taken under the IRB.
Authors’ Contributions
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data Availability Statement
All the data related to this article will be available on request to the corresponding author*.
Supplemental Material
Supplemental material for this article is available online.
Declaration on AI Use
COPILOT webtool was occasionally used for grammar check and improving sentence clarity.
References
Supplementary Material
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