|Ahead of print publication
Detection of novel infiltrating ductal carcinoma-associated BReast CAncer gene 2 mutations which alter the deoxyribonucleic acid-binding ability of BReast CAncer gene 2 protein
Lateef Ullah1, Yasir Hameed2, Samina Ejaz2, Anam Raashid2, Jamshed Iqbal3, Inam Ullah1, Syeda Abida Ejaz4
1 Department of Genetics, Hazara University, Mansehra, Pakistan
2 Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
3 Department of Pharmacy, Comsats University, Pakistan
4 Department of Pharmacy, Women Institute of Learning Sciences, Abbottabad; Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan
|Date of Submission||14-Oct-2019|
|Date of Decision||25-Nov-2019|
|Date of Acceptance||07-Jan-2020|
|Date of Web Publication||28-Oct-2020|
Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur
Source of Support: None, Conflict of Interest: None
Background: BReast CAncer gene 2 (BRCA2), a tumor suppressor gene located on chromosome 13q, encodes a 384-kDa protein which activates homologous recombination pathway to repair ssDNA damage.
Aims and Objectives: Keeping in view the high prevalence of breast cancer in Pakistan and significant association of BRCA2 with breast cancer, this project was initiated to investigate the mutational status of BRCA2 in Pakistani breast cancer patients.
Materials and Methods: For this purpose, blood samples of 45 individuals, including 24 female patients (infiltrating ductal carcinoma of breast), who visited Institute of Nuclear Medicine, Oncology and Radiotherapy Hospital and Ayub Medical Complex, Abbottabad, Khyber Pakhtunkhwa, and 21 normal female residents of the area were collected and processed to extract deoxyribonucleic acid (DNA). Different regions of BRCA2 exon 11 were amplified through polymerase chain reaction (PCR), and PCR-amplified products of one (NF45) normal sample and four cancerous (205BC, 215BC, 218BC, 222BC) samples were subjected to DNA sequence analysis.
Results: Analysis of retrieved sequences revealed one novel nonsense mutation in sample 205BC. The observed mutation (delA21587) shifted the normal frame of amino acid (N905I, T906L, K907R, E908N, L909F, H910M, E911K, T912Q, and D913T) in encoded mutant protein and converted L914 into premature termination codon. In case of sample 222BC, another novel substitution mutation (A>G24962) was observed, which altered codon of I (isoleucine) into the codon for M (methionine) at position 2040 in resultant mutant protein.
Conclusion: The results reflect the unique mutational profile of BRCA2 in Pakistani infiltrating ductal carcinoma patients and suggest an extension of study on a large scale.
Keywords: BReast CAncer gene 2, homologous recombination pathway, infiltrating ductal carcinoma, nonsense mutation, premature termination codon, substitution mutation
|How to cite this URL:|
Ullah L, Hameed Y, Ejaz S, Raashid A, Iqbal J, Ullah I, Ejaz SA. Detection of novel infiltrating ductal carcinoma-associated BReast CAncer gene 2 mutations which alter the deoxyribonucleic acid-binding ability of BReast CAncer gene 2 protein. J Can Res Ther [Epub ahead of print] [cited 2020 Dec 2]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=299463
| > Introduction|| |
Human breast is composed of different lobes and ducts, responsible for milk production in females. Malignant cell formation in lobes and ducts gives rise to breast cancer. Infiltrating ductal carcinoma of the breast is considered worldwide as a major subtype (90%) of breast cancer. According to “Oncology Practice,” female mortality rate due to infiltrating ductal carcinoma of the breast is expected to be about 25.3/100,000 women in Columbia during 2018. In 2019, an estimated 268,600 new cases of invasive breast cancer and 62,930 new cases of noninvasive (in situ) breast cancer are expected to be diagnosed in the US. Furthermore, an estimated 41,760 US women are expected to die in 2019 from breast cancer. According to the DAWN news in Pakistan annually, 90,000 new cases are reported and 40,000 deaths occur because of breast cancer.
Infiltrating ductal carcinoma usually promoted by genetic alterations which are the outcome of various factors such as hormonal imbalance, age, environmental factors, and family history. Genetic alterations mostly target BRCA2 “BReast CAncer gene 2” which is a tumor suppressor gene and located on chromosome 13q. The BRCA2 gene is highly associated with infiltrating ductal carcinoma. In earlier studies, a strong relationship has been documented between BRCA2 mutations and infiltrating ductal carcinoma. Acquired or genetically inherited BRCA2 mutations can alter the BRCA2 protein structure and result in aberrant deoxyribonucleic acid (DNA) repair process. The defective DNA repair process ultimately leads to genetic abnormalities and many physiological disorders including infiltrating ductal carcinoma. A lot of work has been done worldwide to explore the types, location, nature, and consequences of BRCA2 mutations prevalent in infiltrating ductal carcinoma patients in order to make a way for possible early diagnosis and treatment of this disease. The earlier known worldwide and Pakistani population-specific BRCA2 mutations have been cited elsewhere.,,
The BRCA2 gene consists of 27 exons and codes for the BRCA2 protein of molecular weight 384 kDa. The BRCA2 protein is a multidomain protein. The transactivation domain of BRCA2 is used to interact with ataxia-telangiectasia mutated (ATM) and many other proteins such as X-ray repair cross-complementing protein 1 and recA DNA repair 51 (RAD51) proteins during DNA repair process. The RAD51 has the ability to bind to any of the 8 BCR repeats of the BRCA2 protein to perform its function. Mutated BRCA2 protein with only single BCR repeat 3 is capable of binding with RAD51. DNA-binding domain helps in the binding of BRCA2 with the target DNA. A C-terminal domain with two nuclear localization signals is required for the transport of BRCA2 protein into the nucleus. The RAD51 protein is the key player involved in the DNA repair process. Usually, BRCA2 and RAD51 complex is present in an inactive form. Followed by damage to the DNA the transactivation domain of BRCA2 is phosphorylated by different proteins such as ataxia telangiectasia and Rad3-related protein and ATM which ultimately lead to activation of BRCA2-RAD51 complex and initiate the process of DNA repair. Activated BRCA2 protein further interacts with its different partner proteins such as p21 (also known as cyclin-dependent kinase inhibitor 1), The Growth Arrest and DNA Damage 45, MutS homolog protein 4, and MutS homolog protein 6 in order to complete the process of DNA repair.
Breast cancer prevalence is increasing day by day in Pakistan, and no recent study has been carried out to establish a noteworthy relationship between BRCA2 mutations and the high prevalence of infiltrating ductal carcinoma in Pakistan. Previously published data regarding BRCA2 mutations in Pakistani infiltrating ductal carcinoma patients are not enough to declare mutated BRCA2 as the causative factor of infiltrating ductal carcinoma in Pakistan. Hence, keeping in view the information regarding the significant association of BRCA2 known worldwide, this study was initiated.
It was hypothesized that breast cancer patients of Pakistan may harbor some novel or already existing BRCA2 mutations being genetically unique from other global populations. This study aimed to update the mutational status of BRCA2 in infiltrating ductal carcinoma female patients of Abbottabad, Khyber Pakhtunkhwa, Pakistan.
| > Methodology|| |
This study involved both wet-lab experiments and dry-lab experiments.
Analytic wet bench process
A total of 45 samples, including infiltrating ductal carcinoma female patients (n = 24) who visited Institute of Nuclear Medicine, Oncology and Radiotherapy Hospital and Ayub Medical Complex and normal females (n = 21) resident of the area, were collected from Abbottabad, Khyber Pakhtunkhwa, Pakistan. Blood was collected from all participants after obtaining consent by following the standard method and ethical guidelines mentioned in the Helsinki Declaration, the year 2013.
Patients satisfying the following criteria were included in the study:
- Patients diagnosed with an invasive infiltrating ductal carcinoma and who received no chemotherapeutic treatment
- Patients who signed the consent form
- Patients of all age groups (40–60 years).
Patients who refused to sign consent form, going through the chemotherapy treatment, and found positive for hepatitis C virus/hepatitis B virus infection were excluded from the study.
Genomic DNA was extracted from whole blood using an organic method with slight modifications in an earlier reported method. Isopropanol-precipitated DNA was resuspended in 0.5 M TE buffer (pH 8.0) and stored at −70°C for polymerase chain reaction (PCR) amplification.
Polymerase chain reaction amplification
Different regions of BRCA2 exon 11 were amplified using three different sets of primers: F1R1, F2R2, and F3R3 [Table 1]. The PCR reaction mixture (50 μl) contained 50–120 ng genomic DNA, 1 X PCR buffer, 200 μM dNTPs, 20 picomoles of both reverse and forward primers, 2 mM of MgSO4, and 1.25 U of Taq DNA polymerase (Thermo Fisher Scientific, Boston, MA, USA).
PCR amplification was performed in MyGene TmL series Peltier thermal cycler (UNIEQUIP). PCR reaction was started with initial heating to 95°C for 1 min followed by 35 cycles at 95°C for 30 s, with specific annealing temperature (57°C) for 1 min and final extension period of 10 min at 72°C. After amplification, PCR products were analyzed on 2% agarose gel electrophoresis.
PCR amplicons were purified by using (Thermo Fisher, purification kit, Cat#T1030S). Purified amplicons were sent to the Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan, for bidirectional DNA sequence analysis.
Dry-lab experiments involved comparative and mutational analysis. During comparative analysis, gene-related information was obtained from the ENSEMBL database (available at: https://asia.ensembl.org/index.html). To identify sequence similarity across all genomes, BLAST (available at: https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used. Pairwise sequence alignment tools (available at: https://www.ebi.ac.uk/Tools/psa/) helped to compare and contrast the retrieved sequences. During mutational analysis, EXPASY translation tool (available at: https://web.expasy.org/translate) was used to translate the cDNA into protein. Subcellular localization of resultant proteins was found out using ProtComp 9.0 (available at: http://www.softberry.com/berry.phtml?topic = protcomp). Construction of three-dimensional (3D) models of resultant proteins was done using PHYRE2 (available at: www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id = index). Effects of exonic and intronic mutations were analyzed using Human Splicing Finder 3.1 (available at: www.umd.be/HSF3/).
| > Results|| |
In this study, a total of 45 participants (24 female infiltrating ductal carcinoma patients and 21 normal females) were analyzed for BRCA2 mutations. Due to financial limitations, only five samples, including four (205BC, 215BC, 218BC, and 222BC) infiltrating ductal carcinoma patients and one normal (NF45) individual sample, were subjected to DNA sequence analysis. DNA sequencing analysis revealed mutations in only two infiltrating ductal carcinoma patients. To explore the effect of detected mutations on encoded BRCA2 protein, bioinformatics analysis was carried out using various web-based tools. For convenience samples found positive for BRCA2 mutations were referred as case no. 1 (222BC) and case no. 2 (205BC) and the proteins encoded by the corresponding mutated BRCA2 genes were categorized as mutant 1 and mutant 2 proteins, respectively.
The frameshift mutation delA21587 noticed in case no. 1 changes N905I, N906L, K907R, E908N, L909F, H910M, E911K, T912Q, D913T, and introduced premature termination codon at position 914. Hence, it resulted in a truncated BRCA2 protein [Figure 1].
|Figure 1: Nature, location, and type of BReast CAncer gene 2 mutations observed in case no. 1 and 2 and the encoded mutant proteins. del = Deletion, A = Adenine, G = Guanine, N = Asparagine, I = Isoleucine, T = Tryptophan, K = Lysine, R = Arginine, E = Glutamic acid, L = Leucine, F = Proline, H = Histidine, M = Methionine, Q = Glutamate, D = Aspartic acid, X = Stop codon|
Click here to view
The substitution mutation (A>G24962) in case no. 2 causes a change of one amino acid (I2040M) in encoded BRCA2 protein [Figure 1].
Functional impact of observed mutations in mutant proteins
Taking the known sequence of all other coding exons and adding studied exon 11 sequence, a full-length coding sequence was generated. Bioinformatics analysis was performed to observe the functional impact of noticed mutations on encoded proteins.
To analyze the functional impact of observed mutations, mutant 1 protein was compared with normal BRCA2 protein [Figure 2]. This comparison revealed that in mutant 1 protein, the introduction of the premature termination codon at position 914 resulted in loss of RAD51-binding sequence, DNA-binding domain, and nuclear localization signals. Moreover, the encoded BRCA2 protein was unable to regulate the activity of other partner proteins such as FA Complementation Group D2 and Tumor Protein 53 due to the absence of interaction sites [Figure 2].
|Figure 2: Functional impact of observed mutations on encoded mutant 1 and 2 BReast CAncer gene 2 proteins. N = Asparagine, I = Isoleucine, T = Tryptophan, K = Lysine, R = Arginine, E = Glutamic acid, L = Leucine, F = Proline, H = Histidine, M = Methionine, Q = Glutamate, D = Aspartic acid, X = Stop codon|
Click here to view
To analyze the functional impact of observed mutations, mutant 2 protein was compared with normal BRCA2 proteins [Figure 2]. This comparison revealed a single amino acid change (I2040M), and no functional difference was noticed in mutant 2 and normal BRCA2 protein. The mutant 1 and mutant 2 proteins were further subjected to bioinformatics analysis to predict pI, molecular weight, subcellular localization, and 3D structure [Table 2]. Results indicated slight changes in pI and molecular weight with no changes in subcellular localization and 3D model of mutant 2 protein as compared to the wild-type BRCA2 protein [Table 2]. Major changes were observed in pI, molecular weight, subcellular localization, and 3D model of mutant 1 protein in comparison to normal BRCA2 protein. In mutant 1 protein, the introduction of the premature termination codon at position 914 resulted in a cytoplasmic truncated protein lacking different functional domains with low pI and molecular weight [Table 2].
|Table 2: Characteristics of normal and mutant BReast CAncer gene 2 proteins|
Click here to view
| > Discussion|| |
BRCA2 gene is identified as a tumor suppressor gene. Mutations in the BRCA2 gene are known to increase the risk of infiltrating ductal carcinoma. In the present study to investigate the mutational status of BRCA2 gene, different regions of exon 11 were amplified through PCR using three pairs of primers (F1R1, F2R2, and F3R3).
The positivity ratio of PCR amplification [Figure 3] with primer pairs F1R1 and F3R3 was higher in cancerous samples (91.66% with F1R1 and 37.5% with F3R3) than the normal ones (90.43% with F1R1 and 23.8% with F3R3). Contrary to this positivity ratio of PCR amplification with primer pair F2R2 was slightly higher in normal samples (90.43%) than observed in case of the cancerous samples (83.33%). No amplification with specific primer indicates that either the population of Abbottabad, Pakhtunkhwa, Pakistan, possessed unique genetic alteration or the complete absence and the presence of highly mutated target regions.
|Figure 3: Percentage positivity ratio of polymerase chain reaction amplification with primers (F1R1, F2R2, and F3R3). F = Forward, R = Reverse, the polymerase chain reaction percentage positivity ratio was calculated using the following formula (no. of samples found positive for polymerase chain reaction amplification/total no. of samples subjected to polymerase chain reaction amplification ×100)|
Click here to view
By keeping in view the budget limitations, a total of five samples, i.e., one normal (NF45) and four infiltrating ductal carcinoma female patients (205BC, 215BC, 218BC, and 222BC), were subjected to DNA sequence analysis. Sequence analysis revealed mutations in two samples (205BC and 222BC).
Mutational analysis of BRCA2 has been conducted worldwide and revealed occurrence of many population-specific nonsense and substitution mutations in infiltrating ductal carcinoma patients. The mutations identified in B Brazilians and Hispanic population included E49X, Q906X, E824X, Q756X, Q742X, C1654X, E2183X, W2586X, N2706X, Q2894X, R3128X, E1593D and E3002K. While L992X, Q619X and E1912X have been detected in Pakistani population. All observed mutations resulted in a truncated BRCA2 protein with altered BRCT domain and DNA-binding ability.,,,
In case no. 1 (222BC), novel nonsense mutation delA21587 was observed, which shifted the normal frame of amino acid in encoded mutant 1 protein and generated a premature termination codon at the 914th position. Due to the introduction of the premature termination codon, the resultant mutant 1 protein lacked BRCT domains that are required for RAD51 binding in ssDNA repair. Moreover, DNA-binding domain (required for binding to the target DNA), nuclear localization signals (required for transport of BRCA2 into the nucleus), and C-terminal domain (required for the regulatory function of BRCA2) were missing in mutant 1 protein [Figure 2]. Low pI, molecular weight, and abnormal 3D structure of mutant 1 protein [Table 2] as compared with normal BRCA2 protein suggest the role of observed BRCA2 mutations in the dysregulated DNA repair process which may further promote the pathogenesis of infiltrating ductal carcinoma of the breast.
In case no. 2, novel substitution mutation (A>G24962) was observed that substituted a hydrophobic I (isoleucine) with hydrophobic M (methionine) at position 2040 in the nonfunctional domain of resultant mutant 2 protein [Figure 2]. Slight changes in pI and molecular weight of mutant 2 protein were observed while no changes have been noticed in a 3D model of mutant 2 protein [Table 2]. In light of available literature, the substitution of isoleucine and methionine is a well-tolerated substitution which does not alter the domain structure and function of the BRCA2 protein.
The findings of the present study have strengthened the initial hypothesis regarding the occurrence of unique BRCA 2 mutations in the Pakistani infiltrating ductal carcinoma patients. Results of our study have revealed that the Pakistani infiltrating ductal carcinoma patients harbor few novel BRCA 2 mutations. One novel mutation delA21587 resulted defective BRCA2 protein while other novel missense mutation A>G 24962 had no affect the structure and functionality of the encoded BRCA 2 protein. No PCR amplification with specific primer indicates the absence of target region or presence of genetically unique alterations into the target regionand suggests further screening.
| > Conclusion|| |
The results of the present study reflect the unique mutational profile of BRCA 2 in Pakistani breast infiltrating ductal carcinoma patients and suggest an extension of study on a large scale. There is a need to further extend the study on a large scale to explore the functional impact of noticed BRCA 2 mutations through different wet -lab experiments. Isolation and characterization of mutant BRCA2 isoforms/variants and recording variations in BRCA 2 protein levels will also be helpful for future applications as diagnostic and prognostic markers.
We are thankful to the cancer patients for their support during blood sample collection.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
DedgeM. Breast cancer deaths projected for 2018. 2018.
Smith RA, Andrews KS, Brooks D, Fedewa SA, Manassaram-Baptiste D, Saslow D, et al
. Cancer screening in the United States, 2019: A review of current American Cancer Society guidelines and current issues in cancer screening. CA Cancer J Clin 2019;69:184-210.
Faiza I. Over 40,000 die of Breast Cancer Every year in Pakistan. DAWN News; 2017.
Zhu Y, Wu J, Zhang C, Sun S, Zhang J, Liu W, et al
. BRCA mutations and survival in breast cancer: An updated systematic review and meta-analysis. Oncotarget 2016;7:70113-27.
Yang RL, Mick R, Lee K, Graves HL, Nathanson KL, Domchek SM, et al
. DCIS in BRCA1 and BRCA2 mutation carriers: Prevalence, phenotype, and expression of oncodrivers C-MET and HER3. J Transl Med 2015;13:335.
Stoppa-Lyonnet D. The biological effects and clinical implications of BRCA mutations: Where do we go from here? Eur J Hum Genet 2016;24 Suppl 1:S3-9.
Nahleh Z, Otoukesh S, Dwivedi AK, Mallawaarachchi I, Sanchez L, Saldivar JS, et al
. Clinical and pathological characteristics of Hispanic BRCA-associated breast cancers in the American-Mexican border city of El Paso, TX. Am J Cancer Res 2015;5:466-71.
Carraro DM, Koike Folgueira MA, Garcia Lisboa BC, Ribeiro Olivieri EH, Vitorino Krepischi AC, de Carvalho AF, et al
. Comprehensive analysis of BRCA1, BRCA2 and TP53 germline mutation and tumor characterization: A portrait of early-onset breast cancer in Brazil. PLoS One 2013;8:e57581.
Rashid MU, Zaidi A, Torres D, Sultan F, Benner A, Naqvi B, et al
. Prevalence of BRCA1 and BRCA2 mutations in Pakistani breast and ovarian cancer patients. Int J Cancer 2006;119:2832-9.
Galkin VE, Esashi F, Yu X, Yang S, West SC, Egelman EH. BRCA2 BRC motifs bind RAD51-DNA filaments. Proc Natl Acad Sci U S A 2005;102:8537-42.
Venkitaraman AR. Functions of BRCA1 and BRCA2 in the biological response to DNA damage. J Cell Sci 2001;114:3591-8.
Tan SC, Yiap BC. DNA, RNA, and protein extraction: The past and the present. J Biomed Biotechnol 2009.
Silver DP, Livingston DM. Mechanisms of BRCA1 tumor suppression. Cancer Discov 2012;2:679-84.
Spitzer E, Abbaszadegan MR, Schmidt F, Hauser A, Buwitt U, Lauter FR, et al
. Detection of BRCA1 and BRCA2 mutations in breast cancer families by a comprehensive two-stage screening procedure. Int J Cancer 2000;85:474-81.
Ohmura T, Ueda T, Hashimoto Y, Imoto T. Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme. Protein Eng 2001;14:421-5.
Lara K, Consigliere N, Pérez J, Porco A. BRCA1 and BRCA2 mutations in breast cancer patients from Venezuela. Biol Res 2012;45:117-30.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]