Significant association of BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan

Significant association of BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Significant association of BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan Yumna Khan, Najeeb Ullah Khan, Ijaz Ali, Samiullah Khan, Aakif Ullah Khan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2625327/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jun, 2023 Read the published version in Molecular Biology Reports → Version 1 posted 3 You are reading this latest preprint version Abstract Background: Single Nucleotide polymorphism (SNPs) in BRCA1 , BRCA2 and TP53 has been widely associated with breast cancer risk in different ethnicities with inconsistent results. There is no such study conducted so far in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. Therefore, this study was conducted to check BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan. Methods: This study, consisting 140 breast cancer patients and 80 gender and age matched healthy controls were subjected to confirm BRCA1 , BRCA2 and TP53 polymorphism. Clinicopathological data and blood samples were taken from all the participants. DNA was extracted and SNPs were confirmed using T-ARMS-PCR protocol. Results: Our data indicated that BRCA1 , BRCA2 , and TP53 selected SNPs risk allele and risk allele containing genotypes displayed significant association ( P < 0.05) with breast cancer risk in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. Conclusion: All the three selected SNPs of BRCA1 , BRCA2 and TP53 showed significant association with breast cancer risk in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. However, more investigation will be required on large data sets to confirm the selected SNPs and other SNPs in the selected and other related genes with the risk of breast cancer. Breast cancer Risk association BRCA1 BRCA2 TP53 Polymorphisms Figures Figure 1 Figure 2 Figure 3 Introduction Breast cancer is the second leading cause of death and the most common diagnosed cancer in women worldwide [ 1 ]. Large scale population studies including GWAS, have primarily focused on the genetic predispositions associated with chronic disease amongst the Caucasian population. However, amongst all developing countries Pakistan has one of the highest rates of breast cancer, accounting for approximately 38% of total cancer diagnoses within the country. With the advent of more effective sequencing procedures, the ability to assess cancer risk and genetic diversity at a subpopulation level has allowed clinical researchers to develop innovative and personalized treatment protocols specific to their communities. With respect to breast cancer, a continuing need in developing countries is to elucidate the genetic predispositions that associate with cancer susceptibility, onset, development, progression, relapse, and drug response. Subsequently, in Pakistan there has been an enormous effort to collect genomic data from various tribal populations within the country in order to develop epidemiologic risk factors linked to breast cancer development [ 2 ]. To date, relatively few studies have identified the genetic risk factors for breast and ovarian cancer development within the Pakistani population [ 3 , 4 ]. Notable study by Liede et al . [ 4 ] focused on the distribution of BRCA1/2 mutations across numerous Pakistani tribes, including those of Punjabi, Muhajir, Path, Sindhi, Memon, and Parsi descent. However, this study was performed in early 2002, with little to no in-depth follow up, and before the enhanced molecular classification of breast cancer (i.e, Luminal A, HER2, TNBC) [ 5 ]. In this study, identification of known genetic risk alleles for breast cancer such as BRCA1 , BRCA2 , and TP53 was performed solely in patients located in the Khyber Pakhtunkhwa region of Pakistan, so as to better understand the prevalence and genetic risk factors associated with breast cancer development in those of Pathan or Pashtun ancestry. Breast cancer is the result of cumulative mutations within oncogenes and tumor suppressor genes that result in the clonal development of progressively malignant cells [ 6 , 7 ]. Traditionally, breast cancer was categorized by the presence or absence of estrogen receptor-alpha (ER𝛼), progesterone receptor (PR), and human epidermal growth factor receptor (EGFR/HER2) [ 8 ]. Recently, molecular subdivision of breast cancer facilitated a denser subcategorization profile that includes Luminal A, Luminal B, HER2, and Basal-like (which can be further stratified into BL1/BL2, stem cell-like, androgen, immunomodulatory, HER2-enriched. Unfortunately, mutations in the DNA repair genes responsible for maintaining genomic stability such as BRCA1 , BRCA2 and TP53 have not been included in such molecular categorization. Although it is widely accepted that mutations in DNA repair genes are causally linked to the onset of breast cancer development, and are more prevalent in triple negative breast cancer, as defined by the absence of ER𝛼, PR, and HER2, and are thus more difficult to treat clinically [ 9 , 10 ]. BRCA1 has numerous roles in facilitating DNA repair, particularly in cell cycle checkpoint regulation, single strand annealing (SSA), homologous recombination (HR), and non-homologous end joining (NHEJ) [ 11 ]. BRCA2 is involved in maintaining genomic stability by facilitating homologous recombination [ 12 ]. Specifically, BRCA2 co-localizes with BRCA1 and maintains genomic stability through homology-directed DNA repair (HDDR) processes that in turn controls cell cycle progression [ 13 , 14 ]. Approximately 20% of all familial breast cancer cases worldwide are attributed to mutations in BRCA1 and BRCA2 [ 15 , 16 ]. BRCA1 mutations (i.e., reference SNPs rs8170 and rs2363956) are associated with ER𝛼-negative staining in breast tumors [ 15 – 17 ]whereas, mutations in BRCA2 are more often correlated with ER𝛼-positive status [ 11 , 18 ]. TP53 is a transcriptional regulator and tumor suppressor gene located on chromosome 17p13.1 and nuclear phosphoprotein (p53) that plays a vital role in cell cycle regulation, DNA damage repair, and cell survival under genotoxic stress (i.e., exposure to DNA damaging agents, chemotherapeutic drugs, and molecules that that cause DNA nicking or double stranded DNA breakpoint thereby reducing DNA integrity [ 19 ]. Due to its ability to protect the genome by responding to genotoxic stress and other stress signals, it is also dubbed as “The guardian of the genome” [ 20 ]. TP53 mutations are present in 30–50% of malignancy in human and these mutations occur in cancers with BRCA1 and BRCA2 mutations with greater frequency as compared to sporadic tumors with wild type BRCA1 and BRCA2 [ 21 ]. Breast cancer is a heterogeneous disease characterized by variant pathological features, diverse response to therapeutics and extensive differences in long-term survival [ 22 ]. One of the reasons for this heterogeneity in cancer incidence is the key genetic polymorphisms in gate keeper genes such as TP53 [ 23 ]. The inter-individual inherited polymorphisms may also be an important factor regarding the predisposition for the development of cancer as well the variation of chemo and radiation therapy responses [ 24 , 25 ]. A common polymorphism in BRCA1 (rs1799950), reported in a number of familial breast cancers [ 26 , 27 ] is located in the ring finger domain [ 28 ]. This domain is crucial to BRCA1 function because of its interaction with numerous genes such as, RAD51, p53, and pRB which are all related to DNA repair [ 28 , 29 ]. Recently, it was reported that rs1799950 is significantly associated with a greater risk for developing breast cancer [ 30 ]. Similarly, a polymorphism in BRCA2 (rs144848), causes change in the amino acid sequence resulting in A to C substitution i.e asparagine to histidine transition in exon 10 [ 31 ] and was found to be associated with an increased risk of breast cancer [ 32 ]. Likewise, a non-synonymous polymorphism in codon 72 is the most common SNP in the TP53 and occurs in the non-conserved proline-rich region of exon 4 [ 33 ]. The proline rich domain has been revealed to be vital for apoptotic function of p53 [ 34 ]. Several lines of evidence showed that Arg72Pro can play an important role in apoptosis and cancer formation in humans and is supported by observation that iASPP, a specific cellular inhibitor of p53, binds to the proline rich domain of p53 and interacts more strongly with Pro72 variant leading to inhibition of apoptosis [ 35 ]. In the present study we have randomly selected the following polymorphisms ( BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) that have been reported to increase breast cancer risk. There is no such evidence from our region. Different results between studies might actually reflect inherent characteristics of the population being analyzed, as there are variations in the relative frequencies of the two polymorphic alleles in various populations. We aimed to investigate the association of selected polymorphisms of BRCA1 , BRCA2 and TP53 (rs1799950, rs144848 and rs1042522, respectively) with breast cancer risk in the population of Khyber Pakhtunkhwa, Pakistan. The association between genetic variants, demographic, and clinicopathological characteristics of breast cancer patients were also assessed. Materials And Methods Study population, ethical consideration and sample collection In the present case-control study, a total of 220 samples (140 breast cancer patients and 80 healthy controls) were recruited from Institute of Radiotherapy and Nuclear Medicine (IRNUM) Hospital Peshawar, Pakistan. An ethical approval for the study was obtained from IRNUM as well as from the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar (UAP) and written consent was taken from the patients and control subjects. Under standard biosafety protocol and ethical consideration, about 3cc blood samples were collected from all subjects during October 2020 to January 2021, with demographic as well as all clinical parameters on prescribed proforma. The collected samples were stored in EDTA vials at -20ºC at IBGE for further processing. Dna Isolation, Snps Genotyping And Gel Electrophoresis DNA isolation, SNPs Genotyping and Gel electrophoresis DNA extraction and SNP genotyping was done with the procedure previously adopted in our lab [ 36 , 37 ]. Primer blast software was used to design the specific primers for each SNP (Table 1). Homozygous and heterozygous mutations were detected by using two sets of outer and inner primers (forward and reverse). The PCR mixture of 10 µL was prepared, consisting of 5 µL master mix (Thermo scientific DreamTaq Master Mix (2X), 0.5 µL of each forward and reverse primer, 1 µL of template DNA, and 3 µL of ddH2O. The PCR conditions were; Initial denaturation 95ºC for 5min, followed by 35 cycles of denaturation at 95ºC for 30sec, annealing for 30sec at 62ºC, 62ºC and 65.9ºC for BRAC1 , BRAC2 and TP53 respectively, extension 72ºC for 30sec and final extension at 72ºC for 5min. The amplified PCR products were run and confirmed with 1KB ladder (Thermo scientific) on 2% agarose gel. Statistical analysis Statistical analysis was carried out using SPSS software version 16. The alleles and genotypes were compared between the cases and controls using chi-square test. The association between genetic variants and breast cancer was determined using the Mantel-Haenszel test to assess odds ratio and 95% confidence intervals. The association between demographic, clinicopathological characteristics and genetic variants of breast cancer patients was also analyzed using chi-square test. P -values less than 0.05 were considered statistically significant. Results Association between demographic, clinicopathological characteristics and genetic variants A total of 140 breast cancer patients were recruited in this study. The demographic, clinicopathological and genotypic characteristics of patients are presented in Table 2. The BRCA1/2 and TP53 genotypes were analyzed according to demographic and clinicopathological characteristics of breast cancer patients. We did not find any association among BRCA1/2 genotypes and grade, stage, nodal status and metastasis. Significant associations were among the BRCA1/2 genotypes with family history, nulliparity and menstrual status of patients. BRCA1 (rs1799950) was found to be associated with HER2 ( p = 0.019) and TNBC ( p = 0.000) displayed negative correlation with HER2 ( p = 0.019) but exhibited positive affiliation with TNBC ( p = 0.000) while BRCA2 (rs144848) showed association with Luminal A ( p = 0.000) and Luminal B ( p = 0.015) subtypes. In relation to TP53 (rs1042522), no association was observed among genotypes and family history while nulliparity ( p = 0.004), menstrual status ( p = 0.022), high nuclear grade ( p = 0.002), stage ( p = 0.000), nodal status ( p = 0.001) and metastasis ( p = 0.003) displayed significant association with genotypes. TP53 (rs1042522) was found to be associated with HER2 ( p = 0.034) and TNBC ( p = 0.013) was found to be inversely associated with HER2 ( p = 0.034) and positively associated with TNBC ( p = 0.013). Allelic and genotypic frequencies of BRCA1 (rs1799950) Allelic and genotypic frequencies of BRCA1 (rs1799950) were first assessed utilizing an ARMS-PCR protocol on all case samples, including healthy controls (Fig. 1). Our results indicated a statistically significant association of risk allele and risk allele containing genotypes with increased risk of breast cancer (Table 3). The frequency of risk allele (C) was significantly higher in breast cancer patients (33.57%) as compared to controls (18.12%) and was noted with an increased risk of breast cancer (OR = 2.283, 95% CI = 1.423–3.662, P = 0.001). Similarly, the risk allele containing genotypes CC (OR = 4.449, 95% CI = 1.447–13.676, P = 0.005) and TC (OR = 1.926, 95% CI = 1.035–3.585, P = 0.037) were significantly associated with the high risk of breast cancer. Allelic and genotypic frequencies of BRCA2 polymorphism (rs144848) The allelic and genotypic frequencies of BRCA2 (rs144848) was done using ARMS-PCR (Fig. 2). The results showed high frequency of major allele (A) in controls (78.7%) than in patients (47.85%), while the frequency of minor allele (C) was significantly higher in breast cancer patients (52.14%) as compared to controls (21.25%) and was associated with an increased risk of breast cancer (OR = 4.038, 95% CI = 2.586–6.304, P = 0.000). The frequencies of AA (wild), AC (heterozygous) and CC (mutant) genotypes were 62.5%, 32.5% and 5% in controls and 22.8%, 50% and 27.14% in patients, respectively. Our results indicated that individuals carrying risk allele containing genotypes CC (OR = 14.84, 95% CI = 4.835–45.574, P = 0.000) and AC (OR = 4.207, 95% CI = 2.236–7.915, P = 0.000) were at increased risk of breast cancer (Table 4). Allelic and genotypic frequencies of TP53 polymorphism (rs1042522) The allelic and genotypic frequency of TP53 was done using T-ARMS-PCR (Fig. 3). Our results displayed high frequency of major allele (G) (73.75%) in controls than in breast cancer patients (51.4%), while the minor allele (C) was significantly higher in breast cancer patients (48.5%) as compared to controls (26.25%) and was significantly linked with an increased risk of breast cancer (OR = 2.653, 95% CI = 1.738–4.051, P = 0.000). The genotypic distribution GG (wild), GC (heterozygous) and CC (mutant) in the control group was 55%, 37.5% and 7.5%, while this distribution in the patient group was 24.2%, 54.3% and 21.4%, respectively. The risk genotype CC (OR = 6.471, 95% CI = 2.418–17.313, P = 0.000) and GC (OR = 3.278, 95% CI = 1.772–6.067, P = 0.000) were significantly associated with the high risk of breast cancer (Table 5). Discussion In Pakistan, breast cancer is the most common cancer in women with a 2.5% greater incidence rate as compared to India and Iran [ 38 ]. The key elements that play an important role in the development of breast cancer include genetics, environment, endogenous and exogenous hormones in females, reproductive experience and change in immune status [ 38 ]. Genetic polymorphism is the most common type of genetic variation and recently, researchers have focused on polymorphisms that are statistically significantly associated with breast cancer risk amongst the general population. Researchers have identified that genetic polymorphisms are one of the causes of individual difference in cancer incidence [ 23 ]. There is no such work done about the Pushtun population of Khyber Pakhtunkhwa, Pakistan. The current study was conducted to investigate the association of selected polymorphisms of BRCA1 , BRCA2 and TP53 (rs1799950, rs144848 and rs1042522, respectively) with breast cancer risk in the population of Khyber Pakhtunkhwa, Pakistan and to analyze the association among genetic variants, demographic, and clinicopathological characteristics of breast cancer patients. The result of our study displayed a significant association of rs1799950 heterozygous ( p = 0.037) as well as homozygous mutant genotype ( p = 0.005) with breast cancer risk. Though, the mutant genotype was found to be more associated (OR = 4.449, 95% C.I = 1.447–13.676). Previously, similar studies showed the association of rs1799950 ( BRCA1 ) with Breast cancer risk. The homozygous mutant genotype was found to be associated with breast cancer risk in the Saudi population [ 39 ] while the heterozygous genotype was found to be associated with breast cancer risk in Sudanese [ 40 ]. rs1799950 genotype exhibited a positive relationship with family history, nulliparity, menstrual status, ER- and PR- status, but displayed negative correlation with HER2 abundance. This is the first study in the Pakistani population to confirm this, thus more research is needed to investigate the genotypic distribution of rs1799950 and its association with demographic and clinicopathological characteristics of breast cancer patients. Though, it has been vastly documented that BRCA1 mutation is associated with negative hormone receptor status [41,42] which supports the current result. Similarly, our results revealed that the minor allele frequency of BRCA2 was significantly higher in breast cancer patients (52.14%) as compared to controls (21.25%) and strong association was observed between homozygous mutant genotype and breast cancer risk ( p = 0.000, OR = 14.844, 95% C.I = 4.835–45.574) as compared to heterozygous genotype AC ( p = 0.000, OR = 4.207, 95% C.I = 2.237–7.915). Many studies have suggested that this SNP is a susceptibility locus for cancers [ 43 ]. A meta-analysis was performed including 40 relevant studies, out of which 22 studies focused on breast cancer in order to assess the association between rs144848 and breast cancer risk [ 44 ]. Their results showed significant association between rs144848 and cancer risk in all genetic models. In the present study, BRCA2 SNP displayed a positive relationship with Luminal A and Luminal B subtypes. A prior study also demonstrated the association of ER + subtype in Kazakh population, which was the only predominant histological type in BRCA2 carriers [ 18 ]. Moreover, in an earlier study BRCA2 mutations were frequently seen in people with family history [ 45 ] similar to our study which also showed association with family history. Further, well-designed studies are warranted to confirm the current results and more investigations are needed to examine the genotypic distribution of rs144848 and its association with demographic and clinicopathological characteristics of breast cancer patients. In relation to rs1042522 ( TP53 ), several studies have been conducted to evaluate its potential role as a risk factor of breast cancer but the results have been inconclusive. The outcome of existing study is in line with those that detected higher prevalence of homozygous mutant genotype CC in patients with breast cancer as reported in [ 46 , 47 ] while in contrast some authors failed to demonstrate a potential role of rs1042522 in breast cancer and no association was found between CC genotype [ 48 , 49 ]. Our results displayed, strong association of homozygous mutant genotype with breast cancer risk ( p = 0.000, OR = 6.471, 95% C.I = 2.418–17.313) as compared to heterozygous genotype GC ( p = 0.000, OR = 3.278, 95% C.I = 1.772–6.067). A previous report unveiled that major allele was more prominent in Pakistani sporadic breast cancer patients than normal subjects [ 50 ] while in current research, minor allele was found to be associated and was present in high frequency in breast cancer patients (48.5%) than in controls (26.25%). The difference could be due to exposure of particular carcinogens, diet, reproductive pattern, geography and different lifestyle. This SNP was found to be associated with high grade and TNBC [51] which supports the outcome of the current study. Declarations Conflict of interest The authors declare no conflict of interest regarding to publish this paper. Author/s contribution: Y.K. and N.K designed this study. Y.K, N.K, SK conducted the analysis, produced the figures, and drafted the manuscript. A.K, and B.A co-wrote the manuscript. IA, AI and B.A reviewed the manuscript and provided advice on the content of this manuscript. Compliance with Ethical Standards Funding There is no funding received for this study. Ethical approval and Informed consent All the procedures performed in this study were in accordance with the ethical standards of the IBGE, UAP and IRNUM hospital research committees and with the 1964 Helsinki declaration. Informed consent was obtained from all the participants while explaining the objectives of the study. References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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Clinical and pathologic characteristics of patients with BRCA-positive and BRCA-negative breast cancer. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26: 4282–4288. doi:10.1200/JCO.2008.16.6231 Karp SE, Tonin PN, Bégin LR, Martinez JJ, Zhang JC, Pollak MN, et al. Influence of BRCA1 mutations on nuclear grade and estrogen receptor status of breast carcinoma in Ashkenazi Jewish women. Cancer. 1997;80: 435–441. doi:10.1002/(sici)1097-0142(19970801)80:33.0.co;2-y Lord CJ, Ashworth A. RAD51, BRCA2 and DNA repair: a partial resolution. Nature structural & molecular biology. United States; 2007. pp. 461–462. doi:10.1038/nsmb0607-461 Xu G-P, Zhao Q, Wang D, Xie W-Y, Zhang L-J, Zhou H, et al. The association between BRCA1 gene polymorphism and cancer risk: a meta-analysis. Oncotarget. 2018;9: 8681–8694. doi:10.18632/oncotarget.24064 Mehrgou A, Akouchekian M. The importance of BRCA1 and BRCA2 genes mutations in breast cancer development. Med J Islam Repub Iran. 2016;30: 369. Buyru N, Tigli H, Dalay N. P53 codon 72 polymorphism in breast cancer. Oncol Rep. 2003;10: 711–714. Damin APS, Frazzon APG, Damin DC, Roehe A, Hermes V, Zettler C, et al. Evidence for an association of TP53 codon 72 polymorphism with breast cancer risk. Cancer Detect Prev. 2006;30: 523–529. doi:10.1016/j.cdp.2006.09.007 Suspitsin EN, Buslov KG, Grigoriev MY, Ishutkina JG, Ulibina JM, Gorodinskaya VM, et al. Evidence against involvement of p53 polymorphism in breast cancer predisposition. International journal of cancer. United States; 2003. pp. 431–433. doi:10.1002/ijc.10834 Mabrouk I, Baccouche S, El-Abed R, Mokdad-Gargouri R, Mosbah A, Saïd S, et al. No evidence of correlation between p53 codon 72 polymorphism and risk of bladder or breast carcinoma in Tunisian patients. Ann N Y Acad Sci. 2003;1010: 764–770. doi:10.1196/annals.1299.137 Aziz I, Rashid MU, Sultan F, Shakoori AR. Frequency of Pro Allele on Codon 72 of TP53 in Female Breast Cancer Patients of Pakistan: Molecular Stress or Geography. Pak J Zool. 2013;45: 1437–1446. Abubakar M, Guo C, Koka H, Sung H, Shao N, Guida J, et al. Clinicopathological and epidemiological significance of breast cancer subtype reclassification based on p53 immunohistochemical expression. NPJ breast cancer. 2019;5: 20. doi:10.1038/s41523-019-0117-7 Tables Tables 1 to 5 are available in the Supplementary Files section Supplementary Files Table1.pdf Table2.pdf Table3.pdf Table4.pdf Table5.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Jun, 2023 Read the published version in Molecular Biology Reports → Version 1 posted Reviewers agreed at journal 07 Mar, 2023 Editor assigned by journal 27 Feb, 2023 First submitted to journal 25 Feb, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2625327","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":181513028,"identity":"1b4eb7f8-e786-4d8d-b46f-f3eb68e1389c","order_by":0,"name":"Yumna Khan","email":"","orcid":"","institution":"The University of Agriculture Peshawar","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yumna","middleName":"","lastName":"Khan","suffix":""},{"id":181513029,"identity":"bf24c60c-e07c-4f09-b99f-840fa1d7e7dd","order_by":1,"name":"Najeeb Ullah Khan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYHCCBAbGhoQEBgbmAxA+kGJsIE4LWwLRWhigWngMiNNizn7g4YOPO9Ly+Gf3fPzMu4NBju9GAtvDGXi0WPYkJBvOPJNTLHHn7GZp3jMMxpI3EtgNN+DRYnAgIU2at60iseFG7gYggyFxA9AWyQf4tJx/kP77L1DL/Bs5j38DtdQT1nIjIY2ZsS0HaHgOG8iWBAOQFrwOu/EgWbL3TFrixhtpZpZzz0gAPfaw3RCf9w3O5yR++LkjOXHejeTHN97usJHnO5587GEPHi3A6EhAsBkbJEBkG14NDAzsB5C1gCk2AlpGwSgYBaNghAEAP2BcEhNUy8IAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2568-9484","institution":"University of Agriculture Peshawar","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Najeeb","middleName":"Ullah","lastName":"Khan","suffix":""},{"id":181513030,"identity":"59d7b781-0ee7-478c-bc74-79b09ea0f321","order_by":2,"name":"Ijaz Ali","email":"","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ijaz","middleName":"","lastName":"Ali","suffix":""},{"id":181513031,"identity":"23a6c538-a25b-43e9-a3e0-5080a9b503b5","order_by":3,"name":"Samiullah Khan","email":"","orcid":"","institution":"IRNUM Hospital Peshawar","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Samiullah","middleName":"","lastName":"Khan","suffix":""},{"id":181513032,"identity":"4d291e84-6812-4ab8-86cb-0ed81fc805ac","order_by":4,"name":"Aakif Ullah Khan","email":"","orcid":"","institution":"IRNUM Hospital Peshawar","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aakif","middleName":"Ullah","lastName":"Khan","suffix":""},{"id":181513033,"identity":"1b14a439-ca15-4cfc-b152-58479427085d","order_by":5,"name":"Aqib Iqbal","email":"","orcid":"","institution":"The University of Agriculture 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1","display":"","copyAsset":false,"role":"figure","size":264562,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2625327/v1/2364a8bb3d75ad8cb5c17ffc.png"},{"id":34169400,"identity":"a9b16b9e-58dc-475f-92f9-adc1c71484b4","added_by":"auto","created_at":"2023-03-13 14:35:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171169,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2625327/v1/943cf8dea2ea510b6d301fb8.png"},{"id":34173331,"identity":"5b1f985e-d98b-452a-a72c-5344a284e7f7","added_by":"auto","created_at":"2023-03-13 14:59:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":161967,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2625327/v1/6b6b51aaa5038861fff370c8.png"},{"id":44730830,"identity":"faf3783e-abc3-496d-8399-f3e3e1612f08","added_by":"auto","created_at":"2023-10-16 21:34:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":833226,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2625327/v1/d8149591-9a32-4d2b-bfb5-64bf1feb5a51.pdf"},{"id":34173333,"identity":"98cbddf3-faaa-4ee1-b720-4ca558a3709f","added_by":"auto","created_at":"2023-03-13 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14:43:51","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":101731,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2625327/v1/8d5810470eb4cf656a921b7c.pdf"}],"financialInterests":"","formattedTitle":"Significant association of BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the second leading cause of death and the most common diagnosed cancer in women worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Large scale population studies including GWAS, have primarily focused on the genetic predispositions associated with chronic disease amongst the Caucasian population. However, amongst all developing countries Pakistan has one of the highest rates of breast cancer, accounting for approximately 38% of total cancer diagnoses within the country. With the advent of more effective sequencing procedures, the ability to assess cancer risk and genetic diversity at a subpopulation level has allowed clinical researchers to develop innovative and personalized treatment protocols specific to their communities. With respect to breast cancer, a continuing need in developing countries is to elucidate the genetic predispositions that associate with cancer susceptibility, onset, development, progression, relapse, and drug response. Subsequently, in Pakistan there has been an enormous effort to collect genomic data from various tribal populations within the country in order to develop epidemiologic risk factors linked to breast cancer development [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To date, relatively few studies have identified the genetic risk factors for breast and ovarian cancer development within the Pakistani population [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Notable study by Liede \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] focused on the distribution of \u003cem\u003eBRCA1/2\u003c/em\u003e mutations across numerous Pakistani tribes, including those of Punjabi, Muhajir, Path, Sindhi, Memon, and Parsi descent. However, this study was performed in early 2002, with little to no in-depth follow up, and before the enhanced molecular classification of breast cancer (i.e, Luminal A, HER2, TNBC) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In this study, identification of known genetic risk alleles for breast cancer such as \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e, and \u003cem\u003eTP53\u003c/em\u003e was performed solely in patients located in the Khyber Pakhtunkhwa region of Pakistan, so as to better understand the prevalence and genetic risk factors associated with breast cancer development in those of Pathan or Pashtun ancestry.\u003c/p\u003e \u003cp\u003eBreast cancer is the result of cumulative mutations within oncogenes and tumor suppressor genes that result in the clonal development of progressively malignant cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Traditionally, breast cancer was categorized by the presence or absence of estrogen receptor-alpha (ER\u0026#120572;), progesterone receptor (PR), and human epidermal growth factor receptor (EGFR/HER2) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recently, molecular subdivision of breast cancer facilitated a denser subcategorization profile that includes Luminal A, Luminal B, HER2, and Basal-like (which can be further stratified into BL1/BL2, stem cell-like, androgen, immunomodulatory, HER2-enriched. Unfortunately, mutations in the DNA repair genes responsible for maintaining genomic stability such as \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e have not been included in such molecular categorization. Although it is widely accepted that mutations in DNA repair genes are causally linked to the onset of breast cancer development, and are more prevalent in triple negative breast cancer, as defined by the absence of ER\u0026#120572;, PR, and HER2, and are thus more difficult to treat clinically [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eBRCA1\u003c/em\u003e has numerous roles in facilitating DNA repair, particularly in cell cycle checkpoint regulation, single strand annealing (SSA), homologous recombination (HR), and non-homologous end joining (NHEJ) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eBRCA2\u003c/em\u003e is involved in maintaining genomic stability by facilitating homologous recombination [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Specifically, \u003cem\u003eBRCA2\u003c/em\u003e co-localizes with \u003cem\u003eBRCA1\u003c/em\u003e and maintains genomic stability through homology-directed DNA repair (HDDR) processes that in turn controls cell cycle progression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Approximately 20% of all familial breast cancer cases worldwide are attributed to mutations in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eBRCA1\u003c/em\u003e mutations (i.e., reference SNPs rs8170 and rs2363956) are associated with ER\u0026#120572;-negative staining in breast tumors [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR16\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e17\u003c/span\u003e]whereas, mutations in \u003cem\u003eBRCA2\u003c/em\u003e are more often correlated with ER\u0026#120572;-positive status [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTP53 is a transcriptional regulator and tumor suppressor gene located on chromosome 17p13.1 and nuclear phosphoprotein (p53) that plays a vital role in cell cycle regulation, DNA damage repair, and cell survival under genotoxic stress (i.e., exposure to DNA damaging agents, chemotherapeutic drugs, and molecules that that cause DNA nicking or double stranded DNA breakpoint thereby reducing DNA integrity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Due to its ability to protect the genome by responding to genotoxic stress and other stress signals, it is also dubbed as \u0026ldquo;The guardian of the genome\u0026rdquo; [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. TP53 mutations are present in 30\u0026ndash;50% of malignancy in human and these mutations occur in cancers with BRCA1 and BRCA2 mutations with greater frequency as compared to sporadic tumors with wild type BRCA1 and BRCA2 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBreast cancer is a heterogeneous disease characterized by variant pathological features, diverse response to therapeutics and extensive differences in long-term survival [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. One of the reasons for this heterogeneity in cancer incidence is the key genetic polymorphisms in gate keeper genes such as TP53 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The inter-individual inherited polymorphisms may also be an important factor regarding the predisposition for the development of cancer as well the variation of chemo and radiation therapy responses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A common polymorphism in \u003cem\u003eBRCA1\u003c/em\u003e (rs1799950), reported in a number of familial breast cancers [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e] is located in the ring finger domain [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This domain is crucial to \u003cem\u003eBRCA1\u003c/em\u003e function because of its interaction with numerous genes such as, RAD51, p53, and pRB which are all related to DNA repair [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Recently, it was reported that rs1799950 is significantly associated with a greater risk for developing breast cancer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, a polymorphism in \u003cem\u003eBRCA2\u003c/em\u003e (rs144848), causes change in the amino acid sequence resulting in A to C substitution i.e asparagine to histidine transition in exon 10 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and was found to be associated with an increased risk of breast cancer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Likewise, a non-synonymous polymorphism in codon 72 is the most common SNP in the TP53 and occurs in the non-conserved proline-rich region of exon 4 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The proline rich domain has been revealed to be vital for apoptotic function of p53 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Several lines of evidence showed that Arg72Pro can play an important role in apoptosis and cancer formation in humans and is supported by observation that iASPP, a specific cellular inhibitor of p53, binds to the proline rich domain of p53 and interacts more strongly with Pro72 variant leading to inhibition of apoptosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study we have randomly selected the following polymorphisms (\u003cem\u003eBRCA1\u003c/em\u003e (rs1799950), \u003cem\u003eBRCA2\u003c/em\u003e (rs144848) and \u003cem\u003eTP53\u003c/em\u003e (rs1042522) that have been reported to increase breast cancer risk. There is no such evidence from our region. Different results between studies might actually reflect inherent characteristics of the population being analyzed, as there are variations in the relative frequencies of the two polymorphic alleles in various populations. We aimed to investigate the association of selected polymorphisms of \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e (rs1799950, rs144848 and rs1042522, respectively) with breast cancer risk in the population of Khyber Pakhtunkhwa, Pakistan. The association between genetic variants, demographic, and clinicopathological characteristics of breast cancer patients were also assessed.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population, ethical consideration and sample collection\u003c/h2\u003e \u003cp\u003eIn the present case-control study, a total of 220 samples (140 breast cancer patients and 80 healthy controls) were recruited from Institute of Radiotherapy and Nuclear Medicine (IRNUM) Hospital Peshawar, Pakistan. An ethical approval for the study was obtained from IRNUM as well as from the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar (UAP) and written consent was taken from the patients and control subjects. Under standard biosafety protocol and ethical consideration, about 3cc blood samples were collected from all subjects during October 2020 to January 2021, with demographic as well as all clinical parameters on prescribed proforma. The collected samples were stored in EDTA vials at -20\u0026ordm;C at IBGE for further processing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDna Isolation, Snps Genotyping And Gel Electrophoresis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eDNA isolation, SNPs Genotyping and Gel electrophoresis\u003c/div\u003e \u003cp\u003eDNA extraction and SNP genotyping was done with the procedure previously adopted in our lab [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Primer blast software was used to design the specific primers for each SNP (Table\u0026nbsp;1). Homozygous and heterozygous mutations were detected by using two sets of outer and inner primers (forward and reverse). The PCR mixture of 10 \u0026micro;L was prepared, consisting of 5 \u0026micro;L master mix (Thermo scientific DreamTaq Master Mix (2X), 0.5 \u0026micro;L of each forward and reverse primer, 1 \u0026micro;L of template DNA, and 3 \u0026micro;L of ddH2O. The PCR conditions were; Initial denaturation 95\u0026ordm;C for 5min, followed by 35 cycles of denaturation at 95\u0026ordm;C for 30sec, annealing for 30sec at 62\u0026ordm;C, 62\u0026ordm;C and 65.9\u0026ordm;C for \u003cem\u003eBRAC1\u003c/em\u003e, \u003cem\u003eBRAC2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e respectively, extension 72\u0026ordm;C for 30sec and final extension at 72\u0026ordm;C for 5min. The amplified PCR products were run and confirmed with 1KB ladder (Thermo scientific) on 2% agarose gel.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was carried out using SPSS software version 16. The alleles and genotypes were compared between the cases and controls using chi-square test. The association between genetic variants and breast cancer was determined using the Mantel-Haenszel test to assess odds ratio and 95% confidence intervals. The association between demographic, clinicopathological characteristics and genetic variants of breast cancer patients was also analyzed using chi-square test. \u003cem\u003eP\u003c/em\u003e-values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAssociation between demographic, clinicopathological characteristics and genetic variants\u003c/h2\u003e \u003cp\u003eA total of 140 breast cancer patients were recruited in this study. The demographic, clinicopathological and genotypic characteristics of patients are presented in Table\u0026nbsp;2. The \u003cem\u003eBRCA1/2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e genotypes were analyzed according to demographic and clinicopathological characteristics of breast cancer patients. We did not find any association among \u003cem\u003eBRCA1/2\u003c/em\u003e genotypes and grade, stage, nodal status and metastasis. Significant associations were among the \u003cem\u003eBRCA1/2\u003c/em\u003e genotypes with family history, nulliparity and menstrual status of patients. \u003cem\u003eBRCA1\u003c/em\u003e (rs1799950) was found to be associated with HER2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019) and TNBC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) displayed negative correlation with HER2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019) but exhibited positive affiliation with TNBC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) while \u003cem\u003eBRCA2\u003c/em\u003e (rs144848) showed association with Luminal A (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) and Luminal B (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015) subtypes. In relation to \u003cem\u003eTP53\u003c/em\u003e (rs1042522), no association was observed among genotypes and family history while nulliparity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), menstrual status (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022), high nuclear grade (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), stage (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000), nodal status (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and metastasis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) displayed significant association with genotypes. \u003cem\u003eTP53\u003c/em\u003e (rs1042522) was found to be associated with HER2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034) and TNBC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) was found to be inversely associated with HER2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034) and positively associated with TNBC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eAllelic and genotypic frequencies of\u003c/b\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eBRCA1\u003c/span\u003e\u003cb\u003e(rs1799950)\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAllelic and genotypic frequencies of \u003cem\u003eBRCA1\u003c/em\u003e (rs1799950) were first assessed utilizing an ARMS-PCR protocol on all case samples, including healthy controls (Fig.\u0026nbsp;1). Our results indicated a statistically significant association of risk allele and risk allele containing genotypes with increased risk of breast cancer (Table\u0026nbsp;3). The frequency of risk allele (C) was significantly higher in breast cancer patients (33.57%) as compared to controls (18.12%) and was noted with an increased risk of breast cancer (OR\u0026thinsp;=\u0026thinsp;2.283, 95% CI\u0026thinsp;=\u0026thinsp;1.423\u0026ndash;3.662, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). Similarly, the risk allele containing genotypes CC (OR\u0026thinsp;=\u0026thinsp;4.449, 95% CI\u0026thinsp;=\u0026thinsp;1.447\u0026ndash;13.676, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) and TC (OR\u0026thinsp;=\u0026thinsp;1.926, 95% CI\u0026thinsp;=\u0026thinsp;1.035\u0026ndash;3.585, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037) were significantly associated with the high risk of breast cancer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAllelic and genotypic frequencies of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eBRCA2\u003c/span\u003e \u003cb\u003epolymorphism (rs144848)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe allelic and genotypic frequencies of \u003cem\u003eBRCA2\u003c/em\u003e (rs144848) was done using ARMS-PCR (Fig.\u0026nbsp;2). The results showed high frequency of major allele (A) in controls (78.7%) than in patients (47.85%), while the frequency of minor allele (C) was significantly higher in breast cancer patients (52.14%) as compared to controls (21.25%) and was associated with an increased risk of breast cancer (OR\u0026thinsp;=\u0026thinsp;4.038, 95% CI\u0026thinsp;=\u0026thinsp;2.586\u0026ndash;6.304, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000). The frequencies of AA (wild), AC (heterozygous) and CC (mutant) genotypes were 62.5%, 32.5% and 5% in controls and 22.8%, 50% and 27.14% in patients, respectively. Our results indicated that individuals carrying risk allele containing genotypes CC (OR\u0026thinsp;=\u0026thinsp;14.84, 95% CI\u0026thinsp;=\u0026thinsp;4.835\u0026ndash;45.574, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) and AC (OR\u0026thinsp;=\u0026thinsp;4.207, 95% CI\u0026thinsp;=\u0026thinsp;2.236\u0026ndash;7.915, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) were at increased risk of breast cancer (Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAllelic and genotypic frequencies of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eTP53\u003c/span\u003e \u003cb\u003epolymorphism (rs1042522)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe allelic and genotypic frequency of \u003cem\u003eTP53\u003c/em\u003e was done using T-ARMS-PCR (Fig.\u0026nbsp;3). Our results displayed high frequency of major allele (G) (73.75%) in controls than in breast cancer patients (51.4%), while the minor allele (C) was significantly higher in breast cancer patients (48.5%) as compared to controls (26.25%) and was significantly linked with an increased risk of breast cancer (OR\u0026thinsp;=\u0026thinsp;2.653, 95% CI\u0026thinsp;=\u0026thinsp;1.738\u0026ndash;4.051, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000). The genotypic distribution GG (wild), GC (heterozygous) and CC (mutant) in the control group was 55%, 37.5% and 7.5%, while this distribution in the patient group was 24.2%, 54.3% and 21.4%, respectively. The risk genotype CC (OR\u0026thinsp;=\u0026thinsp;6.471, 95% CI\u0026thinsp;=\u0026thinsp;2.418\u0026ndash;17.313, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) and GC (OR\u0026thinsp;=\u0026thinsp;3.278, 95% CI\u0026thinsp;=\u0026thinsp;1.772\u0026ndash;6.067, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) were significantly associated with the high risk of breast cancer (Table\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn Pakistan, breast cancer is the most common cancer in women with a 2.5% greater incidence rate as compared to India and Iran [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The key elements that play an important role in the development of breast cancer include genetics, environment, endogenous and exogenous hormones in females, reproductive experience and change in immune status [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Genetic polymorphism is the most common type of genetic variation and recently, researchers have focused on polymorphisms that are statistically significantly associated with breast cancer risk amongst the general population. Researchers have identified that genetic polymorphisms are one of the causes of individual difference in cancer incidence [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. There is no such work done about the Pushtun population of Khyber Pakhtunkhwa, Pakistan. The current study was conducted to investigate the association of selected polymorphisms of \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e (rs1799950, rs144848 and rs1042522, respectively) with breast cancer risk in the population of Khyber Pakhtunkhwa, Pakistan and to analyze the association among genetic variants, demographic, and clinicopathological characteristics of breast cancer patients.\u003c/p\u003e \u003cp\u003eThe result of our study displayed a significant association of rs1799950 heterozygous (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037) as well as homozygous mutant genotype (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) with breast cancer risk. Though, the mutant genotype was found to be more associated (OR\u0026thinsp;=\u0026thinsp;4.449, 95% C.I\u0026thinsp;=\u0026thinsp;1.447\u0026ndash;13.676). Previously, similar studies showed the association of rs1799950 (\u003cem\u003eBRCA1\u003c/em\u003e) with Breast cancer risk. The homozygous mutant genotype was found to be associated with breast cancer risk in the Saudi population [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e] while the heterozygous genotype was found to be associated with breast cancer risk in Sudanese [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. rs1799950 genotype exhibited a positive relationship with family history, nulliparity, menstrual status, ER- and PR- status, but displayed negative correlation with HER2 abundance. This is the first study in the Pakistani population to confirm this, thus more research is needed to investigate the genotypic distribution of rs1799950 and its association with demographic and clinicopathological characteristics of breast cancer patients. Though, it has been vastly documented that \u003cem\u003eBRCA1\u003c/em\u003e mutation is associated with negative hormone receptor status [41,42] which supports the current result.\u003c/p\u003e \u003cp\u003eSimilarly, our results revealed that the minor allele frequency of \u003cem\u003eBRCA2\u003c/em\u003e was significantly higher in breast cancer patients (52.14%) as compared to controls (21.25%) and strong association was observed between homozygous mutant genotype and breast cancer risk (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000, OR\u0026thinsp;=\u0026thinsp;14.844, 95% C.I\u0026thinsp;=\u0026thinsp;4.835\u0026ndash;45.574) as compared to heterozygous genotype AC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000, OR\u0026thinsp;=\u0026thinsp;4.207, 95% C.I\u0026thinsp;=\u0026thinsp;2.237\u0026ndash;7.915). Many studies have suggested that this SNP is a susceptibility locus for cancers [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A meta-analysis was performed including 40 relevant studies, out of which 22 studies focused on breast cancer in order to assess the association between rs144848 and breast cancer risk [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Their results showed significant association between rs144848 and cancer risk in all genetic models. In the present study, \u003cem\u003eBRCA2\u003c/em\u003e SNP displayed a positive relationship with Luminal A and Luminal B subtypes. A prior study also demonstrated the association of ER\u0026thinsp;+\u0026thinsp;subtype in Kazakh population, which was the only predominant histological type in \u003cem\u003eBRCA2\u003c/em\u003e carriers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, in an earlier study \u003cem\u003eBRCA2\u003c/em\u003e mutations were frequently seen in people with family history [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e] similar to our study which also showed association with family history. Further, well-designed studies are warranted to confirm the current results and more investigations are needed to examine the genotypic distribution of rs144848 and its association with demographic and clinicopathological characteristics of breast cancer patients.\u003c/p\u003e \u003cp\u003eIn relation to rs1042522 (\u003cem\u003eTP53\u003c/em\u003e), several studies have been conducted to evaluate its potential role as a risk factor of breast cancer but the results have been inconclusive. The outcome of existing study is in line with those that detected higher prevalence of homozygous mutant genotype CC in patients with breast cancer as reported in [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e] while in contrast some authors failed to demonstrate a potential role of rs1042522 in breast cancer and no association was found between CC genotype [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Our results displayed, strong association of homozygous mutant genotype with breast cancer risk (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000, OR\u0026thinsp;=\u0026thinsp;6.471, 95% C.I\u0026thinsp;=\u0026thinsp;2.418\u0026ndash;17.313) as compared to heterozygous genotype GC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000, OR\u0026thinsp;=\u0026thinsp;3.278, 95% C.I\u0026thinsp;=\u0026thinsp;1.772\u0026ndash;6.067). A previous report unveiled that major allele was more prominent in Pakistani sporadic breast cancer patients than normal subjects [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e50\u003c/span\u003e] while in current research, minor allele was found to be associated and was present in high frequency in breast cancer patients (48.5%) than in controls (26.25%). The difference could be due to exposure of particular carcinogens, diet, reproductive pattern, geography and different lifestyle. This SNP was found to be associated with high grade and TNBC [51] which supports the outcome of the current study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest regarding to publish this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor/s contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.K.\u0026nbsp;and\u0026nbsp;N.K\u0026nbsp;designed\u0026nbsp;this\u0026nbsp;study.\u0026nbsp;Y.K,\u0026nbsp;N.K,\u0026nbsp;SK conducted\u0026nbsp;the\u0026nbsp;analysis,\u0026nbsp;produced\u0026nbsp;the\u0026nbsp;figures, and drafted the manuscript. A.K, and B.A co-wrote the manuscript. IA, AI and B.A reviewed the manuscript and provided advice on the content of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no funding received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and Informed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the procedures performed in this study were in accordance with the ethical standards of the IBGE, UAP and IRNUM hospital research committees and with the 1964 Helsinki declaration. Informed consent was obtained from all the participants while explaining the objectives of the study. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68: 394\u0026ndash;424. doi:10.3322/caac.21492\u003c/li\u003e\n \u003cli\u003ePervez S, Jabbar AA, Haider G, Ashraf S, Qureshi MA, Lateef F, et al. Karachi Cancer Registry (KCR): Age-Standardized Incidence Rate by Age-Group and Gender in a Mega City of Pakistan. Asian Pacific J Cancer Prev. 2020;21: 3251\u0026ndash;3258. doi:10.31557/APJCP.2020.21.11.3251\u003c/li\u003e\n \u003cli\u003eFarooq A, Naveed AK, Azeem Z, Ahmad T. Breast and Ovarian Cancer Risk due to Prevalence of BRCA1 and BRCA2 Variants in Pakistani Population: A Pakistani Database Report. J Oncol. 2011;2011: 632870. doi:10.1155/2011/632870\u003c/li\u003e\n \u003cli\u003eLiede A, Malik IA, Aziz Z, Rios Pd P de los, Kwan E, Narod SA. Contribution of BRCA1 and BRCA2 mutations to breast and ovarian cancer in Pakistan. 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Hum Mol Genet. 1997;6: 285\u0026ndash;289. doi:10.1093/hmg/6.2.285\u003c/li\u003e\n \u003cli\u003eTommasi S, Pilato B, Pinto R, Monaco A, Bruno M, Campana M, et al. Molecular and in silico analysis of BRCA1 and BRCA2 variants. Mutat Res. 2008;644: 64\u0026ndash;70. doi:10.1016/j.mrfmmm.2008.07.005\u003c/li\u003e\n \u003cli\u003eWang Q, Zhang H, Fishel R, Greene MI. BRCA1 and cell signaling. Oncogene. 2000;19: 6152\u0026ndash;6158. doi:10.1038/sj.onc.1203974\u003c/li\u003e\n \u003cli\u003eJohnson N, Fletcher O, Palles C, Rudd M, Webb E, Sellick G, et al. Counting potentially functional variants in BRCA1, BRCA2 and ATM predicts breast cancer susceptibility. Hum Mol Genet. 2007;16: 1051\u0026ndash;1057. doi:10.1093/hmg/ddm050\u003c/li\u003e\n \u003cli\u003ePharoah PDP, Antoniou A, Bobrow M, Zimmern RL, Easton DF, Ponder BAJ. Polygenic susceptibility to breast cancer and implications for prevention. 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Oncotarget. 2018;9: 8681\u0026ndash;8694. doi:10.18632/oncotarget.24064\u003c/li\u003e\n \u003cli\u003eMehrgou A, Akouchekian M. The importance of BRCA1 and BRCA2 genes mutations in breast cancer development. Med J Islam Repub Iran. 2016;30: 369.\u003c/li\u003e\n \u003cli\u003eBuyru N, Tigli H, Dalay N. P53 codon 72 polymorphism in breast cancer. Oncol Rep. 2003;10: 711\u0026ndash;714.\u003c/li\u003e\n \u003cli\u003eDamin APS, Frazzon APG, Damin DC, Roehe A, Hermes V, Zettler C, et al. Evidence for an association of TP53 codon 72 polymorphism with breast cancer risk. Cancer Detect Prev. 2006;30: 523\u0026ndash;529. doi:10.1016/j.cdp.2006.09.007\u003c/li\u003e\n \u003cli\u003eSuspitsin EN, Buslov KG, Grigoriev MY, Ishutkina JG, Ulibina JM, Gorodinskaya VM, et al. Evidence against involvement of p53 polymorphism in breast cancer predisposition. International journal of cancer. 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NPJ breast cancer. 2019;5: 20. doi:10.1038/s41523-019-0117-7\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 5 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Breast cancer, Risk association, BRCA1, BRCA2, TP53, Polymorphisms","lastPublishedDoi":"10.21203/rs.3.rs-2625327/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2625327/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eSingle Nucleotide polymorphism (SNPs) in \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e has been widely associated with breast cancer risk in different ethnicities with inconsistent results. There is no such study conducted so far in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. Therefore, this study was conducted to check \u003cem\u003eBRCA1\u003c/em\u003e(rs1799950), \u003cem\u003eBRCA2\u003c/em\u003e (rs144848) and \u003cem\u003eTP53\u003c/em\u003e (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e This study, consisting 140 breast cancer patients and 80 gender and age matched healthy controls were subjected to confirm \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e polymorphism. Clinicopathological data and blood samples were taken from all the participants. DNA was extracted and SNPs were confirmed using T-ARMS-PCR protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur data indicated that \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e, and \u003cem\u003eTP53\u003c/em\u003e selected SNPs risk allele and risk allele containing genotypes displayed significant association (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) with breast cancer risk in the Pashtun population of Khyber Pakhtunkhwa, Pakistan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eAll the three selected SNPs of \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e showed significant association with breast cancer risk in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. However, more investigation will be required on large data sets to confirm the selected SNPs and other SNPs in the selected and other related genes with the risk of breast cancer.\u003c/p\u003e","manuscriptTitle":"Significant association of BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-13 14:35:45","doi":"10.21203/rs.3.rs-2625327/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-03-07T12:47:16+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-27T13:27:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2023-02-25T07:52:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3d21721b-3197-4f93-847e-7080c75da00b","owner":[],"postedDate":"March 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:18:03+00:00","versionOfRecord":{"articleIdentity":"rs-2625327","link":"https://doi.org/10.1007/s11033-023-08463-9","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2023-06-10 21:09:00","publishedOnDateReadable":"June 10th, 2023"},"versionCreatedAt":"2023-03-13 14:35:45","video":"","vorDoi":"10.1007/s11033-023-08463-9","vorDoiUrl":"https://doi.org/10.1007/s11033-023-08463-9","workflowStages":[]},"version":"v1","identity":"rs-2625327","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2625327","identity":"rs-2625327","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}