Somatic genetic variants associated with aggressiveness of urothelial bladder cancer: a prospective cohort study

Somatic genetic variants associated with aggressiveness of urothelial bladder cancer: a prospective cohort study | 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 Somatic genetic variants associated with aggressiveness of urothelial bladder cancer: a prospective cohort study Ajith Peiris Malalasekera, Praveenan Somasundaram, Nilaksha Neththikumara, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6553154/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Bladder cancer risk assessment using single nucleotide variants (SNVs) has focused on Caucasian and Chinese patients, with a significant gap in under-represented South Asian populations. This study examines the association of somatic SNVs with clinicopathological data in a Sri Lankan urothelial bladder cancer (UBC) cohort. Methods DNA was extracted from tumour specimens of 41 patients. Fifty SNVs were genotyped using the Agena platform. Risk association analyses were conducted to identify variants associated with aggressive features (high tumour grade or muscle invasiveness). Results Six variants were linked to tumour aggressiveness. Among them, rs1495741G > A, rs1799964T > C, and rs10775480T > C were associated with tumour aggressiveness. Notably, rs1495741G > A and rs10775480T > C showed alignment with previous literature on germline variants, as their minor allele genotypes were linked to tumour aggressiveness in our cohort. Conclusions This study provides valuable insights into the association between SNVs and aggressiveness of UBC and identifies potential markers for tumour aggressiveness in South Asian populations. urothelial bladder cancer single nucleotide variants somatic tumour aggressiveness prognostic indicators Introduction Bladder cancer is a leading cause of cancer morbidity and mortality, resulting in approximately 186,000 deaths each year, and over 3,000,000 disability-adjusted life-years[ 1 ]. The management of bladder cancer focuses on preventing tumour progression and identifying patients at high risk for aggressive disease to provide early interventions[ 2 ]. Molecular diagnostics have shown promise in predicting tumour aggressiveness more accurately than traditional prognostic factors[ 3 ]. While germline variants have been extensively studied, somatic variants have received less attention. Somatic variants accumulate with age and contribute to the rich genomic landscape of bladder cancer. Several studies have investigated somatic variants associated with tumour aggressiveness, but there is minimal research in the Asian context. Here, we report the identification of somatic variants associated with the aggressiveness of urothelial bladder cancer for the first time in a Sri Lankan cohort. Methods A prospective study was conducted involving 41 patients with median (IQR) age of 66 (58–70) years undergoing transurethral tumour resection for the first time for urothelial bladder cancer. DNA was extracted from freshly cut sections of formalin-free paraffin-embedded tumour tissue. Genotyping was performed using a panel of 50 selected single nucleotide variants (SNVs) sourced from the Genome-Wide Association Studies (GWAS) Catalogue ( https://www.ebi.ac.uk/gwas/ ), COSMIC database ( https://cancer.sanger.ac.uk/cosmic ), published Asian SNV articles, and the phase I study. SNP genotyping was carried out on a Multiplex Agena platform. Association testing for tumour aggressiveness was conducted on a case-control basis, with tumours considered aggressive if they exhibited features of being muscle-invasive (pT2 or higher) or high grade (grade 3) (Table 1 ). Table 1 Criterion for assigning aggressive disease Tumour stage Tumour Grade Other criterion Not aggressive (controls) pTa (26/41; 63.4%) Low (1 or 2) (19/41; 46.3%) Aggressive (cases) ≥pT2 (15/41; 36.6%) High (3) (22/41; 53.7%) CIS was present A minimum genotype percentage of 75% (35 out of 50 selected SNVs) was established as the threshold for inclusion. Allele and genotype frequencies for each SNV were calculated using an online calculator ( https://wpcalc.com/en/equilibrium-hardy-weinberg/ ). The conformity to the Hardy-Weinberg equilibrium (HWE) was assessed by testing for deviations, employing a Pearson’s Chi-square test. The p-value was calculated with a degree of freedom of 1 ( https://www.socscistatistics.com/pvalues/chidistribution.aspx ). Markers significantly deviating from HWE (HW p-value < 0.005) were excluded, resulting in the exclusion of 2 SNVs. Additionally, SNVs with a minor allele frequency below 0.01 were excluded, resulting in the exclusion of 10 SNVs (Supporting information 1 and 2). Binary logistic regression analysis was performed to investigate the association between the selected SNVs and tumour aggressiveness. Two-by-two contingency tables were utilized when only two genotypes were observed. In cases where three genotypes were identified, a simple 3x2 contingency table was employed, followed by logistic regression analysis. No assumptions were made regarding the genetic model of inheritance (Supporting information 3,4,5). SNPstats, accessible from https://www.snpstats.net/start.htm , was employed for logistic regression analysis using all the aforementioned models. Results Twenty-three SNVs were ultimately selected for the association analysis. This analysis revealed six variants that demonstrated a significant link with the aggressiveness of urothelial bladder cancer. Specifically, rs1495741G > A (analysed using a 2x2 contingency table), rs1799964T > C (analysed using log additive/multiplicative, codominant, and dominant models), and rs10775480T > C (analysed using log additive/multiplicative and recessive models) were found to be positively associated with aggressiveness, characterized by high tumour grade and/or muscle invasiveness (Table 2 ). Table 2 SNVs significantly associated with tumour aggressiveness in bladder cancer SNV (rs ID) Statistical model OR and 95% CI P value Remarks rs1495741G > A Logistic regression (2x2 contingency table) Grade − 6.43 (1.32–31.37) Muscle invasion − 4.76 (1.04–21.77) 0.014 0.039 Risk factor positively linked to aggressive disease – both grade and muscle invasion rs10775480T > C Log additive (multiplicative) Recessive 1.87 (1.28–26.96) 6.50 (1.28–33.04) 0.0094 0.016 Risk factor positively linked to aggressive disease (grade) rs1799964T > C Log additive (multiplicative) Codominant Dominant 3.60 (1.20-10.74) 5.00 (1.15–21.80) 5.60 (1.43–21.95) 0.011 0.032 0.0095 Risk factor positively linked to aggressive disease (grade) Rs2228001 Codominant Dominant 0.13 (0.02–0.82) 0.18 (0.03–0.89) 0.069 00.03 Risk factor protective against aggressive disease (grade) rs17674580 Codominant Dominant Overdominant 0.19 (0.04–0.89) 0.21 (0.05–0.93) 0.21 (0.04–0.94) 0.09 0.033 0.034 Risk factor protective against aggressive disease (grade) rs7238033 Overdominant 0.24 (0.06–0.98) 0.039 Risk factor protective against aggressive disease (grade) Discussion Of the significant SNVs, one notable SNV rs1495741G > A, located on chromosome 8p22 upstream of the 3' end of the NAT2 gene, has been previously identified in GWAS as a risk factor for urothelial bladder cancer [ 4 ]. The slow acetylation phenotype, indicated by its AA genotype, has been linked to an increased risk of bladder cancer, particularly among cigarette-smoking patients, potentially due to its involvement in the detoxification of aromatic monoamines [ 5 ]. In our study of 41 patients, 34 were successfully genotyped, with none having the wild-type GG genotype, 22 (64.7%) having the heterozygous GA genotype, and 12 (35.3%) having the variant homozygous AA genotype. The AA genotype was significantly associated with a higher risk of tumour aggressiveness, including high-grade tumours and muscle invasiveness. Another significant SNV, rs10775480T > C, located on chromosome 18 within an intron of the SLC14A1 gene, is associated with a higher risk of bladder cancer [ 6 ]. Notably, this particular SNV has demonstrated a correlation with urine concentration [ 7 ]. Previous research has underscored a statistically significant decrease in urine specific gravity for individuals with the TT genotype when compared to those with the CC genotype [ 7 ]. Our study shows those possessing the TC (32.3%) and CC (61.3%) genotypes exhibit an elevated risk of developing high-grade urothelial tumours. Additionally, rs1799964T > C, an SNV situated on chromosome 6 and 2kb upstream of the TNF gene, has shown an association with bladder cancer risk. Previous investigations involving patients treated with intravesical bacille Calmette-Guérin (BCG) have identified an augmented risk of recurrence among individuals harbouring SNVs in TNFA rs1799964 TT + TC vs. CC [ 8 ]. In our study, the TC (35%) and CC (12.5%) genotypes were associated with increased tumour aggressiveness, though the CC genotype alone did not reach statistical significance, possibly due to the limited sample size. For both rs10775480T > C and rs1799964T > C, the exact biological mechanisms leading to association with aggressive disease remain unclear and hence warrant further study Three SNVs (rs2228001G > T, rs17674580C > T, and rs7238033T > C) were observed to exhibit a protective effect against tumour aggressiveness based on logistic regression models (Table 2 ). However, these associations did not achieve statistical significance under the log additive/multiplicative model. Conclusion Our research stands as a pioneering endeavour within the limited body of studies examining somatic variants in urothelial bladder tumours. We have successfully identified six variants that exhibited statistical significance, establishing a compelling link with tumour aggressiveness. Employing contingency tables and the widely recognized log additive genetic model, three SNVs (rs1495741G > A, rs10775480T > C, and rs1799964T > C) reached a level of significance, with rs1495741G > A and rs10775480T > C showing an alignment with previous literature on germline variants, particularly concerning their minor allele genotypes and their association with tumour aggressiveness. These findings hold promise in the potential development of a marker panel for subgrouping patients based on their variant patterns, thereby enhancing prognostication and enabling precision treatment. Sri Lankan and genetically similar south Asian populations are grossly underrepresented in collaborative genomic studies and therefore our study can have significant implications for bladder cancer management in such populations. Studies with larger numbers and corresponding blood and normal bladder tissue would be useful to validate these findings. Declarations Ethics approval and consent to participate Ethical approval was obtained from the ethical review committee of the Faculty of Medicine, University of Colombo. (EC-12-088 / 19th October, 2012) The study was conducted in accordance with the ethical standards of the relevant institutional ethics committee and the Helsinki Declaration of 1975, as revised in 2000. All study participants have granted their informed consent as per the institutional review protocols. A.P.M is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Consent for publication The manuscript does not contain data from any individual person. All authors have granted consent for publication of the final paper Availability of data and materials All relevant data has been anonymised and provided under supplementary information. Competing interests The authors have no relevant financial or non-financial interests to disclose regarding the publication of this article. Funding This work was supported by the following grants. APM received the grant numbers AP/3/2011/PG/14 from the University of Colombo, Sri Lanka (https://cmb.ac.lk/) and the grant number UGC/ICD/RG/2011/07 from the University Grants Commission, Sri Lanka (https://www.ugc.ac.lk/). The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors' contributions Conceptualization, ideas of the work was by A.P.M, K.W, R.W.J, M.D.S.L, V.H.W.D; Data Curation was carried out by A.P.M, S.A.S.G, N.D.P, A.A, P.S; Investigation A.P.M, K.W. Methodology A.P.M, P.S, N.N, Nirmala S, Nilakshi S, D.E, V.H.W.D; Formal Analysis was by A.P.M, P.S, N.N, Nirmala S, Nilakshi S; Writing – Original Draft Preparation was done by A.P.M, P.S, N.N, Nirmala S, Nilakshi S, D.E; Writing – Review & Editing involved all authors. Funding Acquisition by A.P.M, K.W, R.W.J, V.H.W.D; Supervision M.D.S.L, R.W.J, V.H.W.D Acknowledgements The kind assistance provided by all patients and their families is greatly appreciated. We thank Dr Suranga Wijeyarathne and Dr Munipriya Willaraarachchi for their assistance in sample and data collection. References Ebrahimi H, Amini E, Pishgar F, Moghaddam SS, Nabavizadeh B, Rostamabadi Y, et al. Global, Regional and National Burden of Bladder Cancer, 1990 to 2016: Results from the GBD Study 2016. J Urol. 2019;201(5):893–901. Denzinger S, Fritsche HM, Otto W, Blana A, Wieland WF, Burger M. Early versus deferred cystectomy for initial high-risk pT1G3 urothelial carcinoma of the bladder: do risk factors define feasibility of bladder-sparing approach? Eur Urol. 2008;53(1):146–52. Cazier JB, Rao SR, McLean CM, Walker AK, Wright BJ, Jaeger EE, et al. Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden. Nat Commun. 2014;5:3756. Rothman N, Garcia-Closas M, Chatterjee N, Malats N, Wu X, Figueroa JD, et al. A multi-stage genome-wide association study of bladder cancer identifies multiple susceptibility loci. Nat Genet. 2010;42(11):978–84. Marcus PM, Vineis P, Rothman N. NAT2 slow acetylation and bladder cancer risk: a meta-analysis of 22 case-control studies conducted in the general population. Pharmacogenetics. 2000;10(2):115–22. Rafnar T, Vermeulen SH, Sulem P, Thorleifsson G, Aben KK, Witjes JA, et al. European genome-wide association study identifies SLC14A1 as a new urinary bladder cancer susceptibility gene. Hum Mol Genet. 2011;20(21):4268–81. Koutros S, Baris D, Fischer A, Tang W, Garcia-Closas M, Karagas MR, et al. Differential urinary specific gravity as a molecular phenotype of the bladder cancer genetic association in the urea transporter gene, SLC14A1. Int J Cancer. 2013;133(12):3008–13. Lima L, Oliveira D, Ferreira JA, Tavares A, Cruz R, Medeiros R et al. The role of functional polymorphisms in immune response genes as biomarkers of bacille Calmette-Guérin (BCG) immunotherapy outcome in bladder cancer: establishment of a predictive profile in a Southern Europe population. BJU International. 2015;116(5):753 – 63. Additional Declarations No competing interests reported. Supplementary Files SupplementaryData1.docx The 23 SNVs conforming to the Hardy Weinburg Equilibrium and to other criteria. SupplementaryData2.docx Details of all the SNVs included in the association analysis of the SNVs in relation to aggressiveness in bladder cancer Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6553154","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454389687,"identity":"5086e45c-493b-49ef-a6ec-9d547a873ba2","order_by":0,"name":"Ajith Peiris Malalasekera","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYFCCgw3MDBVQNmMDiGRuJqSlsZnhDJBmg2thJKQFqIKxjRQt/I2H2x8XzrPL45/fwPjp5g4Gef4GxmYDfFokDgAdNnNbcrHEMQZm6dwzDIYzDjA2J+C1BqSFdxtzYsMxBjbm3DYGxg1Ahx3Ap0MerGVOfeJ8qBZ7gloMwFoaDidugGpJBGnB6zBDoJbZPMeOJ248ltgM9ItE8ozDBLwvd+P4g888NdWJ8w4fPvg5d4eNbX9782EJfFqAQQZjgSMFqJgZr3og4G8gpGIUjIJRMApGPAAAtTVOue4PSYEAAAAASUVORK5CYII=","orcid":"","institution":"University of Colombo","correspondingAuthor":true,"prefix":"","firstName":"Ajith","middleName":"Peiris","lastName":"Malalasekera","suffix":""},{"id":454389688,"identity":"f77fae79-ad9e-4d8c-b4db-63fac913b173","order_by":1,"name":"Praveenan Somasundaram","email":"","orcid":"","institution":"University of Colombo","correspondingAuthor":false,"prefix":"","firstName":"Praveenan","middleName":"","lastName":"Somasundaram","suffix":""},{"id":454389689,"identity":"c34682b2-ffbd-4a94-ac12-8f3697ed4716","order_by":2,"name":"Nilaksha Neththikumara","email":"","orcid":"","institution":"University of Colombo","correspondingAuthor":false,"prefix":"","firstName":"Nilaksha","middleName":"","lastName":"Neththikumara","suffix":""},{"id":454389691,"identity":"f158c9b9-ad12-4f3a-a371-b884e1d3bc05","order_by":3,"name":"Nirmala Sirisena","email":"","orcid":"","institution":"University of Colombo","correspondingAuthor":false,"prefix":"","firstName":"Nirmala","middleName":"","lastName":"Sirisena","suffix":""},{"id":454389693,"identity":"8805d8f6-1530-4e3c-b068-a2a4feadbdf4","order_by":4,"name":"Nilakshi Samaranayake","email":"","orcid":"","institution":"University of Colombo","correspondingAuthor":false,"prefix":"","firstName":"Nilakshi","middleName":"","lastName":"Samaranayake","suffix":""},{"id":454389694,"identity":"61804859-d490-48e2-b536-0e3c6c1cf3e4","order_by":5,"name":"Dileepa Ediriweera","email":"","orcid":"","institution":"University of Kelaniya","correspondingAuthor":false,"prefix":"","firstName":"Dileepa","middleName":"","lastName":"Ediriweera","suffix":""},{"id":454389698,"identity":"40aa501f-142c-496c-98e4-247297912e34","order_by":6,"name":"Yasith Mathangasinghe","email":"","orcid":"","institution":"University of Colombo","correspondingAuthor":false,"prefix":"","firstName":"Yasith","middleName":"","lastName":"Mathangasinghe","suffix":""},{"id":454389700,"identity":"1b67f2c9-2ee8-43b2-83ee-db9e2282bf3e","order_by":7,"name":"Serozsha AS Goonewardena","email":"","orcid":"","institution":"National Hospital of Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"Serozsha","middleName":"AS","lastName":"Goonewardena","suffix":""},{"id":454389701,"identity":"eac805f0-bd2f-4b8b-a10c-3ac8d7a7dec6","order_by":8,"name":"Neville D Perera","email":"","orcid":"","institution":"National Hospital of Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"Neville","middleName":"D","lastName":"Perera","suffix":""},{"id":454389707,"identity":"dacc0547-4470-4f87-88b3-3340dace0809","order_by":9,"name":"Anuruddha Abeygunasekara","email":"","orcid":"","institution":"Colombo South Teaching Hospital","correspondingAuthor":false,"prefix":"","firstName":"Anuruddha","middleName":"","lastName":"Abeygunasekara","suffix":""},{"id":454389708,"identity":"a7eac5aa-5688-43e8-9e5f-1d9d61e0d80e","order_by":10,"name":"Rohan W. 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W.","lastName":"Dissanayake","suffix":""}],"badges":[],"createdAt":"2025-04-29 06:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6553154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6553154/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83193031,"identity":"5d056023-ad35-41f1-9e64-a3d094fb55e1","added_by":"auto","created_at":"2025-05-21 04:16:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":575072,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6553154/v1/0bb1e70b-2ce4-4f19-b35f-3113c10fccc0.pdf"},{"id":82545949,"identity":"5877c179-d8e6-46ee-9b44-058593634ebb","added_by":"auto","created_at":"2025-05-12 18:12:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":70898,"visible":true,"origin":"","legend":"\u003cp\u003e1. The 23 SNVs conforming to the Hardy Weinburg Equilibrium and to other criteria.\u003c/p\u003e","description":"","filename":"SupplementaryData1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6553154/v1/944a07eb92f95ac554d0311e.docx"},{"id":82545204,"identity":"bc00474c-d2a4-4663-9e24-17029f068a47","added_by":"auto","created_at":"2025-05-12 18:04:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":190427,"visible":true,"origin":"","legend":"\u003cp\u003e2. Details of all the SNVs included in the association analysis of the SNVs in relation to aggressiveness in bladder cancer\u003c/p\u003e","description":"","filename":"SupplementaryData2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6553154/v1/20884f8de5413b4daa213d7c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Somatic genetic variants associated with aggressiveness of urothelial bladder cancer: a prospective cohort study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBladder cancer is a leading cause of cancer morbidity and mortality, resulting in approximately 186,000 deaths each year, and over 3,000,000 disability-adjusted life-years[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The management of bladder cancer focuses on preventing tumour progression and identifying patients at high risk for aggressive disease to provide early interventions[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Molecular diagnostics have shown promise in predicting tumour aggressiveness more accurately than traditional prognostic factors[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While germline variants have been extensively studied, somatic variants have received less attention. Somatic variants accumulate with age and contribute to the rich genomic landscape of bladder cancer. Several studies have investigated somatic variants associated with tumour aggressiveness, but there is minimal research in the Asian context. Here, we report the identification of somatic variants associated with the aggressiveness of urothelial bladder cancer for the first time in a Sri Lankan cohort.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eA prospective study was conducted involving 41 patients with median (IQR) age of 66 (58\u0026ndash;70) years undergoing transurethral tumour resection for the first time for urothelial bladder cancer. DNA was extracted from freshly cut sections of formalin-free paraffin-embedded tumour tissue. Genotyping was performed using a panel of 50 selected single nucleotide variants (SNVs) sourced from the Genome-Wide Association Studies (GWAS) Catalogue (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/gwas/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/gwas/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), COSMIC database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cancer.sanger.ac.uk/cosmic\u003c/span\u003e\u003cspan address=\"https://cancer.sanger.ac.uk/cosmic\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), published Asian SNV articles, and the phase I study. SNP genotyping was carried out on a Multiplex Agena platform. Association testing for tumour aggressiveness was conducted on a case-control basis, with tumours considered aggressive if they exhibited features of being muscle-invasive (pT2 or higher) or high grade (grade 3) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCriterion for assigning aggressive disease\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTumour stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumour Grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOther criterion\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNot aggressive (controls)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epTa\u003c/p\u003e \u003cp\u003e(26/41; 63.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow (1 or 2)\u003c/p\u003e \u003cp\u003e(19/41; 46.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggressive (cases)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;pT2\u003c/p\u003e \u003cp\u003e(15/41; 36.6%)\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh (3)\u003c/p\u003e \u003cp\u003e(22/41; 53.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCIS was present\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA minimum genotype percentage of 75% (35 out of 50 selected SNVs) was established as the threshold for inclusion. Allele and genotype frequencies for each SNV were calculated using an online calculator (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wpcalc.com/en/equilibrium-hardy-weinberg/\u003c/span\u003e\u003cspan address=\"https://wpcalc.com/en/equilibrium-hardy-weinberg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The conformity to the Hardy-Weinberg equilibrium (HWE) was assessed by testing for deviations, employing a Pearson\u0026rsquo;s Chi-square test. The p-value was calculated with a degree of freedom of 1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.socscistatistics.com/pvalues/chidistribution.aspx\u003c/span\u003e\u003cspan address=\"https://www.socscistatistics.com/pvalues/chidistribution.aspx\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Markers significantly deviating from HWE (HW p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.005) were excluded, resulting in the exclusion of 2 SNVs. Additionally, SNVs with a minor allele frequency below 0.01 were excluded, resulting in the exclusion of 10 SNVs (Supporting information 1 and 2). Binary logistic regression analysis was performed to investigate the association between the selected SNVs and tumour aggressiveness. Two-by-two contingency tables were utilized when only two genotypes were observed. In cases where three genotypes were identified, a simple 3x2 contingency table was employed, followed by logistic regression analysis. No assumptions were made regarding the genetic model of inheritance (Supporting information 3,4,5). SNPstats, accessible from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.snpstats.net/start.htm\u003c/span\u003e\u003cspan address=\"https://www.snpstats.net/start.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, was employed for logistic regression analysis using all the aforementioned models.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTwenty-three SNVs were ultimately selected for the association analysis. This analysis revealed six variants that demonstrated a significant link with the aggressiveness of urothelial bladder cancer. Specifically, rs1495741G\u0026thinsp;\u0026gt;\u0026thinsp;A (analysed using a 2x2 contingency table), rs1799964T\u0026thinsp;\u0026gt;\u0026thinsp;C (analysed using log additive/multiplicative, codominant, and dominant models), and rs10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C (analysed using log additive/multiplicative and recessive models) were found to be positively associated with aggressiveness, characterized by high tumour grade and/or muscle invasiveness (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSNVs significantly associated with tumour aggressiveness in bladder cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNV (rs ID)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStatistical model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOR and 95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRemarks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ers1495741G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLogistic regression (2x2 contingency table)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade \u0026minus;\u0026thinsp;6.43 (1.32\u0026ndash;31.37)\u003c/p\u003e \u003cp\u003eMuscle invasion \u0026minus;\u0026thinsp;4.76 (1.04\u0026ndash;21.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003cp\u003e0.039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRisk factor \u003cb\u003epositively\u003c/b\u003e linked to aggressive disease \u0026ndash; both grade and muscle invasion\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ers10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog additive (multiplicative)\u003c/p\u003e \u003cp\u003eRecessive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.87 (1.28\u0026ndash;26.96)\u003c/p\u003e \u003cp\u003e6.50 (1.28\u0026ndash;33.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0094\u003c/p\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRisk factor \u003cb\u003epositively\u003c/b\u003e linked to aggressive disease (grade)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ers1799964T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog additive (multiplicative)\u003c/p\u003e \u003cp\u003eCodominant\u003c/p\u003e \u003cp\u003eDominant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.60 (1.20-10.74)\u003c/p\u003e \u003cp\u003e5.00 (1.15\u0026ndash;21.80)\u003c/p\u003e \u003cp\u003e5.60 (1.43\u0026ndash;21.95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003cp\u003e0.032\u003c/p\u003e \u003cp\u003e0.0095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRisk factor \u003cb\u003epositively\u003c/b\u003e linked to aggressive disease (grade)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRs2228001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCodominant\u003c/p\u003e \u003cp\u003eDominant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13 (0.02\u0026ndash;0.82)\u003c/p\u003e \u003cp\u003e0.18 (0.03\u0026ndash;0.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003cp\u003e00.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRisk factor \u003cb\u003eprotective\u003c/b\u003e against aggressive disease (grade)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ers17674580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCodominant\u003c/p\u003e \u003cp\u003eDominant\u003c/p\u003e \u003cp\u003eOverdominant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19 (0.04\u0026ndash;0.89)\u003c/p\u003e \u003cp\u003e0.21 (0.05\u0026ndash;0.93)\u003c/p\u003e \u003cp\u003e0.21 (0.04\u0026ndash;0.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003cp\u003e0.033\u003c/p\u003e \u003cp\u003e0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRisk factor \u003cb\u003eprotective\u003c/b\u003e against aggressive disease (grade)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ers7238033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverdominant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.24 (0.06\u0026ndash;0.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRisk factor \u003cb\u003eprotective\u003c/b\u003e against aggressive disease (grade)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOf the significant SNVs, one notable SNV rs1495741G\u0026thinsp;\u0026gt;\u0026thinsp;A, located on chromosome 8p22 upstream of the 3' end of the \u003cem\u003eNAT2\u003c/em\u003e gene, has been previously identified in GWAS as a risk factor for urothelial bladder cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The slow acetylation phenotype, indicated by its AA genotype, has been linked to an increased risk of bladder cancer, particularly among cigarette-smoking patients, potentially due to its involvement in the detoxification of aromatic monoamines [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In our study of 41 patients, 34 were successfully genotyped, with none having the wild-type GG genotype, 22 (64.7%) having the heterozygous GA genotype, and 12 (35.3%) having the variant homozygous AA genotype. The AA genotype was significantly associated with a higher risk of tumour aggressiveness, including high-grade tumours and muscle invasiveness.\u003c/p\u003e \u003cp\u003eAnother significant SNV, rs10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C, located on chromosome 18 within an intron of the \u003cem\u003eSLC14A1\u003c/em\u003e gene, is associated with a higher risk of bladder cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notably, this particular SNV has demonstrated a correlation with urine concentration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous research has underscored a statistically significant decrease in urine specific gravity for individuals with the TT genotype when compared to those with the CC genotype [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Our study shows those possessing the TC (32.3%) and CC (61.3%) genotypes exhibit an elevated risk of developing high-grade urothelial tumours.\u003c/p\u003e \u003cp\u003eAdditionally, rs1799964T\u0026thinsp;\u0026gt;\u0026thinsp;C, an SNV situated on chromosome 6 and 2kb upstream of the \u003cem\u003eTNF\u003c/em\u003e gene, has shown an association with bladder cancer risk. Previous investigations involving patients treated with intravesical bacille Calmette-Gu\u0026eacute;rin (BCG) have identified an augmented risk of recurrence among individuals harbouring SNVs in \u003cem\u003eTNFA\u003c/em\u003e rs1799964 TT\u0026thinsp;+\u0026thinsp;TC vs. CC [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In our study, the TC (35%) and CC (12.5%) genotypes were associated with increased tumour aggressiveness, though the CC genotype alone did not reach statistical significance, possibly due to the limited sample size. For both rs10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C and rs1799964T\u0026thinsp;\u0026gt;\u0026thinsp;C, the exact biological mechanisms leading to association with aggressive disease remain unclear and hence warrant further study\u003c/p\u003e \u003cp\u003eThree SNVs (rs2228001G\u0026thinsp;\u0026gt;\u0026thinsp;T, rs17674580C\u0026thinsp;\u0026gt;\u0026thinsp;T, and rs7238033T\u0026thinsp;\u0026gt;\u0026thinsp;C) were observed to exhibit a protective effect against tumour aggressiveness based on logistic regression models (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, these associations did not achieve statistical significance under the log additive/multiplicative model.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur research stands as a pioneering endeavour within the limited body of studies examining somatic variants in urothelial bladder tumours. We have successfully identified six variants that exhibited statistical significance, establishing a compelling link with tumour aggressiveness. Employing contingency tables and the widely recognized log additive genetic model, three SNVs (rs1495741G\u0026thinsp;\u0026gt;\u0026thinsp;A, rs10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C, and rs1799964T\u0026thinsp;\u0026gt;\u0026thinsp;C) reached a level of significance, with rs1495741G\u0026thinsp;\u0026gt;\u0026thinsp;A and rs10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C showing an alignment with previous literature on germline variants, particularly concerning their minor allele genotypes and their association with tumour aggressiveness. These findings hold promise in the potential development of a marker panel for subgrouping patients based on their variant patterns, thereby enhancing prognostication and enabling precision treatment. Sri Lankan and genetically similar south Asian populations are grossly underrepresented in collaborative genomic studies and therefore our study can have significant implications for bladder cancer management in such populations. Studies with larger numbers and corresponding blood and normal bladder tissue would be useful to validate these findings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the ethical review committee of the Faculty of Medicine, University of Colombo. (EC-12-088 / 19th October, 2012)\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the ethical standards of the relevant institutional ethics committee and the Helsinki Declaration of 1975, as revised in 2000.\u003c/p\u003e\n\u003cp\u003eAll study participants have granted their informed consent as per the institutional review protocols.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA.P.M is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript does not contain data from any individual person.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have granted consent for publication of the final paper\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data has been anonymised and provided under supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose regarding the publication of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following grants. APM received the grant numbers AP/3/2011/PG/14 from the University of Colombo, Sri Lanka (https://cmb.ac.lk/) and \u0026nbsp;the grant number UGC/ICD/RG/2011/07 from the University Grants Commission, Sri Lanka (https://www.ugc.ac.lk/). The funders did \u003cu\u003enot\u003c/u\u003e play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, ideas of the work was by A.P.M, K.W, R.W.J, M.D.S.L, V.H.W.D; Data Curation was carried out by A.P.M, S.A.S.G, N.D.P, A.A, P.S; Investigation A.P.M, K.W. Methodology A.P.M, P.S, N.N, Nirmala S, Nilakshi S, D.E, V.H.W.D; Formal Analysis was by A.P.M, P.S, N.N, Nirmala S, Nilakshi S; \u0026nbsp;Writing \u0026ndash; Original Draft Preparation was done by A.P.M, P.S, N.N, Nirmala S, Nilakshi S, D.E; Writing \u0026ndash; Review \u0026amp; Editing involved all authors. Funding Acquisition by A.P.M, K.W, R.W.J, V.H.W.D; Supervision M.D.S.L, R.W.J, V.H.W.D\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe kind assistance provided by all patients and their families is greatly appreciated. We thank Dr Suranga Wijeyarathne and Dr Munipriya Willaraarachchi for their assistance in sample and data collection.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEbrahimi H, Amini E, Pishgar F, Moghaddam SS, Nabavizadeh B, Rostamabadi Y, et al. Global, Regional and National Burden of Bladder Cancer, 1990 to 2016: Results from the GBD Study 2016. J Urol. 2019;201(5):893\u0026ndash;901.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenzinger S, Fritsche HM, Otto W, Blana A, Wieland WF, Burger M. Early versus deferred cystectomy for initial high-risk pT1G3 urothelial carcinoma of the bladder: do risk factors define feasibility of bladder-sparing approach? Eur Urol. 2008;53(1):146\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCazier JB, Rao SR, McLean CM, Walker AK, Wright BJ, Jaeger EE, et al. Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden. Nat Commun. 2014;5:3756.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRothman N, Garcia-Closas M, Chatterjee N, Malats N, Wu X, Figueroa JD, et al. A multi-stage genome-wide association study of bladder cancer identifies multiple susceptibility loci. Nat Genet. 2010;42(11):978\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcus PM, Vineis P, Rothman N. NAT2 slow acetylation and bladder cancer risk: a meta-analysis of 22 case-control studies conducted in the general population. Pharmacogenetics. 2000;10(2):115\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRafnar T, Vermeulen SH, Sulem P, Thorleifsson G, Aben KK, Witjes JA, et al. European genome-wide association study identifies SLC14A1 as a new urinary bladder cancer susceptibility gene. Hum Mol Genet. 2011;20(21):4268\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoutros S, Baris D, Fischer A, Tang W, Garcia-Closas M, Karagas MR, et al. Differential urinary specific gravity as a molecular phenotype of the bladder cancer genetic association in the urea transporter gene, SLC14A1. Int J Cancer. 2013;133(12):3008\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLima L, Oliveira D, Ferreira JA, Tavares A, Cruz R, Medeiros R et al. The role of functional polymorphisms in immune response genes as biomarkers of bacille Calmette-Gu\u0026eacute;rin (BCG) immunotherapy outcome in bladder cancer: establishment of a predictive profile in a Southern Europe population. BJU International. 2015;116(5):753\u0026thinsp;\u0026ndash;\u0026thinsp;63. \u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"urothelial bladder cancer, single nucleotide variants, somatic, tumour aggressiveness, prognostic indicators","lastPublishedDoi":"10.21203/rs.3.rs-6553154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6553154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBladder cancer risk assessment using single nucleotide variants (SNVs) has focused on Caucasian and Chinese patients, with a significant gap in under-represented South Asian populations. This study examines the association of somatic SNVs with clinicopathological data in a Sri Lankan urothelial bladder cancer (UBC) cohort.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eDNA was extracted from tumour specimens of 41 patients. Fifty SNVs were genotyped using the Agena platform. Risk association analyses were conducted to identify variants associated with aggressive features (high tumour grade or muscle invasiveness).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSix variants were linked to tumour aggressiveness. Among them, rs1495741G\u0026thinsp;\u0026gt;\u0026thinsp;A, rs1799964T\u0026thinsp;\u0026gt;\u0026thinsp;C, and rs10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C were associated with tumour aggressiveness. Notably, rs1495741G\u0026thinsp;\u0026gt;\u0026thinsp;A and rs10775480T\u0026thinsp;\u0026gt;\u0026thinsp;C showed alignment with previous literature on germline variants, as their minor allele genotypes were linked to tumour aggressiveness in our cohort.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study provides valuable insights into the association between SNVs and aggressiveness of UBC and identifies potential markers for tumour aggressiveness in South Asian populations.\u003c/p\u003e","manuscriptTitle":"Somatic genetic variants associated with aggressiveness of urothelial bladder cancer: a prospective cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-12 18:04:34","doi":"10.21203/rs.3.rs-6553154/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a296c12e-a3e2-44f0-972f-d5a707be9bf2","owner":[],"postedDate":"May 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-21T04:08:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-12 18:04:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6553154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6553154","identity":"rs-6553154","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}