Whole Exome Sequencing Studies the Association of Rare Variants in Key TGF-β1/SMAD Pathway Gene with Stress Urinary Incontinence Susceptibility

Whole Exome Sequencing Studies the Association of Rare Variants in Key TGF-β1/SMAD Pathway Gene with Stress Urinary Incontinence Susceptibility | 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 Whole Exome Sequencing Studies the Association of Rare Variants in Key TGF-β1/SMAD Pathway Gene with Stress Urinary Incontinence Susceptibility Zhihua Wan, Ting Wang, Ge Chen, Hui Yu, Xujuan Shan, Yuling Tao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7586877/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 : Stress urinary incontinence (SUI) is a prevalent urological problem that is common among middle-aged and older women. Previous studies have shown that TGF-β1/SMAD pathway might play an important role in the pathogenesis of SUI. But the effect of polymorphisms in key genes in TGF-β1/SMAD pathway on the susceptibility to SUI remains unknown. Methods : Whole-exome sequencing (WES) was initially performed in 72 SUI women and 72 matched controls from Jiangxi Province, China. We analyzed rare variants in 7 key genes in TGF-β1/SMAD pathway that were predicted to be disease-causing and present exclusively in the cases. The potential pathogenicity of these candidate variants was assessed using the SIFT, Mutation Taster, and FATHMM prediction tools, and the detected variants were validated by Sanger sequencing. A validation cohort of 397 controls and 361 SUI patients was genotyped for these prioritized variants using both Massarray and KASP platforms. Results : WES revealed 111 variants in 7 key genes in TGFβ1/SMAD pathway among SUI cases. Following selection criteria (rare, predicted disease-causing, and absent in controls), four candidate missense variants were identified as potentially pathogenic for SUI: rs200111443 in the TGFBR2 gene, rs184408275 in the SMAD3 gene, rs1318674011 in the SMAD7 gene, and rs569594975 in the TGFB1 gene. Bioinformatic tools predicted the pathogenicity of these variants: SIFT and FATHMM classified rs200111443 as damaging, while FATHMM classified rs1318674011 as damaging. All four candidate variants were subsequently validated using Sanger sequencing. In the validation cohort (361 patients, 397 controls), none of the four candidate variants showed statistically significant associations with SUI susceptibility via Massarray/KASP genotyping (all p > 0.05). Notably, the rare missense variant (rs569594975) in TGFB1 gene was uniquely detected in SUI cases but absent in controls, suggesting a potential role in the pathogenesis of SUI which requires further investigation. Conclusions : Initial WES identified rare, potentially pathogenic missense variants in key TGFβ1/SMAD pathway genes exclusively among SUI cases. Subsequent large-scale validation using both Massarray and KASP genotyping assays in an independent cohort demonstrated no statistically significant association between the candidate variants (TGFBR2 rs200111443, SMAD3 rs184408275, SMAD7 rs1318674011, TGFB1 rs569594975) and SUI susceptibility. Notably, TGFB1 rs569594975 was uniquely detected in SUI patients, warranting functional investigation to clarify its biological relevance. Stress urinary incontinence TGF-β1/SMAD pathway Pathogenic variants Whole-exome sequencing Rare variants Figures Figure 1 Figure 2 Figure 3 1. Introduction Stress urinary incontinence (SUI), defined by the International Continence Society as the involuntary loss of urine upon physical exertion, sneezing, or coughing [ 1 ] , is a prevalent health issue significantly affecting middle-aged and older women. Its reported prevalence varies widely, ranging from 30% to 60%, depending on the studied population and case definition criteria [ 2 ] . Beyond its physical manifestation, SUI exerts a substantial negative impact on daily activities, sexual function, and mental well-being [ 3 – 5 ] . The etiology and pathogenesis of SUI are complex and multifactorial. Established environmental and lifestyle risk factors include advanced age, elevated body mass index (BMI), multiparity (particularly vaginal deliveries), menopausal status, and physical inactivity [ 6 , 7 ] . Importantly, growing evidence underscores a significant contribution of host genetic susceptibility to SUI development [ 8 ] . This is supported by familial aggregation studies, such as the work by Hannestad YS et al., which found a higher prevalence of urinary incontinence among relatives of affected women, suggesting a heritable predisposition [ 9 ] . More definitively, seminal research utilizing the nationwide Swedish Twin Register, conducted by Daniel Altman et al., provided robust quantitative evidence, estimating the relative contribution of genetic factors to SUI liability and confirming that genetic effects play a substantial role in its occurrence [ 10 ] . The extracellular matrix (ECM), composed primarily of collagen and elastic fibers, plays a crucial role in pelvic floor support and is implicated in the pathogenesis of SUI [ 11 ] . Deficiencies in collagen synthesis or degradation of elastin are recognized contributors to pelvic floor connective tissue defects and dysfunction, representing a major pathogenic mechanism underlying SUI [ 12 ] . Transforming growth factor β (TGF-β), particularly TGF-β1, is a pleiotropic cytokine pivotal in numerous physiological and pathological processes, including ECM regulation [ 13 ] . Central to this regulation is the TGF-β1/SMAD signaling pathway. Upon ligand binding, TGF-β1 first interacts with the type II receptor (TβR-II). This complex then recruits and activates the type I receptor (TβR-I). Activated TβR-I subsequently phosphorylates the receptor-regulated SMADs (R-SMADs), Smad2 and Smad3. Phosphorylated Smad2/3 then forms a complex with the common mediator Smad4 (Co-SMAD). This Smad complex translocates into the nucleus where it regulates the transcription of target genes involved in ECM synthesis and remodeling [ 13 – 15 ] . Importantly, Smad7 acts as a key negative regulator of this pathway by stably associating with TβR-I, thereby preventing the phosphorylation and activation of Smad2 and Smad3 [ 16 ] . Given its fundamental role in ECM homeostasis, the TGF-β1/SMAD pathway has been strongly implicated in the pathogenesis of SUI [ 17 , 18 ] . Whole-exome sequencing (WES) is widely applied to detect genetic variations associated with human diseases [ 19 ] . Although WES has been widely applied to investigate the genetic basis of various disorders, systematic analysis of genetic variants within the TGF-β1/SMAD pathway for SUI susceptibility using this approach remains lacking, as confirmed by our literature review [ 20 – 22 ] . Based on this gap, we hypothesized that rare variants in key TGF-β1/SMAD pathway genes (TGFB1, TGFBR1, TGFBR2, SMAD2, SMAD3, SMAD4, SMAD7) confer risk for SUI. To test this hypothesis, we employed a two-stage design: First, WES was performed on 72 case-control pairs to identify and prioritize candidate variants. Subsequently, prioritized variants were validated using dual-platform genotyping (Massarray and KASP) in an independent cohort (361 patients, 397 controls). Collectively, this comprehensive analysis provided no robust evidence for statistically significant associations between the prioritized rare variants and SUI susceptibility. Despite this overall null finding, the TGFB1 variant rs569594975 was observed exclusively within the case group, warranting further investigation. 2. Material and methods 2.1 Sample collection Women with SUI who visited the Department of Women's Health and Department of Gynecology in Jiangxi Provincial Maternal and Child Health Hospital and Gao 'an Maternal and Child Health Care Hospital between 1 September 2020 and 31 March 2022 were selected as cases. SUI was defined as an involuntary loss of urine occurring as result of an increase in intra-abdominal pressure due to effort or exertion or on sneezing or coughing according to the International Continence Society. The inclusion criteria for SUI cases were: 1) age ≥ 18 years old; 2) with symptoms of SUI; 3) provocation stress test (defined as an observed transurethral loss of urine simultaneous with a cough or Valsalva maneuver); 4) Signed informed consent. The exclusion criteria were: 1) pregnancy or lactation; 2) surgical history of urinary incontinence; 3) overactive bladder or other types of urinary incontinence; 4) pelvic organ prolapse (POP-Q) ≥ II degree; 5) used hormone drugs in the past one year; 6) history of pelvic surgery; 7) acute urinary tract infection; 8) diabetes; 9) history of severe trauma to the brain, spine or pelvis. Women without SUI (ICIQ-short form score equal to zero and a negative cough stress test) who visited the Department of Health Care in the two hospitals during the same period were recruited as controls. 480 SUI women and 480 controls were enrolled as study subjects. Sociodemographic characteristics, menstruation situation, history of pregnancy and childbearing, history of chronic diseases of these subjects were obtained by face to face interview. ICIQ-short form was fulfilled by each participant. 3 mL anticoagulant peripheral venous blood samples of each participant were collected. Samples were immediately centrifuged to separate serum from blood cells and were subsequently frozen at − 80°C until further processing. 2.2 Whole-exome sequencing Genomic DNA samples were isolated from participants’ peripheral blood by an E.Z.N.A. Blood DNA Kit (omega BIO.TEK). To assure the quality, the concentration and integrity of all the extracted DNA samples were detected by a Nanodrop-1000 spectrophotometer (Thermo Fisher, USA) and gel electrophoresis, respectively. DNA samples of 72 SUI women and 72 matched controls were subjected to exome capture using a BGI Exon Kit based on the manufacturer’s protocols. The combinatorial probe anchor ligation (cPAL) method was used to construct DNA libraries. Each resulting qualified captured library was sequenced on BGISEQ-500 platforms. The sequencing data of each sample was mapped to the human reference genome (GRCh38/hg38) using Barrows Wheeler Aligner. Variant calls was done using GATK ( https://software.broadinstitute.org/gatk/ ). Variant annotation was performed using SnpEff software. 2.3 Selection of candidate genetic variants The selection flowchart of candidate genetic variants was shown in Fig. 1 . First, variants with an MAF ≥ 0.01 in the 1000 Genomes Project ( http://www.internationalgenome.org/ ), Exome Aggregation Consortium (ExAC) ( http://exac.broadinstitute.org/ ), and dbSNP ( https://www.ncbi.nlm.nih.gov/snp/ ) databases were filtered out. Second, variants with effect prediction of “5/3_prime_UTR”, “intron” or “synonymous” were removed. Third, variants were preserved using overlapping methods by the 72 matched controls. Sanger sequencing validation Candidate genetic variants were confirmed by Sanger sequencing. The Primer Premier 5 software was used to design the primers. 2.4 Massarray and KASP dual-platform verification cohort genotyping A validation cohort of 397 controls and 361 SUI patients was genotyped for these prioritized variants using both Massarray and KASP platforms. The flow of sample selection and variant verification is summarized in Fig. 2 . 2.4.1 Massarray SNP Typing Genomic DNA samples (> 20 ng/µL, OD260/280 = 1.8–2.0) underwent multiplex PCR amplification with locus-specific primers (HotStar Taq, 45 cycles of 94°C/20s, 56°C/30s, 72°C/1m), followed by shrimp alkaline phosphatase (SAP) treatment to dephosphorylate residual nucleotides. Single-base extension reactions employed sequence-specific primers and mass-modified dideoxynucleotides (iPLEX Pro Kit), with products purified by resin desalting and dispensed onto SpectroCHIP arrays. Allele discrimination was achieved via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the Massarray Analyzer 4.0, with genotypes called by TYPER 4.0 software. 2.4.2 KASP genotyping Complementary validation was performed using competitive allele-specific PCR (KASP) chemistry on an ABI 7900HT Real-Time PCR System. Reactions contained 10 ng DNA, 2× KASP Master Mix (LGC), and 72× assay mix with allele-specific FRET cassettes in 5µL volumes. Thermal cycling included an initial 94°C/15m denaturation, 10 touchdown cycles (94°C/20s; 61–55°C/60s), and 29 amplification cycles (94°C/20s; 55°C/60s). Endpoint fluorescence (FAM/HEX) was measured post-PCR, with cluster analysis performed in SDS 2.4 software to assign genotypes. Negative controls confirmed assay specificity throughout the process. 2.5 Statistical analysis Statistical analyses were performed using SPSS software version 18.0 (SPSS, Chicago, IL). Values for continuous variables were presented as means and standard deviations (SDs). Categorical variables were expressed as numbers and percentages. t test and χ 2 test were applied to the analyses for continuous variables and categorical variables, respectively. For all analyses, p values were two-sided, with values less than 0.05 being regarded as statistically significant. 2.6 Ethical considerations Written informed consent was obtained from each participant. The study was approved by the Ethics committee of Jiangxi Provincial Maternal and Child Health Hospital. 3. Results 3.1 Results related to WES 3.1.1 The characteristics of SUI cases and controls in WES This study initially recruited 480 pairs of SUI patients and controls. From this cohort, 72 patients and 72 matched controls were randomly selected for WES analysis. Characteristics of SUI cases and controls were shown in Table 1. The mean age of SUI cases was comparable to the controls (44.17 ± 7.13 years vs. 44.18 ± 7.15 years, p = 0.990). Statistically significant difference was observed between SUI cases and controls in the distribution of educational level ( p = 0.006). In addition, there were no statistically differences in marital status, BMI, physical exercise, drinking, menstruation, number of vaginal deliveries, constipation or chronic cough between SUI cases and controls. 3.1.2 The WES data results After removing the reads with low quality, adapter contamination, and a high proportion of N bases, WES generated averagely 114,287,039 clean reads for each sample. The clean reads of each sample had high Q20 (> 95%) and Q30 (> 90%), which showed high sequencing quality. The average GC content was 51.77%. 100.0% of reads mapped successfully and 76.53% of reads mapped uniquely to the reference human genome GRCh38. After removing the duplicate reads (16.72%), the average sequencing depth was 132.38x. 99.5% of the whole genome excluding gap regions were covered by at least 1x coverage on average per sequencing individual. Seven key genes (namely TGFB1, TGFBR1, TGFBR2, SMAD2, SMAD3, SMAD4 and SMAD7) in TGFβ1/SMAD pathway underwent targeted WES to identify variants in 72 SUI cases. In total, 111 variants were identified, including 20 variants in TGFB1, 13 variants in TGFBR1, 17 variants in TGFBR2, 11 variants in SMAD2, 31 variants in SMAD3, 10 variants in SMAD4 and 9 variants in SMAD7. These variants consisted of 80 introns, 2 3’ primer UTR, 10 5’ primer UTR, 1 upstream gene, 10 synonymous, and 8 missense. After filtering out, 4 missense variants were selected as the candidate variants. 3.1.3 The candidate genetic variants in TGFβ1/SMAD pathway 4 candidate genetic variants were identified as possibly pathogenetic variants of SUI, which were absent in the controls. These variants were all missense variants, including rs200111443 in TGFBR2 gene, rs184408275 in SMAD3 gene, rs1318674011 in SMAD7 gene and rs569594975 in TGFB1 gene, respectively. The variant (rs200111443) was predicted to be damaging by the SIFT and FATH MM prediction tools, and the variant (rs1318674011) was predicted to be damaging by the FATH MM prediction tool. The detail information on the 4 candidate genetic variants was shown in Table 2. 3.1.4 Confirmation of the candidate variants by Sanger sequencing In order to validate the WES results, Sanger sequencing was conducted for the candidate variants. The details of the PCR primers were shown in Table 3. The results were all in accordance with WES (Fig. 3 ) . 3.2 Genetic Association Analysis of Prioritized Variants 3.2.1 Characteristics of SUI cases and controls in validation cohort From an initial cohort of 480 case-control pairs (960 participants), 72 pairs (144 individuals) were randomly selected for WES. Subsequent exclusions due to inadequate DNA quality control (concentration < 20 ng/µL or OD260/280 < 1.8) or incomplete clinical metadata yielded a final validation cohort of 361 SUI patients and 397 controls. The basic information of patients in the SUI case group and the control group is presented in Table 4. 3.2.2 Massarray SNP Typing The four candidate variants prioritized from WES (rs200111443, rs184408275, rs1318674011, rs569594975) were subjected to Massarray SNP typing in the validation cohort (Table 5). Technical constraints prevented successful clustering of rs1318674011 (SMAD7), resulting in no interpretable genotype calls for this variant. The remaining three variants were reliably detected: rs200111443 (TGFBR2) and rs184408275 (SMAD3) showed EAF of 0.001 and 0.008 in SUI cases versus 0.006 and 0.01 in controls, respectively, while rs569594975 (TGFB1) was absent in all samples (0/758 alleles). This outcome suggests platform-specific limitations in resolving certain rare variants, consistent with Massarray’s reduced sensitivity for low-frequency polymorphisms . 3.2.3 KASP validation of candidate variants Given the Massarray failure, rs1318674011 was reanalyzed using KASP genotyping. This orthogonal method achieved 99% call rate, identifying one heterozygous carrier in SUI patients (effect allele frequency, EAF = 0.001) and five samples in controls (EAF = 0.006). Despite achieving robust clustering, no significant association with SUI susceptibility was observed (odds ratio, OR = 0.127, p = 0.217; Table 5). 3.2.4 Contrasting validation outcomes Association analysis in the validation cohort (361 SUI cases, 397 controls) revealed no statistically significant associations for any candidate variant with SUI susceptibility (Table 5). SMAD3 rs184408275 showed no association (OR = 0.945, p = 0.914); TGFBR2 rs200111443 was non-significant (OR = 0.43, p = 0.3); SMAD7 rs1318674011 lacked association (OR = 0.428, p = 0.298); TGFB1 rs569594975 was absent in all validation samples (0/361 cases, 0/397 controls). Notably, the rare missense variant TGFB1 rs569594975 was detected in only a single individual across the entire study cohort (n = 902, EAF = 0.001): specifically, in one SUI case during WES screening (1/72 cases, EAF = 0.0069). This variant was absent in all other participants, including 72 WES controls, 361 validation cohort SUI patients, and 397 validation controls. 4. Discussion The extracellular matrix (ECM), which mainly comprises collagen, elastic fibers, and reticular fibers [ 23 ] , plays a vital role in pelvic floor supporting tissues. Critically, aberrant ECM metabolism is implicated in the pathogenesis of SUI. Furthermore, numerous studies [ 12 , 24 – 28 ] have demonstrated that the TGFβ1/SMAD pathway directly regulates ECM gene expression, including key components such as collagens and fibronectin. Supporting this mechanistic link, Wang et al. [ 17 ] and Li et al. [ 18 ] found that dysregulation of the TGFβ1/SMAD pathway contributes significantly to SUI development in preclinical models. To investigate this relationship in humans, we employed whole-exome sequencing to screen for pathogenic variants in core TGFβ1/SMAD pathway genes. Consequently, we identified four rare genetic variants (TGFB1 rs569594975, TGFBR2 rs200111443, SMAD3 rs184408275, SMAD7 rs1318674011) that may confer susceptibility to SUI. TGF-β1—encoded by the TGFB1 gene—serves as a master regulator of extracellular matrix (ECM) homeostasis, directly controlling collagen biosynthesis and critically maintaining tissue integrity through its widespread remodeling actions [ 29 ] . Consistent with this pivotal role, animal models of SUI demonstrate significant TGF-β1 upregulation in urethral tissues compared to sham controls [ 30 ] , confirming its pathological relevance. In the present human genetic study, we identified the ultra-rare TGFB1 missense variant rs569594975 (Phe239Ile), which localizes to the LAP domain and may alter TGF-β1 activation kinetics. Mechanistically linked, TGF-β1 signals through TGFβRII (TβR-2), a receptor encoded by TGFBR2 that initiates signaling via oligomerization with TGFβRI upon ligand binding, forming an essential ternary complex for SMAD cascade activation [ 31 , 32 ] . Supporting this pathway's disease involvement, TβR-2 expression is elevated in vaginal tissues of SUI rats [ 17 ] . Correspondingly, we detected a TGFBR2 missense variant (rs200111443, Ser46Arg) with concordant damaging predictions (SIFT = 0.018; FATHMM=-1.97), suggesting potential disruption of receptor function. Collectively, these findings imply that TGFB1 and TGFBR2 variants may dysregulate SUI susceptibility through distinct mechanisms: rs569594975 potentially impairing TGF-β1 latency control, and rs200111443 compromising receptor signaling fidelity—hypotheses requiring functional validation. 5. Strengths Our study presents a significant innovation. Firstly, whole-exome sequencing has identified four ultra-rare missense variants within the TGFβ1/SMAD pathway (TGFB1 rs569594975, TGFBR2 rs200111443, SMAD3 rs184408275, SMAD7 rs1318674011), with TGFB1 rs569594975 emerging as a uniquely case-specific finding. Importantly, orthogonal validation through Massarray and KASP genotyping confirmed its exceptional rarity: this variant was observed only once among 902 participants (1/72 WES cases; EAF = 0.0069) and was undetectable in all validation cohorts (0/758 samples; EAF = 0). 6. Limitations This study presents two principal limitations. First, although prioritized variants were validated in an expanded cohort, the discovery-phase WES cohort, comprising 72 cases and 72 controls, lacked sufficient power to definitively characterize ultra-rare variants. For instance, TGFB1 rs569594975 was detected in only one individual among 902 participants; even when considering the combined cohort (n = 902), the statistical effectiveness remains relatively low, thereby precluding robust association testing. Finally, the biological interpretation of the findings remains speculative without functional validation. The predicted disruptive effect of TGFB1 rs569594975 on the structure of the TGF-β1 latency peptide necessitates cell-based assays to confirm any alterations in cytokine activation or secretion. 7. Conclusion This pioneering two-stage study implemented whole-exome sequencing of TGF-β/SMAD pathway genes in 144 participants (72 cases of SUI and 72 matched controls), followed by orthogonal validation using Massarray and KASP genotyping in an expanded cohort of 758 individuals (361 SUI cases and 397 controls). Although no variants demonstrated statistically significant associations with SUI susceptibility, the TGFB1 rs569594975 missense variant exhibited unique characteristics. This ultra-rare variant was identified as a private allele in one discovery case (EAF = 0.0069) but remained undetectable across all validation samples and population databases. Its exclusive occurrence in the SUI discovery cohort, coupled with a non-significant trend in combined analysis (OR = 2.167, p = 0.518), suggests a potential patient-specific genetic susceptibility that may involve the disruption of TGF-β1 latency peptide function. Future studies should prioritize ultra-deep sequencing in expanded cohorts to capture rare susceptibility alleles, coupled with functional validation of prioritized variants, particularly those located in critical protein domains such as the TGF-β1 latency-associated peptide, utilizing in vitro models. Declarations Authors' contribution Zhihua Wan performed the experiments, collected the data, conducted the statistical analysis, and drafted the initial manuscript. Ting Wang conducted the statistical analysis, drafted the initial manuscript, and further revised and edited the manuscript. Ge Chen and Hui Yu collected the data and performed the experiments. Xujuan Shan and Yuling Tao assisted with data and sample collection. Liqun Wang designed the study and reviewed the manuscript. All authors have seen and approved the contents of the submitted manuscript. Funding The author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by the National Natural Science Foundation of China (81860581), Research Talent Cultivation Program of Jiangxi Provincial Maternal and Child Health Hospital (2023C003). Ethical statement This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Jiangxi Maternal and Child Health Hospital (Approval No: 2023-04-001). Written informed consent was obtained from all individual participants included in the study. This study is not a clinical trial. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Abrams P, Cardozo L, Fall M, et al. The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the International Continence Society[J]. Urology, 2003,61(1):37-49. Landefeld C S, Bowers B J, Feld A D, et al. 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Int Urogynecol J, 2017,28(6):947-955. Wrana J L, Attisano L, Carcamo J, et al. TGF beta signals through a heteromeric protein kinase receptor complex[J]. Cell, 1992,71(6):1003-1014. Chen L, Yang T, Lu D, et al. Central role of dysregulation of TGF-beta/Smad in CKD progression and potential targets of its treatment[J]. Biomed Pharmacother, 2018,101:670-681. Tables Tables 1 to 5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table12345.docx 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. 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Wang","suffix":""},{"id":523966735,"identity":"08e15666-049a-447e-ad2e-d0a933f41e85","order_by":2,"name":"Ge Chen","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Chen","suffix":""},{"id":523966736,"identity":"936b378b-b046-4cc6-9658-c7245b718f72","order_by":3,"name":"Hui Yu","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Yu","suffix":""},{"id":523966737,"identity":"9fc68d8a-4a94-4ec1-a07f-dbed65ae7619","order_by":4,"name":"Xujuan Shan","email":"","orcid":"","institution":"Gao'an Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xujuan","middleName":"","lastName":"Shan","suffix":""},{"id":523966738,"identity":"65a4a1f0-919e-4b5a-8d12-8f7114eb4d77","order_by":5,"name":"Yuling Tao","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuling","middleName":"","lastName":"Tao","suffix":""},{"id":523966739,"identity":"f4cf612e-70f5-4852-8ab2-2767c9bbbb50","order_by":6,"name":"Liqun 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1","display":"","copyAsset":false,"role":"figure","size":71884,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of the selection of candidate variants.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7586877/v1/de8127d90f181b0afa462b45.jpeg"},{"id":92733206,"identity":"94a0c30f-8c26-4c1b-b672-b05ebccb28d9","added_by":"auto","created_at":"2025-10-03 16:19:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":161846,"visible":true,"origin":"","legend":"\u003cp\u003eCandidate variants verification flowchart.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7586877/v1/1e30474e8ad704f24ff2ab72.jpeg"},{"id":92733210,"identity":"2136395e-8e04-4b6d-bee9-57fd02107130","added_by":"auto","created_at":"2025-10-03 16:19:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144009,"visible":true,"origin":"","legend":"\u003cp\u003eSanger sequencing validated the variants in key genes in TGFβ1/SMAD pathway. The location of mutation is marked with an arrow.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7586877/v1/5c233eaed528d6e3da71fc43.png"},{"id":104421882,"identity":"7131d5d4-b447-4ec6-a70e-193096c937de","added_by":"auto","created_at":"2026-03-11 13:59:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1127237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7586877/v1/310fb0b3-18a9-49c9-86ce-1ec6add99ead.pdf"},{"id":92734880,"identity":"419379e6-b848-407a-bceb-ccadea316601","added_by":"auto","created_at":"2025-10-03 16:27:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":353998,"visible":true,"origin":"","legend":"","description":"","filename":"table12345.docx","url":"https://assets-eu.researchsquare.com/files/rs-7586877/v1/5c5668edf7563f9a7f933a36.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole Exome Sequencing Studies the Association of Rare Variants in Key TGF-β1/SMAD Pathway Gene with Stress Urinary Incontinence Susceptibility","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStress urinary incontinence (SUI), defined by the International Continence Society as the involuntary loss of urine upon physical exertion, sneezing, or coughing\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, is a prevalent health issue significantly affecting middle-aged and older women. Its reported prevalence varies widely, ranging from 30% to 60%, depending on the studied population and case definition criteria\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Beyond its physical manifestation, SUI exerts a substantial negative impact on daily activities, sexual function, and mental well-being\u003csup\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The etiology and pathogenesis of SUI are complex and multifactorial. Established environmental and lifestyle risk factors include advanced age, elevated body mass index (BMI), multiparity (particularly vaginal deliveries), menopausal status, and physical inactivity\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Importantly, growing evidence underscores a significant contribution of host genetic susceptibility to SUI development\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. This is supported by familial aggregation studies, such as the work by Hannestad YS et al., which found a higher prevalence of urinary incontinence among relatives of affected women, suggesting a heritable predisposition\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. More definitively, seminal research utilizing the nationwide Swedish Twin Register, conducted by Daniel Altman et al., provided robust quantitative evidence, estimating the relative contribution of genetic factors to SUI liability and confirming that genetic effects play a substantial role in its occurrence\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe extracellular matrix (ECM), composed primarily of collagen and elastic fibers, plays a crucial role in pelvic floor support and is implicated in the pathogenesis of SUI\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Deficiencies in collagen synthesis or degradation of elastin are recognized contributors to pelvic floor connective tissue defects and dysfunction, representing a major pathogenic mechanism underlying SUI\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Transforming growth factor β (TGF-β), particularly TGF-β1, is a pleiotropic cytokine pivotal in numerous physiological and pathological processes, including ECM regulation\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Central to this regulation is the TGF-β1/SMAD signaling pathway. Upon ligand binding, TGF-β1 first interacts with the type II receptor (TβR-II). This complex then recruits and activates the type I receptor (TβR-I). Activated TβR-I subsequently phosphorylates the receptor-regulated SMADs (R-SMADs), Smad2 and Smad3. Phosphorylated Smad2/3 then forms a complex with the common mediator Smad4 (Co-SMAD). This Smad complex translocates into the nucleus where it regulates the transcription of target genes involved in ECM synthesis and remodeling\u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Importantly, Smad7 acts as a key negative regulator of this pathway by stably associating with TβR-I, thereby preventing the phosphorylation and activation of Smad2 and Smad3\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Given its fundamental role in ECM homeostasis, the TGF-β1/SMAD pathway has been strongly implicated in the pathogenesis of SUI\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhole-exome sequencing (WES) is widely applied to detect genetic variations associated with human diseases\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Although WES has been widely applied to investigate the genetic basis of various disorders, systematic analysis of genetic variants within the TGF-β1/SMAD pathway for SUI susceptibility using this approach remains lacking, as confirmed by our literature review\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Based on this gap, we hypothesized that rare variants in key TGF-β1/SMAD pathway genes (TGFB1, TGFBR1, TGFBR2, SMAD2, SMAD3, SMAD4, SMAD7) confer risk for SUI. To test this hypothesis, we employed a two-stage design: First, WES was performed on 72 case-control pairs to identify and prioritize candidate variants. Subsequently, prioritized variants were validated using dual-platform genotyping (Massarray and KASP) in an independent cohort (361 patients, 397 controls). Collectively, this comprehensive analysis provided no robust evidence for statistically significant associations between the prioritized rare variants and SUI susceptibility. Despite this overall null finding, the TGFB1 variant rs569594975 was observed exclusively within the case group, warranting further investigation.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Sample collection\u003c/h2\u003e\u003cp\u003eWomen with SUI who visited the Department of Women's Health and Department of Gynecology in Jiangxi Provincial Maternal and Child Health Hospital and Gao 'an Maternal and Child Health Care Hospital between 1 September 2020 and 31 March 2022 were selected as cases. SUI was defined as an involuntary loss of urine occurring as result of an increase in intra-abdominal pressure due to effort or exertion or on sneezing or coughing according to the International Continence Society. The inclusion criteria for SUI cases were: 1) age\u0026thinsp;\u0026ge;\u0026thinsp;18 years old; 2) with symptoms of SUI; 3) provocation stress test (defined as an observed transurethral loss of urine simultaneous with a cough or Valsalva maneuver); 4) Signed informed consent. The exclusion criteria were: 1) pregnancy or lactation; 2) surgical history of urinary incontinence; 3) overactive bladder or other types of urinary incontinence; 4) pelvic organ prolapse (POP-Q)\u0026thinsp;\u0026ge;\u0026thinsp;II degree; 5) used hormone drugs in the past one year; 6) history of pelvic surgery; 7) acute urinary tract infection; 8) diabetes; 9) history of severe trauma to the brain, spine or pelvis. Women without SUI (ICIQ-short form score equal to zero and a negative cough stress test) who visited the Department of Health Care in the two hospitals during the same period were recruited as controls. 480 SUI women and 480 controls were enrolled as study subjects. Sociodemographic characteristics, menstruation situation, history of pregnancy and childbearing, history of chronic diseases of these subjects were obtained by face to face interview. ICIQ-short form was fulfilled by each participant. 3 mL anticoagulant peripheral venous blood samples of each participant were collected. Samples were immediately centrifuged to separate serum from blood cells and were subsequently frozen at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Whole-exome sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA samples were isolated from participants\u0026rsquo; peripheral blood by an E.Z.N.A. Blood DNA Kit (omega BIO.TEK). To assure the quality, the concentration and integrity of all the extracted DNA samples were detected by a Nanodrop-1000 spectrophotometer (Thermo Fisher, USA) and gel electrophoresis, respectively. DNA samples of 72 SUI women and 72 matched controls were subjected to exome capture using a BGI Exon Kit based on the manufacturer\u0026rsquo;s protocols. The combinatorial probe anchor ligation (cPAL) method was used to construct DNA libraries. Each resulting qualified captured library was sequenced on BGISEQ-500 platforms. The sequencing data of each sample was mapped to the human reference genome (GRCh38/hg38) using Barrows Wheeler Aligner. Variant calls was done using GATK (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://software.broadinstitute.org/gatk/\u003c/span\u003e\u003cspan address=\"https://software.broadinstitute.org/gatk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Variant annotation was performed using SnpEff software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Selection of candidate genetic variants\u003c/h2\u003e\u003cp\u003eThe selection flowchart of candidate genetic variants was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. First, variants with an MAF\u0026thinsp;\u0026ge;\u0026thinsp;0.01 in the 1000 Genomes Project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.internationalgenome.org/\u003c/span\u003e\u003cspan address=\"http://www.internationalgenome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Exome Aggregation Consortium (ExAC) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://exac.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"http://exac.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and dbSNP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/snp/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/snp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases were filtered out. Second, variants with effect prediction of \u0026ldquo;5/3_prime_UTR\u0026rdquo;, \u0026ldquo;intron\u0026rdquo; or \u0026ldquo;synonymous\u0026rdquo; were removed. Third, variants were preserved using overlapping methods by the 72 matched controls.\u003c/p\u003e\u003cp\u003eSanger sequencing validation\u003c/p\u003e\u003cp\u003eCandidate genetic variants were confirmed by Sanger sequencing. The Primer Premier 5 software was used to design the primers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Massarray and KASP dual-platform verification cohort genotyping\u003c/h2\u003e\u003cp\u003eA validation cohort of 397 controls and 361 SUI patients was genotyped for these prioritized variants using both Massarray and KASP platforms. The flow of sample selection and variant verification is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Massarray SNP Typing\u003c/h2\u003e\u003cp\u003eGenomic DNA samples (\u0026gt;\u0026thinsp;20 ng/\u0026micro;L, OD260/280\u0026thinsp;=\u0026thinsp;1.8\u0026ndash;2.0) underwent multiplex PCR amplification with locus-specific primers (HotStar Taq, 45 cycles of 94\u0026deg;C/20s, 56\u0026deg;C/30s, 72\u0026deg;C/1m), followed by shrimp alkaline phosphatase (SAP) treatment to dephosphorylate residual nucleotides. Single-base extension reactions employed sequence-specific primers and mass-modified dideoxynucleotides (iPLEX Pro Kit), with products purified by resin desalting and dispensed onto SpectroCHIP arrays. Allele discrimination was achieved via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the Massarray Analyzer 4.0, with genotypes called by TYPER 4.0 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 KASP genotyping\u003c/h2\u003e\u003cp\u003eComplementary validation was performed using competitive allele-specific PCR (KASP) chemistry on an ABI 7900HT Real-Time PCR System. Reactions contained 10 ng DNA, 2\u0026times; KASP Master Mix (LGC), and 72\u0026times; assay mix with allele-specific FRET cassettes in 5\u0026micro;L volumes. Thermal cycling included an initial 94\u0026deg;C/15m denaturation, 10 touchdown cycles (94\u0026deg;C/20s; 61\u0026ndash;55\u0026deg;C/60s), and 29 amplification cycles (94\u0026deg;C/20s; 55\u0026deg;C/60s). Endpoint fluorescence (FAM/HEX) was measured post-PCR, with cluster analysis performed in SDS 2.4 software to assign genotypes. Negative controls confirmed assay specificity throughout the process.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using SPSS software version 18.0 (SPSS, Chicago, IL). Values for continuous variables were presented as means and standard deviations (SDs). Categorical variables were expressed as numbers and percentages. \u003cem\u003et\u003c/em\u003e test and χ\u003csup\u003e2\u003c/sup\u003e test were applied to the analyses for continuous variables and categorical variables, respectively. For all analyses, \u003cem\u003ep\u003c/em\u003e values were two-sided, with values less than 0.05 being regarded as statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Ethical considerations\u003c/h2\u003e\u003cp\u003e Written informed consent was obtained from each participant. The study was approved by the Ethics committee of Jiangxi Provincial Maternal and Child Health Hospital.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Results related to WES\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 The characteristics of SUI cases and controls in WES\u003c/h2\u003e\u003cp\u003eThis study initially recruited 480 pairs of SUI patients and controls. From this cohort, 72 patients and 72 matched controls were randomly selected for WES analysis. Characteristics of SUI cases and controls were shown in Table\u0026nbsp;1. The mean age of SUI cases was comparable to the controls (44.17\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13 years vs. 44.18\u0026thinsp;\u0026plusmn;\u0026thinsp;7.15 years, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.990). Statistically significant difference was observed between SUI cases and controls in the distribution of educational level (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006). In addition, there were no statistically differences in marital status, BMI, physical exercise, drinking, menstruation, number of vaginal deliveries, constipation or chronic cough between SUI cases and controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 The WES data results\u003c/h2\u003e\u003cp\u003eAfter removing the reads with low quality, adapter contamination, and a high proportion of N bases, WES generated averagely 114,287,039 clean reads for each sample. The clean reads of each sample had high Q20 (\u0026gt;\u0026thinsp;95%) and Q30 (\u0026gt;\u0026thinsp;90%), which showed high sequencing quality. The average GC content was 51.77%. 100.0% of reads mapped successfully and 76.53% of reads mapped uniquely to the reference human genome GRCh38. After removing the duplicate reads (16.72%), the average sequencing depth was 132.38x. 99.5% of the whole genome excluding gap regions were covered by at least 1x coverage on average per sequencing individual.\u003c/p\u003e\u003cp\u003eSeven key genes (namely TGFB1, TGFBR1, TGFBR2, SMAD2, SMAD3, SMAD4 and SMAD7) in TGFβ1/SMAD pathway underwent targeted WES to identify variants in 72 SUI cases. In total, 111 variants were identified, including 20 variants in TGFB1, 13 variants in TGFBR1, 17 variants in TGFBR2, 11 variants in SMAD2, 31 variants in SMAD3, 10 variants in SMAD4 and 9 variants in SMAD7. These variants consisted of 80 introns, 2 3\u0026rsquo; primer UTR, 10 5\u0026rsquo; primer UTR, 1 upstream gene, 10 synonymous, and 8 missense. After filtering out, 4 missense variants were selected as the candidate variants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3 The candidate genetic variants in TGFβ1/SMAD pathway\u003c/h2\u003e\u003cp\u003e4 candidate genetic variants were identified as possibly pathogenetic variants of SUI, which were absent in the controls. These variants were all missense variants, including rs200111443 in TGFBR2 gene, rs184408275 in SMAD3 gene, rs1318674011 in SMAD7 gene and rs569594975 in TGFB1 gene, respectively. The variant (rs200111443) was predicted to be damaging by the SIFT and FATH MM prediction tools, and the variant (rs1318674011) was predicted to be damaging by the FATH MM prediction tool. The detail information on the 4 candidate genetic variants was shown in Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.1.4 Confirmation of the candidate variants by Sanger sequencing\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eIn order to validate the WES results, Sanger sequencing was conducted for the candidate variants. The details of the PCR primers were shown in Table\u0026nbsp;3. The results were all in accordance with WES (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) .\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Genetic Association Analysis of Prioritized Variants\u003c/h2\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Characteristics of SUI cases and controls in validation cohort\u003c/h2\u003e\u003cp\u003eFrom an initial cohort of 480 case-control pairs (960 participants), 72 pairs (144 individuals) were randomly selected for WES. Subsequent exclusions due to inadequate DNA quality control (concentration\u0026thinsp;\u0026lt;\u0026thinsp;20 ng/\u0026micro;L or OD260/280\u0026thinsp;\u0026lt;\u0026thinsp;1.8) or incomplete clinical metadata yielded a final validation cohort of 361 SUI patients and 397 controls. The basic information of patients in the SUI case group and the control group is presented in Table\u0026nbsp;4.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 Massarray SNP Typing\u003c/h2\u003e\u003cp\u003eThe four candidate variants prioritized from WES (rs200111443, rs184408275, rs1318674011, rs569594975) were subjected to Massarray SNP typing in the validation cohort (Table\u0026nbsp;5). Technical constraints prevented successful clustering of rs1318674011 (SMAD7), resulting in no interpretable genotype calls for this variant. The remaining three variants were reliably detected: rs200111443 (TGFBR2) and rs184408275 (SMAD3) showed EAF of 0.001 and 0.008 in SUI cases versus 0.006 and 0.01 in controls, respectively, while rs569594975 (TGFB1) was absent in all samples (0/758 alleles). This outcome suggests platform-specific limitations in resolving certain rare variants, consistent with Massarray\u0026rsquo;s reduced sensitivity for low-frequency polymorphisms .\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 KASP validation of candidate variants\u003c/h2\u003e\u003cp\u003eGiven the Massarray failure, rs1318674011 was reanalyzed using KASP genotyping. This orthogonal method achieved 99% call rate, identifying one heterozygous carrier in SUI patients (effect allele frequency, EAF\u0026thinsp;=\u0026thinsp;0.001) and five samples in controls (EAF\u0026thinsp;=\u0026thinsp;0.006). Despite achieving robust clustering, no significant association with SUI susceptibility was observed (odds ratio, OR\u0026thinsp;=\u0026thinsp;0.127, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.217; Table\u0026nbsp;5).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4 Contrasting validation outcomes\u003c/h2\u003e\u003cp\u003eAssociation analysis in the validation cohort (361 SUI cases, 397 controls) revealed no statistically significant associations for any candidate variant with SUI susceptibility (Table\u0026nbsp;5). SMAD3 rs184408275 showed no association (OR\u0026thinsp;=\u0026thinsp;0.945, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.914); TGFBR2 rs200111443 was non-significant (OR\u0026thinsp;=\u0026thinsp;0.43, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3); SMAD7 rs1318674011 lacked association (OR\u0026thinsp;=\u0026thinsp;0.428, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.298); TGFB1 rs569594975 was absent in all validation samples (0/361 cases, 0/397 controls). Notably, the rare missense variant TGFB1 rs569594975 was detected in only a single individual across the entire study cohort (n\u0026thinsp;=\u0026thinsp;902, EAF\u0026thinsp;=\u0026thinsp;0.001): specifically, in one SUI case during WES screening (1/72 cases, EAF\u0026thinsp;=\u0026thinsp;0.0069). This variant was absent in all other participants, including 72 WES controls, 361 validation cohort SUI patients, and 397 validation controls.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe extracellular matrix (ECM), which mainly comprises collagen, elastic fibers, and reticular fibers\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, plays a vital role in pelvic floor supporting tissues. Critically, aberrant ECM metabolism is implicated in the pathogenesis of SUI. Furthermore, numerous studies \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e have demonstrated that the TGFβ1/SMAD pathway directly regulates ECM gene expression, including key components such as collagens and fibronectin. Supporting this mechanistic link, Wang et al. \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003eand Li et al.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e found that dysregulation of the TGFβ1/SMAD pathway contributes significantly to SUI development in preclinical models. To investigate this relationship in humans, we employed whole-exome sequencing to screen for pathogenic variants in core TGFβ1/SMAD pathway genes. Consequently, we identified four rare genetic variants (TGFB1 rs569594975, TGFBR2 rs200111443, SMAD3 rs184408275, SMAD7 rs1318674011) that may confer susceptibility to SUI.\u003c/p\u003e\u003cp\u003eTGF-β1\u0026mdash;encoded by the TGFB1 gene\u0026mdash;serves as a master regulator of extracellular matrix (ECM) homeostasis, directly controlling collagen biosynthesis and critically maintaining tissue integrity through its widespread remodeling actions\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Consistent with this pivotal role, animal models of SUI demonstrate significant TGF-β1 upregulation in urethral tissues compared to sham controls \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, confirming its pathological relevance. In the present human genetic study, we identified the ultra-rare TGFB1 missense variant rs569594975 (Phe239Ile), which localizes to the LAP domain and may alter TGF-β1 activation kinetics.\u003c/p\u003e\u003cp\u003eMechanistically linked, TGF-β1 signals through TGFβRII (TβR-2), a receptor encoded by TGFBR2 that initiates signaling via oligomerization with TGFβRI upon ligand binding, forming an essential ternary complex for SMAD cascade activation\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Supporting this pathway's disease involvement, TβR-2 expression is elevated in vaginal tissues of SUI rats\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Correspondingly, we detected a TGFBR2 missense variant (rs200111443, Ser46Arg) with concordant damaging predictions (SIFT\u0026thinsp;=\u0026thinsp;0.018; FATHMM=-1.97), suggesting potential disruption of receptor function. Collectively, these findings imply that TGFB1 and TGFBR2 variants may dysregulate SUI susceptibility through distinct mechanisms: rs569594975 potentially impairing TGF-β1 latency control, and rs200111443 compromising receptor signaling fidelity\u0026mdash;hypotheses requiring functional validation.\u003c/p\u003e"},{"header":"5. Strengths","content":"\u003cp\u003eOur study presents a significant innovation. Firstly, whole-exome sequencing has identified four ultra-rare missense variants within the TGFβ1/SMAD pathway (TGFB1 rs569594975, TGFBR2 rs200111443, SMAD3 rs184408275, SMAD7 rs1318674011), with TGFB1 rs569594975 emerging as a uniquely case-specific finding. Importantly, orthogonal validation through Massarray and KASP genotyping confirmed its exceptional rarity: this variant was observed only once among 902 participants (1/72 WES cases; EAF\u0026thinsp;=\u0026thinsp;0.0069) and was undetectable in all validation cohorts (0/758 samples; EAF\u0026thinsp;=\u0026thinsp;0).\u003c/p\u003e"},{"header":"6. Limitations","content":"\u003cp\u003eThis study presents two principal limitations. First, although prioritized variants were validated in an expanded cohort, the discovery-phase WES cohort, comprising 72 cases and 72 controls, lacked sufficient power to definitively characterize ultra-rare variants. For instance, TGFB1 rs569594975 was detected in only one individual among 902 participants; even when considering the combined cohort (n\u0026thinsp;=\u0026thinsp;902), the statistical effectiveness remains relatively low, thereby precluding robust association testing. Finally, the biological interpretation of the findings remains speculative without functional validation. The predicted disruptive effect of TGFB1 rs569594975 on the structure of the TGF-β1 latency peptide necessitates cell-based assays to confirm any alterations in cytokine activation or secretion.\u003c/p\u003e"},{"header":"7. Conclusion","content":"\u003cp\u003eThis pioneering two-stage study implemented whole-exome sequencing of TGF-β/SMAD pathway genes in 144 participants (72 cases of SUI and 72 matched controls), followed by orthogonal validation using Massarray and KASP genotyping in an expanded cohort of 758 individuals (361 SUI cases and 397 controls). Although no variants demonstrated statistically significant associations with SUI susceptibility, the TGFB1 rs569594975 missense variant exhibited unique characteristics. This ultra-rare variant was identified as a private allele in one discovery case (EAF\u0026thinsp;=\u0026thinsp;0.0069) but remained undetectable across all validation samples and population databases. Its exclusive occurrence in the SUI discovery cohort, coupled with a non-significant trend in combined analysis (OR\u0026thinsp;=\u0026thinsp;2.167, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.518), suggests a potential patient-specific genetic susceptibility that may involve the disruption of TGF-β1 latency peptide function. Future studies should prioritize ultra-deep sequencing in expanded cohorts to capture rare susceptibility alleles, coupled with functional validation of prioritized variants, particularly those located in critical protein domains such as the TGF-β1 latency-associated peptide, utilizing in vitro models.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhihua Wan performed the experiments, collected the data, conducted the statistical analysis, and drafted the initial manuscript. Ting Wang conducted the statistical analysis, drafted the initial manuscript, and further revised and edited the manuscript. Ge Chen and Hui Yu collected the data and performed the experiments. Xujuan Shan and Yuling Tao assisted with data and sample collection. Liqun Wang designed the study and reviewed the manuscript. All authors have seen and approved the contents of the submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by the National Natural Science Foundation of China (81860581), Research Talent Cultivation Program of Jiangxi Provincial Maternal and Child Health Hospital (2023C003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Jiangxi Maternal and Child Health Hospital (Approval No: 2023-04-001). Written informed consent was obtained from all individual participants included in the study. This study is not a clinical trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbrams P, Cardozo L, Fall M, et al. The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the International Continence Society[J]. Urology, 2003,61(1):37-49.\u003c/li\u003e\n\u003cli\u003eLandefeld C S, Bowers B J, Feld A D, et al. National Institutes of Health state-of-the-science conference statement: prevention of fecal and urinary incontinence in adults[J]. Ann Intern Med, 2008,148(6):449-458.\u003c/li\u003e\n\u003cli\u003eMiner P B J. Economic and personal impact of fecal and urinary incontinence[J]. 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Biomed Pharmacother, 2018,101:670-681.\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":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":"Stress urinary incontinence, TGF-β1/SMAD pathway, Pathogenic variants, Whole-exome sequencing, Rare variants","lastPublishedDoi":"10.21203/rs.3.rs-7586877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7586877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Stress urinary incontinence (SUI) is a prevalent urological problem that is common among middle-aged and older women. Previous studies have shown that TGF-β1/SMAD pathway might play an important role in the pathogenesis of SUI. But the effect of polymorphisms in key genes in TGF-β1/SMAD pathway on the susceptibility to SUI remains unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Whole-exome sequencing (WES) was initially performed in 72 SUI women and 72 matched controls from Jiangxi Province, China. We analyzed rare variants in 7 key genes in \u0026nbsp;TGF-β1/SMAD pathway that were predicted to be disease-causing and present exclusively in the cases. The potential pathogenicity of these candidate variants was assessed using the SIFT, Mutation Taster, and FATHMM prediction tools, and the detected variants were validated by Sanger sequencing. A validation cohort of 397 controls and 361 SUI patients was genotyped for these prioritized variants using both Massarray and KASP platforms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: WES revealed 111 variants in 7 key genes in TGFβ1/SMAD pathway among SUI cases. Following selection criteria (rare, predicted disease-causing, and absent in controls), four candidate missense variants were identified as potentially pathogenic for SUI: rs200111443 in the TGFBR2 gene, rs184408275 in the SMAD3 gene, rs1318674011 in the SMAD7 gene, and rs569594975 in the TGFB1 gene. Bioinformatic tools predicted the pathogenicity of these variants: SIFT and FATHMM classified rs200111443 as damaging, while FATHMM classified rs1318674011 as damaging. All four candidate variants were subsequently validated using Sanger sequencing. In the validation cohort (361 patients, 397 controls), none of the four candidate variants showed statistically significant associations with SUI susceptibility via Massarray/KASP genotyping (all \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Notably, the rare missense variant (rs569594975) in TGFB1 gene was uniquely detected in SUI cases but absent in controls, suggesting a potential role in the pathogenesis of SUI which requires further investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Initial WES identified rare, potentially pathogenic missense variants in key TGFβ1/SMAD pathway genes exclusively among SUI cases. Subsequent large-scale validation using both Massarray and KASP genotyping assays in an independent cohort demonstrated no statistically significant association between the candidate variants (TGFBR2 rs200111443, SMAD3 rs184408275, SMAD7 rs1318674011, TGFB1 rs569594975) and SUI susceptibility. Notably, TGFB1 rs569594975 was uniquely detected in SUI patients, warranting functional investigation to clarify its biological relevance.\u003c/p\u003e","manuscriptTitle":"Whole Exome Sequencing Studies the Association of Rare Variants in Key TGF-β1/SMAD Pathway Gene with Stress Urinary Incontinence Susceptibility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 16:19:01","doi":"10.21203/rs.3.rs-7586877/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":"159a8f56-43f9-4876-b98e-01e6f8b7ae66","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-11T13:58:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 16:19:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7586877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7586877","identity":"rs-7586877","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}