Whole Exome Sequencing in Fetal Cardiac Rhabdomyoma Detected by Ultrasonography: An Analysis of 12 Cases

Whole Exome Sequencing in Fetal Cardiac Rhabdomyoma Detected by Ultrasonography: An Analysis of 12 Cases | 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 in Fetal Cardiac Rhabdomyoma Detected by Ultrasonography: An Analysis of 12 Cases Jing Liu, Jun He, Wanglan Tang, Jing Chen, Yingchun Luo, Haoxian Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7319816/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2025 Read the published version in BMC Pregnancy and Childbirth → Version 1 posted 11 You are reading this latest preprint version Abstract Background Most of fetal cardiac rhabdomyomas (CRs) are associated with tuberous sclerosis (TSC), an autosomal dominant inherited disorder caused by mutations in the TSC1 or TSC2 genes. In this study, we conducted a comprehensive analysis integrating prenatal echocardiographic findings, parental phenotypic characteristics, and genetic profiles of fetuses with CR. Methods 12 fetuses with sonographically identified CR were included. Karyotype and SNP-array/CNV-seq were performed simultaneously with Trio-WES. Results All CRs were observed in the ventricle, interventricular septum, and the atrium, while the left ventricle were the most common areas. All subjects did not detect arrhythmia during pregnancy. Beside CRs, irregular low echo in the brain was detected in Case 12. TSC1 and TSC2 variants were identified in all 12 fetuses (100%). Mutants of TSC1 account for 25% (3/12) and TSC2 account for 75%(9/12). Two-thirds of these variants were de novo . The P/LP variant spectrum in TSC1 / TSC2 includes 4 (34%) nonsense, 4 (33%) missense, 2 (17%) frameshift, 1 (8%) splice and 1 (8%) small deletion. A 780kb deletion in 9q34.13 (arr[hg19] 9q34.13 (132286422–133062068)×1) encompassing the entire of TSC1 gene, and the other two de novo mutants of c.1687G > C and c.1106delT in TSC2 gene had not been reported previously. Conclusion Combination of ultrasound and genetic testing can effectively diagnose the prenatal cases of TSC. Three novel mutations in TSC genes enlarge the mutation spectrum of TSC . cardiac rhabdomyoma TSC1 TSC2 Trio-WES prenatal diagnosis Figures Figure 1 Figure 2 1. INTRODUCTION Rhabdomyoma is the most prevalent fetal cardiac tumor and can be detected via routine prenatal ultrasound from the 15th gestational week onward (Ekmekci et al., 2018 ). The incidence of this tumor ranges from 0.02 to 0.17% in liveborn infants and 0.12% in prenatal fetal studies (Isaacs, 2004 ; Yuan, 2017 ). The majority of fetal cardiac rhabdomyomas (CRs) are associated with tuberous sclerosis (TSC), an autosomal dominant disorder characterized by cellular and tissue dysplasia in multiple organs, which can lead to neurologic and behavioral abnormalities (Józwiak and Kotulska, 2006 ; Chen et al., 2019 ; Pavlicek et al., 2021 ). The two known genes, TSC1 located at 9q34.3 and TSC2 located at 16p13.3, are crucial disease-causing genes for TSC. Loss of function of TSC1/TSC2 leads to hyperactivity of mTORC1, which drives abnormal cellular growth and tumorigenesis (Dibble and Manning, 2013 ; Zhang et al., 2014 ). Next-generation sequencing (NGS) offers deep, high-throughput, and parallel genome sequencing. With its expanding application in prenatal diagnosis, the diagnostic rate for fetuses with CRs has significantly improved, thereby enhancing the capacity for genetic counseling. In the present study, Trio-WES was conducted to identify the genetic causes in 12 fetuses with sonographically detected CRs, and TSC was subsequently diagnosed in all 12 cases. We integrated detailed parental phenotypes, echocardiography, and genetic analysis to conduct a comprehensive assessment of these 12 CR fetuses. The combination of ultrasound and genetic tests enabled us to effectively diagnose prenatal TSC cases. We also identified one novel deletion involving the TSC1 gene and two novel mutations in the TSC2 gene, thereby expanding the spectrum of TSC mutations. 2. MATERIALS AND METHODS 2.1 Ethical compliance The research was in compliance with the Helsinki Declaration and approved by the Ethics Committee of Changsha Hospital for Maternal and Child Health Care. 2.2 Subjects and Sample Collection In this study, we included twelve prenatal cases of cardiac rhabdomyoma which were collected in the prenatal Diagnosis Central of Hunan Provincial Maternal and Child Health Care Hospital from 2017 to 2022. The reasons for prenatal diagnosis were cardiac rhabdomyoma presented in all of these fetuses found by ultrasound. The results of prenatal ultrasound examination are shown in Fig. 1 for part of these fetuses. After obtained the informed consents, amniocentesis cells of each fetuses were collected via amniocentesis for genetic testing, and peripheral blood was collected from parents to determine inheritance patterns of any mutations. Karyotype and SNP-array/CNV seq were performed simultaneously with NGS or before. 2.3 Next-Generation Sequencing Genomic DNA samples from 12 fetuses and their respective parents were isolated using the Qiagen DNA Blood Midi/Mini kit (Qiagen GmbH, Germany). Extracted DNA underwent enzymatic fragmentation targeting ~ 200-bp segments, followed by end repair and 3'-terminal adenine (A) addition. Indexed adapters were ligated to the resulting fragments, and products of approximately 320 bp were size-selected using XP beads. PCR amplification was subsequently performed. Enrichment of exonic regions was achieved through hybridization capture with the Nano WES system (following manufacturer specifications). Captured libraries were eluted, PCR-amplified, and subjected to quality control: enrichment efficiency was assessed via qPCR, while fragment size distribution and concentration were determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA). Final libraries were sequenced on the Illumina NovaSeq6000 platform (150-bp paired-end reads). Base calling and raw data generation from image files utilized CASAVA (v1.82). Variants identified were validated by Sanger sequencing in trios. 2.4 Data analysis Raw sequencing reads underwent alignment to the GRCh37/hg19 reference genome via the Burrows-Wheeler Aligner (BWA). PCR duplicates were subsequently eliminated using Picard v1.57. Variant pathogenicity classification adhered to American College of Medical Genetics and Genomics (ACMG) standards, categorizing variants as: Pathogenic, Likely Pathogenic, Benign, Likely Benign, or Variants of Uncertain Significance (VUS). For copy number variation (CNV) detection, the Sprinkle pipeline (BerryGenomics) was implemented. This integrated:(i) XHMM-based principal component analysis for noise reduction, (ii) GC-content and bias adjustment through CNV Kit's fix module, (iii) Exon-level and large-segment CNV identification via copy number calculation. 2.5 Predicted structure models To assess the structural and functional implications of the G370S and A563P variants in TSC2, computational modeling was performed using VarSite and PyMOL. The wild-type TSC2 protein complex structure (PDB ID: not specified) was retrieved from the Protein Data Bank Europe (PDBe) as the reference framework. PyMOL facilitated: (i) Structural alignment between wild-type and mutant conformations, (ii) In silico mutagenesis to generate variant models, (iii) Visualization of atomic-level perturbations. This integrated approach enabled systematic evaluation of mutation-induced structural perturbations and their potential functional consequences. 3. RESULTS 3.1 General Information and Prenatal ultrasound Results A total of 12 fetuses with CR were included in the study. The average age of the 12 pregnant women was 27.83 years (range 22–31years). The mean GA of CRs detected during routine ultrasound scans was 28.67 ± 2.32 weeks (range 24–32 weeks). All CRs were detected in the ventricle, interventricular septum, and the atrium, especially frequent in the left ventricle (Fig. 1 ). All subjects did not detect arrhythmia during the pregnancy. Beside CRs, irregular low echo in the brain was detected in Case 12 (Fig. 1 F). 3.2 Identification of TSC1 and TSC2 Variants After genetic testing, all 12 fetuses showed normal karyotypes and CNV, but they all carried TSC1 and TSC2 variants (100%). Roughly 25% of these variants had not been reported previously (Fig. 2 A). The P/LP variant spectrum in TSC1 / TSC2 includes 4 (33%) nonsense, 4 (33%) missense, 2 (17%) frameshift, 1 (8%) splice, 1 (8%) small deletion (Fig. 2 B). Mutants of TSC1 account for 25% (3/12) and TSC2 account for 75% (Fig. 2 C). One-thirds of these variants were de novo (Fig. 2 D). CNV seq results revealed a 780 Kb-sized deletion on 9q34.13 (arr[hg19] 9q34.13 (132286422–133062068)×1) in case 1, encompassing the entire of TSC1 gene, which was inherited from mother (Fig. 2 E). In case 5, the de novo frameshift mutant of c.1106delT (p.Gln370Serfs*19) in TSC2 (NM_000548.4) is classified to be pathogenic according to the ACMG guideline (PVS1 + PM2_supporting + PS2). In case 7, the de novo missense mutant of c.1687G > C (p.Ala563Pro) in TSC2 (NM_000548.4) is classified to be likely pathogenic (PS2 + PM2_supporting + PP3). To our knowledge, the three variants have not been reported in previous study or recorded in the disease-related variation databases, such as HGMD or ClinVar. 3.3 Predicting the effect of variation on protein structure and function In silico protein modeling displayed that the c.1106delT variant causes a loss of 1418 amino acids, including the entire Rap-GAP domain (Fig. 2 G, Right side). PyMOL was utilized to analyze the structural consequences of A563P, in the A563P mutant model, the hydrogen bonds between lysine at position 559 and threonine at position 562 were deleted (Fig. 2 F, Right side). 4. DISCUSSION Tuberous sclerosis complex (TSC, OMIM 191100) is an autosomal dominant disorder having an incidence of 1 in 6,000 to 1 in 10,000 live births (Henske et al., 2016 ), 30% are inherited while 70% are de novo mutations. The clinical manifestations are extremely variable among patients, characterized by the development of multiple hamartomas in many internal organs including the heart, lungs, kidneys, and skin, as well as a variety of neurological features including intellectual handicap, autistic disorders, and epilepsy (Henske et al., 2016 ; Martin et al., 2017 ). Cardiac rhabdomyomas was considered as a major criteria diagnostic for TSC (Northrup et al., 2013 ), which could be detected during prenatal period. In our study, we found single or multiple CRs in 12 fetuses, most of the mothers presented with normal manifestation except case 1, of which the mother mentioned occasionally epilepsy before 6 years old. The two known genes associated with TSC were TSC1 and TSC2 , codified in the loci 9p34 and 16p13, respectively. These two genes encode Hamartin and Tuberin which form a GTPase activating protein (GAP) complex, which inhibits the mechanistic target of rapamycin (mTOR) pathway (Dibble and Manning, 2013 ; Zhang et al., 2014 ). mTOR serves as a central regulator of a number of important physiological functions, such as cell growth and proliferation, metabolism, and protein synthesis (Saxton and Sabatini, 2017 ). The mutation of TSC1 or TSC2 leads to dysfunctions of mTOR, while the increased cell growth dysregulation leads to the variety of tumors. The mutation types of TSC1 and TSC2 differ from each other, nonsense or frameshift mutations which lead to protein truncation are seen more in TSC1 , while missense mutations and rearrangements are seen more in TSC2 ( Mayer et al., 1999 ). Of the two genes, mutations of TSC2 account for more than 70 ~ 90% cases (Tyburczy et al., 2015 ). By the end of April 2025, over 6200 different allelic mutations of TSC1/TSC2 from TSC patients have been curated in Leiden Open Variation Database (LOVD, http://chromium.lovd.nl/LOVD2/TSC ), including > 1400 mutations in TSC1 and > 4700 mutations in TSC2, respectively. In our study, the mutations of TSC2 account for 75% of all mutations, but no large deletions and rearrangements were detected. Besides the reported mutations, we find three novel mutations of TSC1 and TSC 2 (Table 1 ). Table 1 States of TSC1 and TSC2 in fetus with Cardiac Rhabdomyoma No. Ages Prenatal weeks gene exon Nucleotide change Codon change Mutation type Inheritance Reported 1 31 31 TSC1 chr9:132286422–133062068, 0.78Mb deletion F N 2 29 32 TSC1 - c.363 + 2T > C - Splicing S Y 3 27 27 TSC1 18 c.2380C > T p.Q794* Nonsense S Y 4 25 31 TSC2 10 c.877A > T p.R293* Nonsense F Y 5 28 26 TSC2 11 c.1106delT p.G370Sfs*19 Frameshift S N 6 30 27 TSC2 13 c.1283_1285 del CCT p.(Ser428del) Small deletion F Y 7 29 29 TSC2 16 c.1687G > C p.A563P Missense S N 8 27 30 TSC2 21 c.2540T > C p.L847P Missense S Y 9 30 28 TSC2 31 c.3750C > A p.Y1250* Nonsense S Y 10 30 24 TSC2 34 c.4255C > T p.Q1419* Nonsense S Y 11 22 28 TSC2 36 c.4643T > C p.L1548P Missense S Y 12 26 31 TSC2 37 c.4868C > T p.T1623I Missense F Y F: familial case, S: sporadic case. N: non-reported, Y: yes. There is sufficient evidence for haploinsufficiency of TSC1 and TSC 2 genes to cause TSC. In our study, the 0.78Mb deletion at 9q34 region detected in case 1 compassing the whole TSC1 gene, which has never been reported before, but there were several smaller CNVs in the database of ClinVar. Variation ID 534520: arr[GRCh38] 9q34.13 (132896229–132928878)×1 compassing partial TSC1 deletion, and Variation ID 1696834: arr[GRCh38] 9q34.13(132691768_133013020)x1, compassing the whole TSC1 gene. A Japanese family with two sibling diagnosis of TSC carried an intragenic deletion (NC_000009.11: g.135777040_135780539del). In Case 1, the fetus presented one echogenicity in LV (7.1×4.4mm) suspicious of rhabdomyomas (Fig. 1 A), whose mother and grandmother also carried the 0.78Mb deletion at 9q34, but both of them presented no obviously phenotypes of TSC except for facial hemangioma. For the other two novel mutations in TSC2 gene, c.1106delT was a frameshift mutant, c.1687G > C was a missense mutant, both were not reported in previous literatures or HGMD database. In case 5, the variant c.1106delT was classified to be pathogenic, several factors were taken into consideration to assess its pathogenicity: (1) this frameshift variant produces a new truncated protein, while the TSC2 is definite causative gene which lead to the disease through the LOF mechanism of (PVS1); (2) the variant is absent in several common population databases, including 1000 Genomes Project, CNGBdb, ExAC, and gnomAD, supports its rarity (PM2_supporting); (3) the variant was validated to be de novo (parentally confirmed) in the patient with disease and no family history (PS2). Based on these evidences, this variant was classified as a pathogenic variant following the ACMG guidelines (PVS1 + PM2_supporting + PS2). Li et al mentioned the nearby mutation of c.1143delG (p.Thr382Profs*7) lead to the same truncated protein in 2011 (Li et al., 2011 ). The silico protein modeling revealed that the c.1106delT variant causes a loss of 1418 amino acids, including the Rap-GAP domain (Fig. 2 G). In case 7, the variant c.1687G > C (p.Ala563Pro) was classified to be likely pathogenic according to several factors, including PS2, PM2_supporting and PP3 (multiple bioinformatics tools predicted deleterious effects). The original alanine has an aliphatic side which chain contained only carbon and hydrogen atoms, while the variant residue is a proline with a rigid side chain restricting the conformation of the protein at this point. In the A563P mutant structure model, the hydrogen bonds between lysine at position 559 and threonine at position 562 were deleted. VarSite diagram illustrated various sequences and structural features of the TSC2 protein, and the newly identified A563P has CADD score above 25.60, indicating its likely deleterious nature. Taken together, it is highly probable that these two novel mutations induce disfunction of TSC2 protein. Collectively, our study identified two novel mutations (c.1687G > C and c.1106delT) in TSC2 gene and a 780 Kb-sized deletion on 9q34.13 covering the TSC1 gene, which expand the landscape of mutation spectrums of TSC1/TSC2 in TSC patients and contribute to the genetic diagnosis and counseling of this disorder. Combination of ultrasound and genetic testing can benefit effective diagnosis of the prenatal TSC cases. Declarations Ethical statement The study was approved by the Ethics Committee of Changsha Hospital for Maternal and Child Health Care (EC20230421-02). Written informed consent was obtained from parents of the patient, who agreed to join this study, with the intent of using the resulting medical data for scientific research and publication. Declaration of Competing Interest The authors report no conflicts of interest. Funding This research was supported by the National Key R&D Program of China (2022YFC2703700, 2024YFC2707100), the Clinical Medical Technology Demonstration Base for Genetic Research of Fetal Congenital Heart Disease in Hunan Province (2021SK4036), the Open Research Fund of Hunan Provincial Key Laboratory of Regional Hereditary Birth Defects Prevention and Control (HPKL2023004), and the Natural Science Foundation of Hunan Province (2025JJ50517, 2023JJ30725). Author Contribution Jing Liu and Zhuo Li had major roles in the design of the study. Jing Liu and Jun He drafted the manuscript. Lin Zhou, Ying Peng, Haoxian Li and Zonglei Zhang performed the functional experiments and analyzed the data. Wanglan Tang, Yingchun Luo, Jing Chen and Shihao Zhou analyzed the clinical data. Zhuo Li and Jun He are the corresponding authors of this manuscript. All authors read and approved the final manuscript. Acknowledgments We thank these families for their contribution. We also thank laboratory staff at Prenatal Diagnosis Center of Hunan Province, Hunan provincial Maternal and Child Health Care Hospital, Jiahui genetic hospital. Data Availability The sequencing data has been deposited in the GSA database (https://ngdc.cncb.ac.cn/gsa-human/, accession number HRA007725) and will be shared at request of qualified investigators. References Chen, J., Wang, J., Sum, H., Gu, X., Hao, X., Fu, Y., et al. (2019). Fetal cardiac tumor: echocardiography, clinical outcome and genetic analysis in 53 cases. Ultrasound in Obstetrics & Gynecology 54(1) , 103-109. doi: 10.1002/uog.19108. Dibble, C.C., and Manning, B.D. (2013). Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nature Cell Biology 15(6) , 555-564. doi: 10.1038/ncb2763. Ekmekci, E., Ozkan, B.O., Yildiz, M.S., and Kocakaya, B. (2018). Prenatal diagnosis of fetal cardiac rhabdomyoma associated with tuberous sclerosis: A case report. Case reports in women's health 19 , e00070. doi: 10.1016/j.crwh.2018.e00070. Henske, E.P., Józwiak, S., Kingswood, J.C., Sampson, J.R., and Thiele, E.A. (2016). Tuberous sclerosis complex. Nature Reviews Disease Primers 2 , 18. doi: 10.1038/nrdp.2016.35. Isaacs, H. (2004). Fetal and neonatal cardiac tumors. Pediatric Cardiology 25(3) , 252-273. doi: 10.1007/s00246-003-0590-4. Józwiak, S., and Kotulska, K. (2006). Are all prenatally diagnosed multiple cardiac rhabdomyomas a sign of tuberous sclerosis? Prenatal Diagnosis 26(9) , 867-869. doi: 10.1002/pd.1506. Li, W., Zhou, L.-h., Gao, B.-d., Li, L.-y., Zhong, C.-g., Gong, F., et al. (2011). Mutation screening and prenatal diagnosis of tuberous sclerosis complex. Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 28(4) , 361-366. doi: 10.3760/cma.j.issn.1003-9406.2011.04.001. Martin, K.R., Zhou, W.D., Bowman, M.J., Shih, J., Au, K.S., Dittenhafer-Reed, K.E., et al. (2017). The genomic landscape of tuberous sclerosis complex. Nature Communications 8 , 13. doi: 10.1038/ncomms15816. Mayer, K., Ballhausen, W., and Rott, H.D. (1999). Mutation screening of the entire coding regions of the TSC1 and the TSC2 gene with the protein truncation test PTT identifies frequent splicing defects. Human Mutation 14(5) , 401-411. doi: 10.1002/(sici)1098-1004(199911)14:53.0.Co;2-r. Northrup, H., Krueger, D.A., Northrup, H., Krueger, D.A., Roberds, S., Smith, K., et al. (2013). Tuberous Sclerosis Complex Diagnostic Criteria Update: Recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference. Pediatric Neurology 49(4) , 243-254. doi: 10.1016/j.pediatrneurol.2013.08.001. Pavlicek, J., Klaskova, E., Kapralova, S., Prochazka, M., Vrtel, R., Gruszka, T., et al. (2021). Fetal heart rhabdomyomatosis: a single-center experience. Journal of Maternal-Fetal & Neonatal Medicine 34(5) , 701-707. doi: 10.1080/14767058.2019.1613365. Saxton, R.A., and Sabatini, D.M. (2017). mTOR Signaling in Growth, Metabolism, and Disease. Cell 168(6) , 960-976. doi: 10.1016/j.cell.2017.02.004. Tyburczy, M.E., Dies, K.A., Glass, J., Camposano, S., Chekaluk, Y., Thorner, A.R., et al. (2015). Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing. Plos Genetics 11(11) , 17. doi: 10.1371/journal.pgen.1005637. Yuan, S.M. (2017). Fetal Primary Cardiac Tumors During Perinatal Period. Pediatrics and Neonatology 58(3) , 205-210. doi: 10.1016/j.pedneo.2016.07.004. Zhang, Y.N., Nicholatos, J., Dreier, J.R., Ricoult, S.J.H., Widenmaier, S.B., Hotamisligil, G.S., et al. (2014). Coordinated regulation of protein synthesis and degradation by mTORC1. Nature 513(7518) , 440-+. doi: 10.1038/nature13492. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7319816","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501611956,"identity":"c440ec1e-f522-4ba0-8053-4e3694103901","order_by":0,"name":"Jing Liu","email":"","orcid":"","institution":"Changsha Hospital for Maternal \u0026 Child Health Care Affiliated to Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Liu","suffix":""},{"id":501611957,"identity":"a7f83c37-8eb6-4a45-911e-b945d9589d61","order_by":1,"name":"Jun He","email":"","orcid":"","institution":"Changsha Hospital for Maternal \u0026 Child Health Care Affiliated to Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"He","suffix":""},{"id":501611958,"identity":"7a37d7ae-f3b1-46b2-b269-b12854c3e265","order_by":2,"name":"Wanglan Tang","email":"","orcid":"","institution":"Maternal and Child Health Hospital of Hunan Province","correspondingAuthor":false,"prefix":"","firstName":"Wanglan","middleName":"","lastName":"Tang","suffix":""},{"id":501611959,"identity":"211b4beb-1a08-4a58-9a1b-a7b10034b346","order_by":3,"name":"Jing Chen","email":"","orcid":"","institution":"Maternal and Child Health Hospital of Hunan Province","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Chen","suffix":""},{"id":501611960,"identity":"de015297-6aa1-40e0-97ae-81d6dea9693a","order_by":4,"name":"Yingchun Luo","email":"","orcid":"","institution":"Maternal and Child Health Hospital of Hunan Province","correspondingAuthor":false,"prefix":"","firstName":"Yingchun","middleName":"","lastName":"Luo","suffix":""},{"id":501611961,"identity":"38ed0685-de01-4a2a-bceb-97ec580da1ad","order_by":5,"name":"Haoxian Li","email":"","orcid":"","institution":"Jiangmen maternity and child health care hospital","correspondingAuthor":false,"prefix":"","firstName":"Haoxian","middleName":"","lastName":"Li","suffix":""},{"id":501611962,"identity":"8050d725-66d2-401f-9548-77456497aa8c","order_by":6,"name":"Zhuo Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACPmYGNiBlwyDBDuYzE9bCBtGSxiDBTLQWMGI4TIoWdvZnj3kqzttLNnMnv2CosE5sYD97gJDD0o15ztxOnM3Mu82C4Ux6YgNPXgIhLcekedtuJ8gBtRgwth1ObJDgMSCghbENqOWcPUTLP6K0MLMBtRxgBDps8wPGBqK0sLFJzjmTnDizmXcbQ8KxdOM2nhz8Wvj5jz+TeFNhZy9xvHfzhw811rL97Gfwa0GxUSKBARJNRAPmD6SoHgWjYBSMgpEDALHdN13C3LhCAAAAAElFTkSuQmCC","orcid":"","institution":"Central South University","correspondingAuthor":true,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Li","suffix":""},{"id":501611963,"identity":"5d4035e1-0a79-4a07-8987-5e1fe2a12e66","order_by":7,"name":"Lingqian Wu","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lingqian","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-08-07 14:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7319816/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7319816/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12884-025-08365-7","type":"published","date":"2025-11-24T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89541126,"identity":"6654c5f6-76c6-44d4-b1d1-2738ff8e3693","added_by":"auto","created_at":"2025-08-21 06:31:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1335071,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of prenatal ultrasound examination for part of these fetuses.\u003c/p\u003e\n\u003cp\u003e(A) Fetal echocardiography at 31weeks gestational age in case 1 showed one echogenicity in LV (7.1×4.4mm) suspicious of rhabdomyomas. (B) Fetal echocardiography at 32 weeks gestational age in case 2 showed one echogenicity in LV (9.3×5.9×6.6mm). (C) Fetal echocardiography at 24 weeks gestational age in case 10 showed one large echogenicity in RV (16.1×10.9×12.5mm). (D) Fetal echocardiography at 24 weeks gestational age in case 10 showed two echogenicity in LV (7.0×4.2×4.8mm) and (8.3×4.6×6.2mm). (E) Fetal echocardiography at 28 weeks gestational age in case 11 showed multiple sizes of high echo, the larger is located in the left ventricular near apex. (F) Irregular low echo in the brain in Case 12.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7319816/v1/b6cada20a405f90f2f3d7c33.png"},{"id":89542159,"identity":"8590f979-1cc4-4151-a3d8-145cfb863dbe","added_by":"auto","created_at":"2025-08-21 06:39:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1077712,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of genetic testing results of \u003cem\u003eTSC1/2\u003c/em\u003e in fetuses with CR.\u003c/p\u003e\n\u003cp\u003e(A) The ratio of the reported sites to the novel sites in \u003cem\u003eTSC1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003egenes. (B) Molecular consequences of mutations classified as pathogenic or possibly pathogenic. (C) The ratio of the mutants in \u003cem\u003eTSC1\u003c/em\u003e and\u003cem\u003e TSC2\u003c/em\u003egenes. (D)The proportion of the origin of \u003cem\u003eTSC1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e variation in fetuses with CR. (E) The ideograph of chromosome 9 in case 1 with 9q34.13-34.3 loss. CNV seq detects a copy number loss at 9qter, the red region shows 9q34.13 (arr[hg19] 9q34.13 (132286422-133062068)×1). (F) Sanger sequencing confirms the \u003cem\u003ede\u003c/em\u003e \u003cem\u003enovo\u003c/em\u003e mutant of c.1687G\u0026gt;C in \u003cem\u003eTSC2\u003c/em\u003e gene in case 7. In the A563Pmutant model was presented at the right side, the hydrogen bonds between lysine at position 559 and threonine at position 562 were deleted. (G) Sanger sequencing confirms the \u003cem\u003ede\u003c/em\u003e \u003cem\u003enovo\u003c/em\u003e mutant of c.1106delT in \u003cem\u003eTSC2\u003c/em\u003e gene in case 5 and its parent. The silico protein modeling revealed that the c.1106delT variant causes a loss of 1418 amino acids, including the Rap-GAP domain.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7319816/v1/d78a99befd296cea37285251.png"},{"id":97178694,"identity":"95a5431d-6488-435b-b8d4-d8dcc50172c2","added_by":"auto","created_at":"2025-12-01 16:12:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3072488,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7319816/v1/736995f4-b220-4090-99a9-dfdc3a1c2c1d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eWhole Exome Sequencing in Fetal Cardiac Rhabdomyoma Detected by Ultrasonography: An Analysis of 12 Cases\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eRhabdomyoma is the most prevalent fetal cardiac tumor and can be detected via routine prenatal ultrasound from the 15th gestational week onward (Ekmekci et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The incidence of this tumor ranges from 0.02 to 0.17% in liveborn infants and 0.12% in prenatal fetal studies (Isaacs, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yuan, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The majority of fetal cardiac rhabdomyomas (CRs) are associated with tuberous sclerosis (TSC), an autosomal dominant disorder characterized by cellular and tissue dysplasia in multiple organs, which can lead to neurologic and behavioral abnormalities (J\u0026oacute;zwiak and Kotulska, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pavlicek et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The two known genes, TSC1 located at 9q34.3 and TSC2 located at 16p13.3, are crucial disease-causing genes for TSC. Loss of function of TSC1/TSC2 leads to hyperactivity of mTORC1, which drives abnormal cellular growth and tumorigenesis (Dibble and Manning, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNext-generation sequencing (NGS) offers deep, high-throughput, and parallel genome sequencing. With its expanding application in prenatal diagnosis, the diagnostic rate for fetuses with CRs has significantly improved, thereby enhancing the capacity for genetic counseling. In the present study, Trio-WES was conducted to identify the genetic causes in 12 fetuses with sonographically detected CRs, and TSC was subsequently diagnosed in all 12 cases. We integrated detailed parental phenotypes, echocardiography, and genetic analysis to conduct a comprehensive assessment of these 12 CR fetuses. The combination of ultrasound and genetic tests enabled us to effectively diagnose prenatal TSC cases. We also identified one novel deletion involving the TSC1 gene and two novel mutations in the TSC2 gene, thereby expanding the spectrum of TSC mutations.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Ethical compliance\u003c/h2\u003e\u003cp\u003e The research was in compliance with the Helsinki Declaration and approved by the Ethics Committee of Changsha Hospital for Maternal and Child Health Care.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Subjects and Sample Collection\u003c/h2\u003e\u003cp\u003eIn this study, we included twelve prenatal cases of cardiac rhabdomyoma which were collected in the prenatal Diagnosis Central of Hunan Provincial Maternal and Child Health Care Hospital from 2017 to 2022. The reasons for prenatal diagnosis were cardiac rhabdomyoma presented in all of these fetuses found by ultrasound. The results of prenatal ultrasound examination are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for part of these fetuses. After obtained the informed consents, amniocentesis cells of each fetuses were collected via amniocentesis for genetic testing, and peripheral blood was collected from parents to determine inheritance patterns of any mutations. Karyotype and SNP-array/CNV seq were performed simultaneously with NGS or before.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Next-Generation Sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA samples from 12 fetuses and their respective parents were isolated using the Qiagen DNA Blood Midi/Mini kit (Qiagen GmbH, Germany). Extracted DNA underwent enzymatic fragmentation targeting\u0026thinsp;~\u0026thinsp;200-bp segments, followed by end repair and 3'-terminal adenine (A) addition. Indexed adapters were ligated to the resulting fragments, and products of approximately 320 bp were size-selected using XP beads. PCR amplification was subsequently performed. Enrichment of exonic regions was achieved through hybridization capture with the Nano WES system (following manufacturer specifications). Captured libraries were eluted, PCR-amplified, and subjected to quality control: enrichment efficiency was assessed via qPCR, while fragment size distribution and concentration were determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA). Final libraries were sequenced on the Illumina NovaSeq6000 platform (150-bp paired-end reads). Base calling and raw data generation from image files utilized CASAVA (v1.82). Variants identified were validated by Sanger sequencing in trios.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Data analysis\u003c/h2\u003e\u003cp\u003eRaw sequencing reads underwent alignment to the GRCh37/hg19 reference genome via the Burrows-Wheeler Aligner (BWA). PCR duplicates were subsequently eliminated using Picard v1.57. Variant pathogenicity classification adhered to American College of Medical Genetics and Genomics (ACMG) standards, categorizing variants as: Pathogenic, Likely Pathogenic, Benign, Likely Benign, or Variants of Uncertain Significance (VUS). For copy number variation (CNV) detection, the Sprinkle pipeline (BerryGenomics) was implemented. This integrated:(i) XHMM-based principal component analysis for noise reduction, (ii) GC-content and bias adjustment through CNV Kit's fix module, (iii) Exon-level and large-segment CNV identification via copy number calculation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Predicted structure models\u003c/h2\u003e\u003cp\u003eTo assess the structural and functional implications of the G370S and A563P variants in TSC2, computational modeling was performed using VarSite and PyMOL. The wild-type TSC2 protein complex structure (PDB ID: not specified) was retrieved from the Protein Data Bank Europe (PDBe) as the reference framework. PyMOL facilitated: (i) Structural alignment between wild-type and mutant conformations, (ii) In silico mutagenesis to generate variant models, (iii) Visualization of atomic-level perturbations. This integrated approach enabled systematic evaluation of mutation-induced structural perturbations and their potential functional consequences.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 General Information and Prenatal ultrasound Results\u003c/h2\u003e\u003cp\u003eA total of 12 fetuses with CR were included in the study. The average age of the 12 pregnant women was 27.83 years (range 22\u0026ndash;31years). The mean GA of CRs detected during routine ultrasound scans was 28.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32 weeks (range 24\u0026ndash;32 weeks). All CRs were detected in the ventricle, interventricular septum, and the atrium, especially frequent in the left ventricle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All subjects did not detect arrhythmia during the pregnancy. Beside CRs, irregular low echo in the brain was detected in Case 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Identification of \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e Variants\u003c/h2\u003e\u003cp\u003eAfter genetic testing, all 12 fetuses showed normal karyotypes and CNV, but they all carried \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e variants (100%). Roughly 25% of these variants had not been reported previously (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The P/LP variant spectrum in \u003cem\u003eTSC1\u003c/em\u003e/\u003cem\u003eTSC2\u003c/em\u003e includes 4 (33%) nonsense, 4 (33%) missense, 2 (17%) frameshift, 1 (8%) splice, 1 (8%) small deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Mutants of \u003cem\u003eTSC1\u003c/em\u003e account for 25% (3/12) and \u003cem\u003eTSC2\u003c/em\u003e account for 75% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). One-thirds of these variants were \u003cem\u003ede novo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). CNV seq results revealed a 780 Kb-sized deletion on 9q34.13 (arr[hg19] 9q34.13 (132286422\u0026ndash;133062068)\u0026times;1) in case 1, encompassing the entire of \u003cem\u003eTSC1\u003c/em\u003e gene, which was inherited from mother (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In case 5, the \u003cem\u003ede novo\u003c/em\u003e frameshift mutant of c.1106delT (p.Gln370Serfs*19) in \u003cem\u003eTSC2\u003c/em\u003e (NM_000548.4) is classified to be pathogenic according to the ACMG guideline (PVS1\u0026thinsp;+\u0026thinsp;PM2_supporting\u0026thinsp;+\u0026thinsp;PS2). In case 7, the \u003cem\u003ede novo\u003c/em\u003e missense mutant of c.1687G\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Ala563Pro) in \u003cem\u003eTSC2\u003c/em\u003e (NM_000548.4) is classified to be likely pathogenic (PS2\u0026thinsp;+\u0026thinsp;PM2_supporting\u0026thinsp;+\u0026thinsp;PP3). To our knowledge, the three variants have not been reported in previous study or recorded in the disease-related variation databases, such as HGMD or ClinVar.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Predicting the effect of variation on protein structure and function\u003c/h2\u003e\u003cp\u003eIn silico protein modeling displayed that the c.1106delT variant causes a loss of 1418 amino acids, including the entire Rap-GAP domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, Right side). PyMOL was utilized to analyze the structural consequences of A563P, in the A563P mutant model, the hydrogen bonds between lysine at position 559 and threonine at position 562 were deleted (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Right side).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eTuberous sclerosis complex (TSC, OMIM 191100) is an autosomal dominant disorder having an incidence of 1 in 6,000 to 1 in 10,000 live births (Henske et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), 30% are inherited while 70% are \u003cem\u003ede\u003c/em\u003e novo mutations. The clinical manifestations are extremely variable among patients, characterized by the development of multiple hamartomas in many internal organs including the heart, lungs, kidneys, and skin, as well as a variety of neurological features including intellectual handicap, autistic disorders, and epilepsy (Henske et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Martin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cardiac rhabdomyomas was considered as a major criteria diagnostic for TSC (Northrup et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which could be detected during prenatal period. In our study, we found single or multiple CRs in 12 fetuses, most of the mothers presented with normal manifestation except case 1, of which the mother mentioned occasionally epilepsy before 6 years old.\u003c/p\u003e\u003cp\u003eThe two known genes associated with TSC were \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e, codified in the loci 9p34 and 16p13, respectively. These two genes encode Hamartin and Tuberin which form a GTPase activating protein (GAP) complex, which inhibits the mechanistic target of rapamycin (mTOR) pathway (Dibble and Manning, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). mTOR serves as a central regulator of a number of important physiological functions, such as cell growth and proliferation, metabolism, and protein synthesis (Saxton and Sabatini, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The mutation of \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e leads to dysfunctions of mTOR, while the increased cell growth dysregulation leads to the variety of tumors. The mutation types of \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e differ from each other, nonsense or frameshift mutations which lead to protein truncation are seen more in \u003cem\u003eTSC1\u003c/em\u003e, while missense mutations and rearrangements are seen more in \u003cem\u003eTSC2 (\u003c/em\u003eMayer et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Of the two genes, mutations of \u003cem\u003eTSC2\u003c/em\u003e account for more than 70\u0026thinsp;~\u0026thinsp;90% cases (Tyburczy et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). By the end of April 2025, over 6200 different allelic mutations of \u003cem\u003eTSC1/TSC2\u003c/em\u003e from TSC patients have been curated in Leiden Open Variation Database (LOVD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://chromium.lovd.nl/LOVD2/TSC\u003c/span\u003e\u003cspan address=\"http://chromium.lovd.nl/LOVD2/TSC\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), including\u0026thinsp;\u0026gt;\u0026thinsp;1400 mutations in TSC1 and \u0026gt;\u0026thinsp;4700 mutations in TSC2, respectively. In our study, the mutations of \u003cem\u003eTSC2\u003c/em\u003e account for 75% of all mutations, but no large deletions and rearrangements were detected. Besides the reported mutations, we find three novel mutations of \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC\u003c/em\u003e2 (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\u003eStates of \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e in fetus with Cardiac Rhabdomyoma\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" 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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAges\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePrenatal weeks\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003egene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eexon\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNucleotide change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCodon change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMutation type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eInheritance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eReported\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e\u003cp\u003e\u003cb\u003echr9:132286422\u0026ndash;133062068, 0.78Mb deletion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.363\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSplicing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.2380C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.Q794*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNonsense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.877A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.R293*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNonsense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003ec.1106delT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.G370Sfs*19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eFrameshift\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.1283_1285 del CCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.(Ser428del)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSmall deletion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003ec.1687G\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.A563P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMissense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.2540T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.L847P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMissense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.3750C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.Y1250*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNonsense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.4255C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.Q1419*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNonsense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.4643T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.L1548P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMissense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec.4868C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ep.T1623I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMissense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003eF: familial case, S: sporadic case.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003eN: non-reported, Y: yes.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThere is sufficient evidence for haploinsufficiency of \u003cem\u003eTSC1\u003c/em\u003eand \u003cem\u003eTSC\u003c/em\u003e2 genes to cause TSC. In our study, the 0.78Mb deletion at 9q34 region detected in case 1 compassing the whole \u003cem\u003eTSC1\u003c/em\u003e gene, which has never been reported before, but there were several smaller CNVs in the database of ClinVar. Variation ID 534520: arr[GRCh38] 9q34.13 (132896229\u0026ndash;132928878)\u0026times;1 compassing partial \u003cem\u003eTSC1\u003c/em\u003e deletion, and Variation ID 1696834: arr[GRCh38] 9q34.13(132691768_133013020)x1, compassing the whole \u003cem\u003eTSC1\u003c/em\u003e gene. A Japanese family with two sibling diagnosis of TSC carried an intragenic deletion (NC_000009.11: g.135777040_135780539del). In Case 1, the fetus presented one echogenicity in LV (7.1\u0026times;4.4mm) suspicious of rhabdomyomas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), whose mother and grandmother also carried the 0.78Mb deletion at 9q34, but both of them presented no obviously phenotypes of TSC except for facial hemangioma.\u003c/p\u003e\u003cp\u003eFor the other two novel mutations in \u003cem\u003eTSC2\u003c/em\u003e gene, c.1106delT was a frameshift mutant, c.1687G\u0026thinsp;\u0026gt;\u0026thinsp;C was a missense mutant, both were not reported in previous literatures or HGMD database. In case 5, the variant c.1106delT was classified to be pathogenic, several factors were taken into consideration to assess its pathogenicity: (1) this frameshift variant produces a new truncated protein, while the \u003cem\u003eTSC2\u003c/em\u003e is definite causative gene which lead to the disease through the LOF mechanism of (PVS1); (2) the variant is absent in several common population databases, including 1000 Genomes Project, CNGBdb, ExAC, and gnomAD, supports its rarity (PM2_supporting); (3) the variant was validated to be de \u003cem\u003enovo\u003c/em\u003e (parentally confirmed) in the patient with disease and no family history (PS2). Based on these evidences, this variant was classified as a pathogenic variant following the ACMG guidelines (PVS1\u0026thinsp;+\u0026thinsp;PM2_supporting\u0026thinsp;+\u0026thinsp;PS2). Li et al mentioned the nearby mutation of c.1143delG (p.Thr382Profs*7) lead to the same truncated protein in 2011 (Li et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The silico protein modeling revealed that the c.1106delT variant causes a loss of 1418 amino acids, including the Rap-GAP domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In case 7, the variant c.1687G\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Ala563Pro) was classified to be likely pathogenic according to several factors, including PS2, PM2_supporting and PP3 (multiple bioinformatics tools predicted deleterious effects). The original alanine has an aliphatic side which chain contained only carbon and hydrogen atoms, while the variant residue is a proline with a rigid side chain restricting the conformation of the protein at this point. In the A563P mutant structure model, the hydrogen bonds between lysine at position 559 and threonine at position 562 were deleted. VarSite diagram illustrated various sequences and structural features of the \u003cem\u003eTSC2\u003c/em\u003e protein, and the newly identified A563P has CADD score above 25.60, indicating its likely deleterious nature. Taken together, it is highly probable that these two novel mutations induce disfunction of \u003cem\u003eTSC2\u003c/em\u003e protein.\u003c/p\u003e\u003cp\u003eCollectively, our study identified two novel mutations (c.1687G\u0026thinsp;\u0026gt;\u0026thinsp;C and c.1106delT) in \u003cem\u003eTSC2 gene\u003c/em\u003e and a 780 Kb-sized deletion on 9q34.13 covering the \u003cem\u003eTSC1\u003c/em\u003e gene, which expand the landscape of mutation spectrums of \u003cem\u003eTSC1/TSC2\u003c/em\u003e in TSC patients and contribute to the genetic diagnosis and counseling of this disorder. Combination of ultrasound and genetic testing can benefit effective diagnosis of the prenatal TSC cases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical statement\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Changsha Hospital for Maternal and Child Health Care (EC20230421-02). Written informed consent was obtained from parents of the patient, who agreed to join this study, with the intent of using the resulting medical data for scientific research and publication.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\n\u003cp\u003eThe authors report no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was supported by the National Key R\u0026amp;D Program of China (2022YFC2703700, 2024YFC2707100), the Clinical Medical Technology Demonstration Base for Genetic Research of Fetal Congenital Heart Disease in Hunan Province (2021SK4036), the Open Research Fund of Hunan Provincial Key Laboratory of Regional Hereditary Birth Defects Prevention and Control (HPKL2023004), and the Natural Science Foundation of Hunan Province (2025JJ50517, 2023JJ30725).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJing Liu and Zhuo Li had major roles in the design of the study. Jing Liu and Jun He drafted the manuscript. Lin Zhou, Ying Peng, Haoxian Li and Zonglei Zhang performed the functional experiments and analyzed the data. Wanglan Tang, Yingchun Luo, Jing Chen and Shihao Zhou analyzed the clinical data. Zhuo Li and Jun He are the corresponding authors of this manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank these families for their contribution. We also thank laboratory staff at Prenatal Diagnosis Center of Hunan Province, Hunan provincial Maternal and Child Health Care Hospital, Jiahui genetic hospital.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe sequencing data has been deposited in the GSA database (https://ngdc.cncb.ac.cn/gsa-human/, accession number HRA007725) and will be shared at request of qualified investigators.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChen, J., Wang, J., Sum, H., Gu, X., Hao, X., Fu, Y., et al. (2019). Fetal cardiac tumor: echocardiography, clinical outcome and genetic analysis in 53 cases. \u003cem\u003eUltrasound in Obstetrics \u0026amp; Gynecology\u003c/em\u003e 54(1)\u003cstrong\u003e,\u003c/strong\u003e 103-109. doi: 10.1002/uog.19108.\u003c/li\u003e\n \u003cli\u003eDibble, C.C., and Manning, B.D. (2013). 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Fetal and neonatal cardiac tumors. \u003cem\u003ePediatric Cardiology\u003c/em\u003e 25(3)\u003cstrong\u003e,\u003c/strong\u003e 252-273. doi: 10.1007/s00246-003-0590-4.\u003c/li\u003e\n \u003cli\u003eJ\u0026oacute;zwiak, S., and Kotulska, K. (2006). Are all prenatally diagnosed multiple cardiac rhabdomyomas a sign of tuberous sclerosis? \u003cem\u003ePrenatal Diagnosis\u003c/em\u003e 26(9)\u003cstrong\u003e,\u003c/strong\u003e 867-869. doi: 10.1002/pd.1506.\u003c/li\u003e\n \u003cli\u003eLi, W., Zhou, L.-h., Gao, B.-d., Li, L.-y., Zhong, C.-g., Gong, F., et al. (2011). Mutation screening and prenatal diagnosis of tuberous sclerosis complex. \u003cem\u003eZhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics\u003c/em\u003e 28(4)\u003cstrong\u003e,\u003c/strong\u003e 361-366. doi: 10.3760/cma.j.issn.1003-9406.2011.04.001.\u003c/li\u003e\n \u003cli\u003eMartin, K.R., Zhou, W.D., Bowman, M.J., Shih, J., Au, K.S., Dittenhafer-Reed, K.E., et al. (2017). The genomic landscape of tuberous sclerosis complex. \u003cem\u003eNature Communications\u003c/em\u003e 8\u003cstrong\u003e,\u003c/strong\u003e 13. doi: 10.1038/ncomms15816.\u003c/li\u003e\n \u003cli\u003eMayer, K., Ballhausen, W., and Rott, H.D. (1999). Mutation screening of the entire coding regions of the \u0026lt;i\u0026gt;TSC1\u0026lt;/i\u0026gt; and the \u0026lt;i\u0026gt;TSC2\u0026lt;/i\u0026gt; gene with the protein truncation test PTT identifies frequent splicing defects. \u003cem\u003eHuman Mutation\u003c/em\u003e 14(5)\u003cstrong\u003e,\u003c/strong\u003e 401-411. doi: 10.1002/(sici)1098-1004(199911)14:5\u0026lt;401::Aid-humu6\u0026gt;3.0.Co;2-r.\u003c/li\u003e\n \u003cli\u003eNorthrup, H., Krueger, D.A., Northrup, H., Krueger, D.A., Roberds, S., Smith, K., et al. (2013). Tuberous Sclerosis Complex Diagnostic Criteria Update: Recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference. \u003cem\u003ePediatric Neurology\u003c/em\u003e 49(4)\u003cstrong\u003e,\u003c/strong\u003e 243-254. doi: 10.1016/j.pediatrneurol.2013.08.001.\u003c/li\u003e\n \u003cli\u003ePavlicek, J., Klaskova, E., Kapralova, S., Prochazka, M., Vrtel, R., Gruszka, T., et al. (2021). Fetal heart rhabdomyomatosis: a single-center experience. \u003cem\u003eJournal of Maternal-Fetal \u0026amp; Neonatal Medicine\u003c/em\u003e 34(5)\u003cstrong\u003e,\u003c/strong\u003e 701-707. doi: 10.1080/14767058.2019.1613365.\u003c/li\u003e\n \u003cli\u003eSaxton, R.A., and Sabatini, D.M. (2017). mTOR Signaling in Growth, Metabolism, and Disease. \u003cem\u003eCell\u003c/em\u003e 168(6)\u003cstrong\u003e,\u003c/strong\u003e 960-976. doi: 10.1016/j.cell.2017.02.004.\u003c/li\u003e\n \u003cli\u003eTyburczy, M.E., Dies, K.A., Glass, J., Camposano, S., Chekaluk, Y., Thorner, A.R., et al. (2015). Mosaic and Intronic Mutations in \u0026lt;i\u0026gt;TSC1\u0026lt;/i\u0026gt;/\u0026lt;i\u0026gt;TSC2\u0026lt;/i\u0026gt; Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing. \u003cem\u003ePlos Genetics\u003c/em\u003e 11(11)\u003cstrong\u003e,\u003c/strong\u003e 17. doi: 10.1371/journal.pgen.1005637.\u003c/li\u003e\n \u003cli\u003eYuan, S.M. (2017). Fetal Primary Cardiac Tumors During Perinatal Period. \u003cem\u003ePediatrics and Neonatology\u003c/em\u003e 58(3)\u003cstrong\u003e,\u003c/strong\u003e 205-210. doi: 10.1016/j.pedneo.2016.07.004.\u003c/li\u003e\n \u003cli\u003eZhang, Y.N., Nicholatos, J., Dreier, J.R., Ricoult, S.J.H., Widenmaier, S.B., Hotamisligil, G.S., et al. (2014). Coordinated regulation of protein synthesis and degradation by mTORC1. \u003cem\u003eNature\u003c/em\u003e 513(7518)\u003cstrong\u003e,\u003c/strong\u003e 440-+. doi: 10.1038/nature13492.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cardiac rhabdomyoma, TSC1, TSC2, Trio-WES, prenatal diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-7319816/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7319816/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMost of fetal cardiac rhabdomyomas (CRs) are associated with tuberous sclerosis (TSC), an autosomal dominant inherited disorder caused by mutations in the \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e genes. In this study, we conducted a comprehensive analysis integrating prenatal echocardiographic findings, parental phenotypic characteristics, and genetic profiles of fetuses with CR.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003e12 fetuses with sonographically identified CR were included. Karyotype and SNP-array/CNV-seq were performed simultaneously with Trio-WES.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAll CRs were observed in the ventricle, interventricular septum, and the atrium, while the left ventricle were the most common areas. All subjects did not detect arrhythmia during pregnancy. Beside CRs, irregular low echo in the brain was detected in Case 12. \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e variants were identified in all 12 fetuses (100%). Mutants of \u003cem\u003eTSC1\u003c/em\u003e account for 25% (3/12) and \u003cem\u003eTSC2\u003c/em\u003e account for 75%(9/12). Two-thirds of these variants were \u003cem\u003ede novo\u003c/em\u003e. The P/LP variant spectrum in \u003cem\u003eTSC1\u003c/em\u003e/\u003cem\u003eTSC2\u003c/em\u003e includes 4 (34%) nonsense, 4 (33%) missense, 2 (17%) frameshift, 1 (8%) splice and 1 (8%) small deletion. A 780kb deletion in 9q34.13 (arr[hg19] 9q34.13 (132286422\u0026ndash;133062068)\u0026times;1) encompassing the entire of \u003cem\u003eTSC1\u003c/em\u003e gene, and the other two \u003cem\u003ede novo\u003c/em\u003e mutants of c.1687G\u0026thinsp;\u0026gt;\u0026thinsp;C and c.1106delT in \u003cem\u003eTSC2\u003c/em\u003e gene had not been reported previously.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eCombination of ultrasound and genetic testing can effectively diagnose the prenatal cases of TSC. Three novel mutations in \u003cem\u003eTSC\u003c/em\u003e genes enlarge the mutation spectrum of \u003cem\u003eTSC\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Whole Exome Sequencing in Fetal Cardiac Rhabdomyoma Detected by Ultrasonography: An Analysis of 12 Cases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 06:31:13","doi":"10.21203/rs.3.rs-7319816/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-24T06:22:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T13:01:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298676833866503881841970065828233395598","date":"2025-09-14T22:52:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259104822685725373266963269062921390137","date":"2025-09-10T00:49:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-16T02:15:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27564347438608092814788136886168800659","date":"2025-08-13T22:51:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-12T06:44:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-12T05:58:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-11T09:36:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-10T13:24:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pregnancy and Childbirth","date":"2025-08-10T13:22:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3ab859c-4be9-4d30-ad84-f05c71b9953e","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:06:38+00:00","versionOfRecord":{"articleIdentity":"rs-7319816","link":"https://doi.org/10.1186/s12884-025-08365-7","journal":{"identity":"bmc-pregnancy-and-childbirth","isVorOnly":false,"title":"BMC Pregnancy and Childbirth"},"publishedOn":"2025-11-24 15:57:56","publishedOnDateReadable":"November 24th, 2025"},"versionCreatedAt":"2025-08-21 06:31:13","video":"","vorDoi":"10.1186/s12884-025-08365-7","vorDoiUrl":"https://doi.org/10.1186/s12884-025-08365-7","workflowStages":[]},"version":"v1","identity":"rs-7319816","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7319816","identity":"rs-7319816","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}