Cemento-Osseous Dysplasia With a NOTCH4 Mutation: a Case Report

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Breimer, Nard G. Janssen, Anne M.L. Jansen, Pieter J. Slootweg This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6689921/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 Purpose Cemento-osseous dysplasia (COD) is a benign fibro-osseous lesion of the jaw that poses diagnostic challenges due to overlapping features with other entities, particularly cemento-ossifying fibroma (COF). While recent studies have identified mutations in the RAS-MAPK pathway in COD, its broader molecular profile remains poorly understood. Here, we report a case of COD harboring a novel NOTCH4 mutation, expanding the spectrum of genetic alterations associated with this lesion. Methods We present a case of a 32-year-old woman with an incidental lesion in the right mandibular angle. The lesion was surgically enucleated and analyzed histologically. Next-generation sequencing (TSO500 panel) was performed. Results Radiographic evaluation showed a lesion without sharp demarcation. Histopathology shows a fibro-osseous lesion with interconnected bony trabeculae lacking osteoblastic rimming, consistent with COD. Molecular analysis detected a NOTCH4 mutation. Conclusion This case identifies a novel NOTCH4 mutation in COD, suggesting possible involvement of the Notch signaling pathway in its pathogenesis. Incorporating molecular profiling into the diagnostic workflow may aid in distinguishing COD from COF and enhance our understanding of fibro-osseous lesions of the jaw. Figures Figure 1 Figure 2 Figure 3 Introduction Fibro-osseous lesions of the jaw pose diagnostic challenges due to their rarity and overlapping histopathological and radiological features, requiring combined expertise in pathology, radiology, and clinical evaluation for accurate classification [ 1 ]. One important entity within this group is cemento-osseous dysplasia (COD), a benign lesion in the tooth-bearing areas of the jaws. It is classified into four subtypes, i.e., periapical, focal, florid, or familial florid cemento-osseous dysplasia subtype, based on the location and distribution of the lesions. COD must be distinguished from cemento-ossifying fibroma (COF) [ 2 ]. A key distinguishing feature lies in radiological assessment, particularly the nature of the lesion's interface with adjacent bone. A sharply demarcated lesion favors the diagnosis of COF, whereas a more diffuse boundary is suggestive of COD. In recent years, molecular analyses have substantially advanced our understanding and classification of fibro-osseous tumors of the jaw by identifying distinct genetic alterations. For instance, identifying SATB2 rearrangements appears to be highly specific for psammomatoid ossifying fibroma, facilitating its distinction from histologically overlapping lesions [ 3 ]. Additionally, DNA methylation-based approaches have emerged as promising diagnostic tools capable of refining the classification of these complex tumors [ 4 ]. Haefiger et al. recently identified activation of the RAS-MAPK pathway in a subset of cemento-osseous dysplasia (COD) cases, highlighting an important molecular aspect of COD pathogenesis [ 5 ]. Nevertheless, significant gaps remain in our understanding of the genetic landscape and molecular drivers underlying COD. In this report, we describe a case of COD harboring a NOTCH4 mutation, thereby expanding the molecular spectrum associated with this lesion and identifying potential avenues for further investigation into its molecular basis. Case A 32-year-old woman was referred to the maxillofacial surgeon by the orthodontist due to a progressive asymmetry of the mandible, which was suspected to be due to unilateral condylar hyperplasia. In order to assess the eventuality of persistent unilateral condylar growth, a SPECT/CT-scan was performed. As a coincidental finding, a circumscript, 10mm large, round metabolic hotspot was found in the right mandibular angle, exactly in the region where a wisdom tooth had been removed approximately ten years ago. Under local anesthesia, the region was explored and enucleated. A granulomatous tumor was sent for histopathological examination. Imaging, Histology, and Molecular Findings A decisive feature for differentiating COD from COF is the character of the interface between the lesion and adjacent bone: sharp in OF and diffuse in COD. In the orthopantomogram (Fig. 1), no distinct demarcation is observed, favoring a diagnosis of COD. The SPECT/CT shows metabolic activity in the region of the radio-opacity (Fig. 2). There is also a network-like arrangement of interconnected bony trabeculae without a well-defined rim of osteoblasts (Fig. 3), a feature that is uncommon in COF but more frequently observed in COD. DNA extraction was performed using the Maxwell RSC DNA FFPE Kit (Promega) following the manufacturer’s instructions. Library preparation was performed following the TruSight Oncology 500 High Throughput (TSO500 HT) protocol, and sequencing was carried out on the NovaSeq X+ platform (Illumina). Variant calling was done using the Franklin platform (Genoox), and variants were classified according to the five-tiered ACMG guidelines (ranging from benign to pathogenic) [6]. For NOTCH4 annotation, NM_004557 was used. Splice predictions were done using Alamut VisualPlus (Sophia Genetics) and SpliceAI [7]. Using the TSO500 assay, a splice site variant was identified in intron 12 of the NOTCH4 gene (c.2021+1G>A). This variant has not been previously reported in public databases or the literature but affects the highly conserved canonical splice donor site. In silico analysis predicts complete disruption of normal splicing at this site, supporting classification as a likely pathogenic variant. Loss of this splice site is expected to result in aberrant splicing, likely leading to exon skipping, a frameshift, and subsequent introduction of a premature stop codon—consistent with a loss-of-function (LoF) mechanism. Discussion This case report describes a COD lesion harboring a NOTCH4 mutation. While the histomorphological features of COD are well established, its molecular pathogenesis has only recently begun to be elucidated. In a recent study of 18 COD cases, somatic mutations were identified in 5 cases (28%), involving genes in the RAS-MAPK signaling pathway, including BRAF , HRAS , KRAS , NRAS , and FGFR3 [ 5 ]. It is noteworthy that their sequencing panel did not cover NOTCH . In contrast, another study of 12 COF cases reported no recurrent gene fusions (in 5 cases) or recurrent pathogenic variants, specifically not in the NOTCH gene family (using whole-exome sequencing in 11 cases) [ 8 ]. However, copy number gains involving chromosome 12 were identified in 3 out of 11 cases (27%) [ 8 ]. Additionally, other fibro-osseous lesions demonstrate distinct molecular signatures. For example, psammomatoid ossifying fibroma is associated with SATB2 rearrangements, while fibrous dysplasia is characterized by GNAS mutations [ 1 , 3 ]. These findings suggest that in selected cases, molecular analyses may assist in distinguishing COD from COF. However, the Notch signaling pathway, implicated in our case via a NOTCH4 mutation, has previously been described in COF, implying there might be an overlap in molecular patterns in COD and COF [ 9 ]. In the DNA-methylation and copy number alteration study by Kleijn et al. the authors did not seem to be able to discriminate between COD and COF [ 4 ]. Splice site mutations in Notch family genes, including NOTCH1 and NOTCH2 , have been shown to result in LoF and contribute to tumor suppression, for example, in various squamous cell carcinomas [ 10 ]. While functional studies for this specific NOTCH4 variant are lacking, canonical splice site variants are frequently deleterious and result in truncated, non-functional protein products. Notch4 is a transmembrane receptor that functions within the Notch signaling pathway, which is critical for regulating stem cell maintenance, cell fate determination, differentiation, proliferation, and apoptosis [ 11 ]. Although Notch signaling was initially characterized as oncogenic in T-cell leukemia, subsequent studies have demonstrated that its role is context-dependent, with evidence supporting both oncogenic and tumor-suppressive functions depending on the cellular and tissue environment [ 11 ]. Notch signaling plays an important role during bone development, bone remodeling, and regeneration, acting as a suppressor of intramembranous bone formation and an inhibitor of osteogenesis [ 12 – 14 ]. Mutations in the NOTCH gene family have been implicated in syndromic diseases with skeletal involvement, such as Adams–Oliver syndrome ( NOTCH1 ), Alagille syndrome ( NOTCH2 ), and Hajdu-Cheney syndrome ( NOTCH2 ) [ 13 , 15 ]. Although the role of NOTCH4 in benign fibro-osseous lesions has not been previously described, its involvement in this COD case suggests a possible novel mechanism of pathogenesis that warrants further investigation. Until molecular characteristics are more clearly defined, radiographic imaging remains the primary diagnostic modality for distinguishing COD from COF. Accurate diagnosis is essential, as COD typically follows a conservative, non-interventional management strategy, whereas COF may require surgical removal [ 5 ]. Declarations Informed consent was obtained from the patient for both participation in the study and publication of their clinical case details (including any accompanying images). Contributions G.B., A.J., N.J. and P.S. wrote the main manuscript and text. N.J. prepared Figs. 1 and 2. G.B. prepared Fig. 3. All authors reviewed the manuscript. Ethics declarations Ethical Approval The study has been approved by the institutional research ethics committee and has been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments. Informed Consent Informed consent was obtained from the participant included in the study. Competing Interests The authors declare no competing interests. References Crane H, Walsh H, Hunter KD. Fibro-osseous lesions of the jaws. Diagn Histopathol. 2024;30:170–8. https://doi.org/10.1016/j.mpdhp.2023.12.004 Vered M, Wright JM. Update from the 5th Edition of the World Health Organization Classification of Head and Neck Tumors: Odontogenic and Maxillofacial Bone Tumours. Head Neck Pathol. 2022;16:63–75. https://doi.org/10.1007/s12105-021-01404-7 Cleven AHG, Szuhai K, van IJzendoorn DGP, Groen E, Baelde H, Schreuder WH, et al. Psammomatoid Ossifying Fibroma Is Defined by SATB2 Rearrangement. Mod Pathol. 2023;36:100013. https://doi.org/10.1016/j.modpat.2022.100013 Kleijn TG, Ameline B, Schreuder WH, Szuhai K, Kooistra W, van Kempen L, et al. Classification of Fibro-Osseous Tumors in the Craniofacial Bones Using DNA Methylation and Copy Number Alterations. Mod Pathol. 2025;38:100717. https://doi.org/10.1016/j.modpat.2025.100717 Haefliger S, Turek D, Andrei V, Alborelli I, Calgua B, Ameline B, et al. Cemento-osseous dysplasia is caused by RAS-MAPK activation. Pathology. 2023;55:324–8. https://doi.org/10.1016/j.pathol.2022.10.006 Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24. https://doi.org/10.1038/gim.2015.30 Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, Darbandi SF, Knowles D, Li YI, et al. Predicting Splicing from Primary Sequence with Deep Learning. Cell. 2019;176:535-548.e24. https://doi.org/10.1016/j.cell.2018.12.015 Gomez RS, El Mouatani A, Duarte-Andrade FF, Pereira TDSF, Guimarães LM, Gayden T, et al. Comprehensive Genomic Analysis of Cemento-Ossifying Fibroma. Mod Pathol. 2024;37:100388. https://doi.org/10.1016/j.modpat.2023.100388 Zhang TH, Liu HC, Liao GQ, Liang YJ, Chu M, Wan CQ, et al. Detection of Notch signaling molecules in cemento-ossifying fibroma of the jaws. J Oral Pathol Med. 2010;39:263–8. https://doi.org/10.1111/j.1600-0714.2009.00842.x Nowell CS, Radtke F. Notch as a tumour suppressor. Nat Rev Cancer. 2017;17:145–59. https://doi.org/10.1038/nrc.2016.145 Radtke F, Raj K. The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer. 2003;3:756–67. https://doi.org/10.1038/nrc1186 Zieba JT, Chen Y-T, Lee BH, Bae Y. Notch Signaling in Skeletal Development, Homeostasis and Pathogenesis. Biomolecules. 2020;10:332. https://doi.org/10.3390/biom10020332 Ballhause TM, Jiang S, Baranowsky A, Brandt S, Mertens PR, Frosch KH, et al. Relevance of notch signaling for bone metabolism and regeneration. Int J Mol Sci. 2021;22:1325. https://doi.org/10.3390/ijms22031325 Dishowitz MI, Terkhorn SP, Bostic SA, Hankenson KD. Notch signaling components are upregulated during both endochondral and intramembranous bone regeneration. J Orthop Res. 2012;30:296–303. https://doi.org/10.1002/jor.21518 Mortier GR, Cohn DH, Cormier-Daire V, Hall C, Krakow D, Mundlos S, et al. Nosology and classification of genetic skeletal disorders: 2019 revision. Am J Med Genet A. 2019;179:2393–419. https://doi.org/10.1002/ajmg.a.61366 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6689921","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":461424910,"identity":"e9ed445a-06e3-4fe9-a9a5-5f186b0d0bb5","order_by":0,"name":"Gerben E. 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Slootweg","email":"","orcid":"","institution":"Radboud University Medical Centre","correspondingAuthor":false,"prefix":"","firstName":"Pieter","middleName":"J.","lastName":"Slootweg","suffix":""}],"badges":[],"createdAt":"2025-05-18 05:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6689921/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6689921/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83914872,"identity":"e2562878-3506-4f0f-9bcc-4be849d8e4f0","added_by":"auto","created_at":"2025-06-04 12:36:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":609632,"visible":true,"origin":"","legend":"\u003cp\u003ePanoramic X-ray showing a circumscript radio-opacity distally of the right lower second molar.\u003c/p\u003e","description":"","filename":"Fig1.OPT.png","url":"https://assets-eu.researchsquare.com/files/rs-6689921/v1/2de704f91b2eddca910e8818.png"},{"id":83914874,"identity":"d77952aa-bdd1-43ee-ad8f-7aab596ea31c","added_by":"auto","created_at":"2025-06-04 12:36:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":179467,"visible":true,"origin":"","legend":"\u003cp\u003eFused SPECT/CT image showing metabolic activity in the region of the radio-opacity.\u003c/p\u003e","description":"","filename":"Fig2.SPECT.png","url":"https://assets-eu.researchsquare.com/files/rs-6689921/v1/d8bc6461e7e2fd1454c71e73.png"},{"id":83914877,"identity":"dcee1466-3f06-4345-90a8-d9135421c020","added_by":"auto","created_at":"2025-06-04 12:36:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1399839,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin-stained micrograph demonstrating a network-like arrangement of interconnected bony trabeculae lacking distinct osteoblastic rimming.\u003c/p\u003e","description":"","filename":"Fig3.HE.png","url":"https://assets-eu.researchsquare.com/files/rs-6689921/v1/e44ae172e115863f1ed36b71.png"},{"id":83916565,"identity":"e7c805c6-ff23-4ad4-9ee8-453a146805a4","added_by":"auto","created_at":"2025-06-04 12:52:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2747237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6689921/v1/12502ea5-32a9-415b-aac0-545dd348de95.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cemento-Osseous Dysplasia With a NOTCH4 Mutation: a Case Report","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFibro-osseous lesions of the jaw pose diagnostic challenges due to their rarity and overlapping histopathological and radiological features, requiring combined expertise in pathology, radiology, and clinical evaluation for accurate classification [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One important entity within this group is cemento-osseous dysplasia (COD), a benign lesion in the tooth-bearing areas of the jaws. It is classified into four subtypes, i.e., periapical, focal, florid, or familial florid cemento-osseous dysplasia subtype, based on the location and distribution of the lesions. COD must be distinguished from cemento-ossifying fibroma (COF) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A key distinguishing feature lies in radiological assessment, particularly the nature of the lesion's interface with adjacent bone. A sharply demarcated lesion favors the diagnosis of COF, whereas a more diffuse boundary is suggestive of COD.\u003c/p\u003e \u003cp\u003eIn recent years, molecular analyses have substantially advanced our understanding and classification of fibro-osseous tumors of the jaw by identifying distinct genetic alterations. For instance, identifying \u003cem\u003eSATB2\u003c/em\u003e rearrangements appears to be highly specific for psammomatoid ossifying fibroma, facilitating its distinction from histologically overlapping lesions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, DNA methylation-based approaches have emerged as promising diagnostic tools capable of refining the classification of these complex tumors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Haefiger et al. recently identified activation of the RAS-MAPK pathway in a subset of cemento-osseous dysplasia (COD) cases, highlighting an important molecular aspect of COD pathogenesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nevertheless, significant gaps remain in our understanding of the genetic landscape and molecular drivers underlying COD.\u003c/p\u003e \u003cp\u003eIn this report, we describe a case of COD harboring a \u003cem\u003eNOTCH4\u003c/em\u003e mutation, thereby expanding the molecular spectrum associated with this lesion and identifying potential avenues for further investigation into its molecular basis.\u003c/p\u003e"},{"header":"Case","content":"\u003cp\u003eA 32-year-old woman was referred to the maxillofacial surgeon by the orthodontist due to a progressive asymmetry of the mandible, which was suspected to be due to unilateral condylar hyperplasia. In order to assess the eventuality of persistent unilateral condylar growth, a SPECT/CT-scan was performed. As a coincidental finding, a circumscript, 10mm large, round metabolic hotspot was found in the right mandibular angle, exactly in the region where a wisdom tooth had been removed approximately ten years ago. Under local anesthesia, the region was explored and enucleated. A granulomatous tumor was sent for histopathological examination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaging, Histology, and Molecular Findings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA decisive feature for differentiating COD from COF is the character of the interface between the lesion and adjacent bone: sharp in OF and diffuse in COD. In the orthopantomogram (Fig. 1), no distinct demarcation is observed, favoring a diagnosis of COD. The SPECT/CT shows metabolic activity in the region of the radio-opacity (Fig. 2). There is also a network-like arrangement of interconnected bony trabeculae without a well-defined rim of osteoblasts (Fig. 3), a feature that is uncommon in COF but more frequently observed in COD.\u003c/p\u003e\n\u003cp\u003eDNA extraction was performed using the Maxwell RSC DNA FFPE Kit (Promega) following the manufacturer\u0026rsquo;s instructions. Library preparation was performed following the TruSight Oncology 500 High Throughput (TSO500 HT) protocol, and sequencing was carried out on the NovaSeq X+ platform (Illumina). Variant calling was done using the Franklin platform (Genoox), and variants were classified according to the five-tiered ACMG guidelines (ranging from benign to pathogenic) [6]. For \u003cem\u003eNOTCH4\u003c/em\u003e annotation, NM_004557 was used. Splice predictions were done using Alamut VisualPlus (Sophia Genetics) and SpliceAI [7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUsing the TSO500 assay, a splice site variant was identified in intron 12 of the\u003cem\u003e\u0026nbsp;NOTCH4\u003c/em\u003e gene (c.2021+1G\u0026gt;A). This variant has not been previously reported in public databases or the literature but affects the highly conserved canonical splice donor site. In silico analysis predicts complete disruption of normal splicing at this site, supporting classification as a likely pathogenic variant. Loss of this splice site is expected to result in aberrant splicing, likely leading to exon skipping, a frameshift, and subsequent introduction of a premature stop codon\u0026mdash;consistent with a loss-of-function (LoF) mechanism.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis case report describes a COD lesion harboring a \u003cem\u003eNOTCH4\u003c/em\u003e mutation. While the histomorphological features of COD are well established, its molecular pathogenesis has only recently begun to be elucidated. In a recent study of 18 COD cases, somatic mutations were identified in 5 cases (28%), involving genes in the RAS-MAPK signaling pathway, including \u003cem\u003eBRAF\u003c/em\u003e, \u003cem\u003eHRAS\u003c/em\u003e, \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eNRAS\u003c/em\u003e, and \u003cem\u003eFGFR3\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is noteworthy that their sequencing panel did not cover \u003cem\u003eNOTCH\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, another study of 12 COF cases reported no recurrent gene fusions (in 5 cases) or recurrent pathogenic variants, specifically not in the \u003cem\u003eNOTCH\u003c/em\u003e gene family (using whole-exome sequencing in 11 cases) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, copy number gains involving chromosome 12 were identified in 3 out of 11 cases (27%) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, other fibro-osseous lesions demonstrate distinct molecular signatures. For example, psammomatoid ossifying fibroma is associated with \u003cem\u003eSATB2\u003c/em\u003e rearrangements, while fibrous dysplasia is characterized by \u003cem\u003eGNAS\u003c/em\u003e mutations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings suggest that in selected cases, molecular analyses may assist in distinguishing COD from COF. However, the Notch signaling pathway, implicated in our case via a \u003cem\u003eNOTCH4\u003c/em\u003e mutation, has previously been described in COF, implying there might be an overlap in molecular patterns in COD and COF [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the DNA-methylation and copy number alteration study by Kleijn et al. the authors did not seem to be able to discriminate between COD and COF [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSplice site mutations in Notch family genes, including \u003cem\u003eNOTCH1\u003c/em\u003e and \u003cem\u003eNOTCH2\u003c/em\u003e, have been shown to result in LoF and contribute to tumor suppression, for example, in various squamous cell carcinomas [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While functional studies for this specific \u003cem\u003eNOTCH4\u003c/em\u003e variant are lacking, canonical splice site variants are frequently deleterious and result in truncated, non-functional protein products.\u003c/p\u003e \u003cp\u003eNotch4 is a transmembrane receptor that functions within the Notch signaling pathway, which is critical for regulating stem cell maintenance, cell fate determination, differentiation, proliferation, and apoptosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although Notch signaling was initially characterized as oncogenic in T-cell leukemia, subsequent studies have demonstrated that its role is context-dependent, with evidence supporting both oncogenic and tumor-suppressive functions depending on the cellular and tissue environment [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notch signaling plays an important role during bone development, bone remodeling, and regeneration, acting as a suppressor of intramembranous bone formation and an inhibitor of osteogenesis [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mutations in the \u003cem\u003eNOTCH\u003c/em\u003e gene family have been implicated in syndromic diseases with skeletal involvement, such as Adams\u0026ndash;Oliver syndrome (\u003cem\u003eNOTCH1\u003c/em\u003e), Alagille syndrome (\u003cem\u003eNOTCH2\u003c/em\u003e), and Hajdu-Cheney syndrome (\u003cem\u003eNOTCH2\u003c/em\u003e) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although the role of \u003cem\u003eNOTCH4\u003c/em\u003e in benign fibro-osseous lesions has not been previously described, its involvement in this COD case suggests a possible novel mechanism of pathogenesis that warrants further investigation.\u003c/p\u003e \u003cp\u003eUntil molecular characteristics are more clearly defined, radiographic imaging remains the primary diagnostic modality for distinguishing COD from COF. Accurate diagnosis is essential, as COD typically follows a conservative, non-interventional management strategy, whereas COF may require surgical removal [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eInformed consent was obtained from the patient for both participation in the study and publication of their clinical case details (including any accompanying images).\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.B., A.J., N.J. and P.S. wrote the main manuscript and text. N.J. prepared Figs. 1 and 2. G.B. prepared Fig. 3. All authors reviewed the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has been approved by the institutional research ethics committee and has been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from the participant included in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCrane H, Walsh H, Hunter KD. Fibro-osseous lesions of the jaws. Diagn Histopathol. 2024;30:170\u0026ndash;8. https://doi.org/10.1016/j.mpdhp.2023.12.004\u003c/li\u003e\n\u003cli\u003eVered M, Wright JM. Update from the 5th Edition of the World Health Organization Classification of Head and Neck Tumors: Odontogenic and Maxillofacial Bone Tumours. Head Neck Pathol. 2022;16:63\u0026ndash;75. https://doi.org/10.1007/s12105-021-01404-7\u003c/li\u003e\n\u003cli\u003eCleven AHG, Szuhai K, van IJzendoorn DGP, Groen E, Baelde H, Schreuder WH, et al. Psammomatoid Ossifying Fibroma Is Defined by SATB2 Rearrangement. Mod Pathol. 2023;36:100013. https://doi.org/10.1016/j.modpat.2022.100013\u003c/li\u003e\n\u003cli\u003eKleijn TG, Ameline B, Schreuder WH, Szuhai K, Kooistra W, van Kempen L, et al. Classification of Fibro-Osseous Tumors in the Craniofacial Bones Using DNA Methylation and Copy Number Alterations. Mod Pathol. 2025;38:100717. https://doi.org/10.1016/j.modpat.2025.100717\u003c/li\u003e\n\u003cli\u003eHaefliger S, Turek D, Andrei V, Alborelli I, Calgua B, Ameline B, et al. Cemento-osseous dysplasia is caused by RAS-MAPK activation. Pathology. 2023;55:324\u0026ndash;8. https://doi.org/10.1016/j.pathol.2022.10.006\u003c/li\u003e\n\u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405\u0026ndash;24. https://doi.org/10.1038/gim.2015.30\u003c/li\u003e\n\u003cli\u003eJaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, Darbandi SF, Knowles D, Li YI, et al. Predicting Splicing from Primary Sequence with Deep Learning. Cell. 2019;176:535-548.e24. https://doi.org/10.1016/j.cell.2018.12.015\u003c/li\u003e\n\u003cli\u003eGomez RS, El Mouatani A, Duarte-Andrade FF, Pereira TDSF, Guimar\u0026atilde;es LM, Gayden T, et al. Comprehensive Genomic Analysis of Cemento-Ossifying Fibroma. Mod Pathol. 2024;37:100388. https://doi.org/10.1016/j.modpat.2023.100388\u003c/li\u003e\n\u003cli\u003eZhang TH, Liu HC, Liao GQ, Liang YJ, Chu M, Wan CQ, et al. Detection of Notch signaling molecules in cemento-ossifying fibroma of the jaws. J Oral Pathol Med. 2010;39:263\u0026ndash;8. https://doi.org/10.1111/j.1600-0714.2009.00842.x\u003c/li\u003e\n\u003cli\u003eNowell CS, Radtke F. Notch as a tumour suppressor. Nat Rev Cancer. 2017;17:145\u0026ndash;59. https://doi.org/10.1038/nrc.2016.145\u003c/li\u003e\n\u003cli\u003eRadtke F, Raj K. The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer. 2003;3:756\u0026ndash;67. https://doi.org/10.1038/nrc1186\u003c/li\u003e\n\u003cli\u003eZieba JT, Chen Y-T, Lee BH, Bae Y. Notch Signaling in Skeletal Development, Homeostasis and Pathogenesis. Biomolecules. 2020;10:332. https://doi.org/10.3390/biom10020332\u003c/li\u003e\n\u003cli\u003eBallhause TM, Jiang S, Baranowsky A, Brandt S, Mertens PR, Frosch KH, et al. Relevance of notch signaling for bone metabolism and regeneration. Int J Mol Sci. 2021;22:1325. https://doi.org/10.3390/ijms22031325 \u003c/li\u003e\n\u003cli\u003eDishowitz MI, Terkhorn SP, Bostic SA, Hankenson KD. Notch signaling components are upregulated during both endochondral and intramembranous bone regeneration. J Orthop Res. 2012;30:296\u0026ndash;303. https://doi.org/10.1002/jor.21518\u003c/li\u003e\n\u003cli\u003eMortier GR, Cohn DH, Cormier-Daire V, Hall C, Krakow D, Mundlos S, et al. Nosology and classification of genetic skeletal disorders: 2019 revision. Am J Med Genet A. 2019;179:2393\u0026ndash;419. https://doi.org/10.1002/ajmg.a.61366\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6689921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6689921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCemento-osseous dysplasia (COD) is a benign fibro-osseous lesion of the jaw that poses diagnostic challenges due to overlapping features with other entities, particularly cemento-ossifying fibroma (COF). While recent studies have identified mutations in the RAS-MAPK pathway in COD, its broader molecular profile remains poorly understood. Here, we report a case of COD harboring a novel NOTCH4 mutation, expanding the spectrum of genetic alterations associated with this lesion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe present a case of a 32-year-old woman with an incidental lesion in the right mandibular angle. The lesion was surgically enucleated and analyzed histologically. Next-generation sequencing (TSO500 panel) was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRadiographic evaluation showed a lesion without sharp demarcation. Histopathology shows a fibro-osseous lesion with interconnected bony trabeculae lacking osteoblastic rimming, consistent with COD. Molecular analysis detected a \u003cem\u003eNOTCH4 \u003c/em\u003emutation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis case identifies a novel NOTCH4 mutation in COD, suggesting possible involvement of the Notch signaling pathway in its pathogenesis. Incorporating molecular profiling into the diagnostic workflow may aid in distinguishing COD from COF and enhance our understanding of fibro-osseous lesions of the jaw.\u003c/p\u003e","manuscriptTitle":"Cemento-Osseous Dysplasia With a NOTCH4 Mutation: a Case Report","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-04 12:36:21","doi":"10.21203/rs.3.rs-6689921/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":"e516e044-b3af-4688-b1b7-d0f7f82d4e6d","owner":[],"postedDate":"June 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-04T12:36:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-04 12:36:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6689921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6689921","identity":"rs-6689921","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}