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The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome
Thesis (PhD)--Stellenbosch University, 2025.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867614129252990976 |
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| access_status_str | Open Access |
| author | Christowitz, Claudia |
| author2 | Engelbrecht, Anna-Mart |
| author_browse | Christowitz, Claudia Engelbrecht, Anna-Mart |
| author_facet | Engelbrecht, Anna-Mart Christowitz, Claudia |
| author_sort | Christowitz, Claudia |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/132152 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:47:07.138Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/132152 The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome Christowitz, Claudia Engelbrecht, Anna-Mart Kotze, Maritha Olivier, Daniel Wilhelm Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. Breast -- Cancer -- Genetic aspects Germ cells -- Mutation Tumor suppressor proteins -- Genetic aspects Blood -- Coagulation p53 protein Functional genomics UCTD Thesis (PhD)--Stellenbosch University, 2025. Christowitz, C. 2025. The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome. Unpublished doctoral dissertation. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/f4c7939a-8955-4a66-b047-ce2b1a72861c ENGLISH ABSTRACT: Introduction: Breast cancer remains a major global health concern for women, with personalized medicine offering improved patient outcomes. The pathology-support genetic testing (PSGT) framework, recognized as international best practice, provides a multidisciplinary pharmacodiagnostic platform in South Africa for assessing inherited, lifestyle-triggered and/or therapy-induced pathologies. Functional genomics can improve next generation sequencing benefits by aiding variant classification and interpretation. This study demonstrated its clinical utility by investigating a rare germline tumour suppressor protein p53 (TP53) variant (NM_001126114.3, c.1018A>G, p.N340D), identified for the first time in a family diagnosed with multiple cancers and the Li-Fraumeni-like syndrome. In silico tools predicted the variant as likely pathogenic, supported by protein modelling revealing structural alterations that could impair protein function. The functional impact of the TP53β N340D variant on cell proliferation, cell death, senescence, migration, immune destruction, coagulation, and cell signalling responses was explored using a translational ex vivo model. Methods: Blood samples were collected from female control individuals and breast cancer patients, with and without the TP53β N340D variant [e.g. TP53β wild-type (WT) control (N = 11), TP53β WT breast cancer (N = 11), TP53β N340D control (N = 2), and TP53β N340D breast cancer (N = 2)]. Patient characteristics were summarized using a health questionnaire and genotypic confirmation was performed using Sanger sequencing. Furthermore, p53 expression was assessed in patient-derived, formalin-fixed paraffin-embedded tissues using immunohistochemistry. Peripheral blood mononuclear cell (PBMC) proliferation, stimulated by lipopolysaccharide (LPS) and phytohemagglutinin-L (PHA-L), was evaluated using a water-soluble tetrazolium 1 (WST-1) assay. Moreover, PBMC cell death, induced by doxorubicin (DXR), gemcitabine, 5-fluorouracil, and etoposide, was assessed using a WST-1 assay, with further evaluation of the DXR response using flow cytometry and western blotting. Senescence was determined by performing a senescence-associated beta-galactosidase assay and western blotting. PBMC migration was assessed with a Transwell assay, and immune destruction was investigated using a 3D spheroid co-culture model. Coagulation parameters in platelet-poor plasma (PPP) were evaluated by thromboelastograph, fluorescence microscopy, and plasminogen activator inhibitor-1 (PAI-1) and thrombomodulin (TM) enzyme-linked immunosorbent assays. Results: The TP53β N340D variant impaired PBMC proliferation following LPS and PHA-L stimulation, as well as DXR-induced cell death. Late apoptosis induction was significantly reduced, with impaired CASP3 and PARP activation, promoting cell survival under DXR-induced stress. An upregulation of senescence was observed in TP53β N340D PBMCs, suggesting a shift from apoptosis to senescence, which may contribute to chemoresistance and cancer recurrence. The TP53β N340D variant also significantly impaired the migratory capacity of adherent PBMCs, potentially impacting immune recruitment. No significant differences in the immune destruction of BT-549 spheroids were observed among the groups. The TP53β N340D variant was associated with a pro-thrombotic phenotype, characterized by enhanced fibrin and clot formation, and increased microclot presence. Lastly, altered PAI-1 and TM levels were observed in TP53β N340D carriers. Conclusions: This study highlights the clinical significance of integrating functional genomics into the PSGT framework in South Africa using collaborative multidisciplinary efforts. According to the ACMG/AMP guidelines, supporting evidence is provided for the pathogenicity of the TP53β N340D variant. AFRIKAANSE OPSOMMING: Inleiding: Borskanker bly 'n groot gesondheidskwessie wêreldwyd vir vroue, met persoonlike medisyne wat verbeterde pasiëntuitkomste bied. Die patologie-ondersteunde genetiese toets (PSGT)-raamwerk, erken as internasionale beste praktyk, bied 'n multidissiplinêre farmakodiagnostiese platform in Suid-Afrika vir die assessering van oorerflike, lewenstyl- geïnduseerde en/of terapie-geïnduseerde patologieë. Funksionele genomika kan die voordele van volgende-generasie-volgorde verbeter deur variantklassifikasie en interpretasie te ondersteun. Hierdie studie het die kliniese bruikbaarheid daarvan gedemonstreer deur 'n seldsame kiemlyn-tumoronderdrukkingsproteïen p53 (TP53) variant (NM_001126114.3, c.1018A>G, p.N340D) te ondersoek, wat vir die eerste keer geïdentifiseer is in 'n familie gediagnoseer met veelvuldige kankers en die Li-Fraumeni-agtige sindroom. In silico- hulpmiddels het die variant as waarskynlik patogeen voorspel, ondersteun deur proteïenmodellering wat strukturele veranderinge getoon het wat proteïenfunksie kan benadeel. Die funksionele impak van die TP53β N340D-variant op selproliferasie, seldood, veroudering, migrasie, immuunvernietiging, stolling en seingweë-reaksies is ondersoek met behulp van 'n translasionele ex vivo-model. Metodes: Bloedmonsters is versamel van vroulike kontrolegroepe en borskankerpasiënte, met en sonder die TP53β N340D-variant [bv. TP53β wild-tipe (WT) kontrole (N = 11), TP53β WT borskanker (N = 11), TP53β N340D kontrole (N = 2), en TP53β N340D borskanker (N = 2)]. Pasientkenmerke is opgesom deur middel van 'n gesondheidsvraelys, en genotipiese bevestiging is uitgevoer met behulp van Sanger volgordebepaling. Verder is p53-uitdrukking in pasiënt-afgeleide, formalien-gefixeerde paraffien-geïnkapsuleerde weefsels geëvalueer deur immunohistochemie. Proliferasie van perifere bloed-mononukleêre selle (PBMC), gestimuleer deur lipopolisakkaried (LPS) en fitohemaglutinien-L (PHA-L), is geëvalueer deur 'n wateroplosbare tetrazolium 1 (WST-1)-toets. Seldood van PBMC, geïnduseer deur doksorubisien (DXR), gemcitabien, 5-fluoroürasiel en etoposied, is ook geëvalueer deur die WST-1-toets, met verdere evaluasie van die DXR-reaksie deur vloeisitometrie en westelike blotting. Veroudering is bepaal deur 'n verouderingsgeassosieerde beta-galaktosidase-toets en westelike blotting uit te voer. PBMC-migrasie is beoordeel met 'n Transwell-toets, en immuunvernietiging is ondersoek met behulp van 'n 3D-sferoïed-kokultuurmodel. Stollingsparameters in plaatarm plasma (PPP) is geëvalueer met tromboelastografie, fluoresserende mikroskopie en plasminogeenaktiveringsinhibeerder-1 (PAI-1) en trombomodulien (TM) ensiemgekoppelde immunosorbentoetse. Resultate: Die TP53β N340D-variant het PBMC-proliferasie ná LPS- en PHA-L-stimulasie, sowel as DXR-geïnduseerde seldood, belemmer. Laat apoptose-induksie is aansienlik verminder, met verminderde CASP3- en PARP-aktiwiteit, wat seloorlewing onder DXR-geïnduseerde stres bevorder. 'n Toename in veroudering is waargeneem in TP53β N340D PBMC's, wat op 'n verskuiwing van apoptose na veroudering dui, wat moontlik chemoweerstandigheid en kankerherhaling kan bevorder. Die TP53β N340D-variant het ook aansienlik die migrasie van aanhegtings-PBMC's aansienlik benadeel, wat moontlik immuunrekrutering kan beïnvloed. Geen beduidende verskille in die immuunvernietiging van BT-549-sferoïede is tussen die groepe waargeneem nie. Die TP53β N340D-variant is geassosieer met 'n pro-trombotiese fenotipe, gekenmerk deur verbeterde fibrien- en stolselvorming, en verhoogde mikroklont- teenwoordigheid. Laastens is veranderde PAI-1- en TM-vlakke waargeneem in TP53β N340D- draers. Gevolgtrekkings: Hierdie studie beklemtoon die kliniese betekenis van die integrasie van funksionele genomika in die PSGT-raamwerk in Suid-Afrika deur gebruik te maak van samewerkende multidissiplinêre pogings. Volgens die ACMG/AMP-riglyne word ondersteunende bewyse verskaf vir die patogenisiteit van die TP53β N340D-variant. Doctoral 2025-05-28T06:38:32Z 2025-05-28T06:38:32Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/132152 en Stellenbosch University xxv, 125 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Breast -- Cancer -- Genetic aspects Germ cells -- Mutation Tumor suppressor proteins -- Genetic aspects Blood -- Coagulation p53 protein Functional genomics UCTD Christowitz, Claudia The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome |
| title | The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome |
| title_full | The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome |
| title_fullStr | The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome |
| title_full_unstemmed | The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome |
| title_short | The functional impact of a rare germline TP53β N340D variant identified in breast cancer patients with the Li-Fraumeni-like syndrome |
| title_sort | functional impact of a rare germline tp53β n340d variant identified in breast cancer patients with the li fraumeni like syndrome |
| topic | Breast -- Cancer -- Genetic aspects Germ cells -- Mutation Tumor suppressor proteins -- Genetic aspects Blood -- Coagulation p53 protein Functional genomics UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/132152 |
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