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dc.contributor.advisorDe Kock, A.
dc.contributor.advisorViljoen, C. D.
dc.contributor.authorDu Plessis, Hanri
dc.date.accessioned2015-10-27T13:35:10Z
dc.date.available2015-10-27T13:35:10Z
dc.date.issued2014-07
dc.identifier.urihttp://hdl.handle.net/11660/1442
dc.description.abstractEnglish: Myeloproliferative neoplasms (MPNs) are clonal haematopoietic stem cell disorders which are characterized by the excessive proliferation of one or more of the myeloid cell lineages. Mutations in several genes have been found to contribute to the pathogenesis of the Philadelphia (Ph) -negative MPNs. These MPNs include polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). The most common genes associated with Ph-negative MPNs are the JAK2 and MPL genes, which also form an integral part of the 2008 World Health Organization (WHO) diagnostic criteria for PV, ET and PMF. Mutations in the JAK2 and MPL genes are, however, not always detected in PV, ET and PMF patients. The ten-eleven-translocation (TET) 2 tumor suppressor gene has been identified to be a potential contributor to the normal development of haematopoietic cells, particularly the myeloid blood cells. While the exact role of TET2 in haematopoiesis is not yet completely understood, mutations in the TET2 gene have recently been found to occur in PV, ET and PMF patients. It has been suggested that mutations occurring in the conserved domains of TET2 could interfere with the catalytic activity of the protein, which ultimately has the potential to result in an inactive tumor suppressor gene. TET2 mutations situated outside of the conserved domains also have the potential to alter protein function. Defective expression of TET2 has previously been shown to alter haematopoietic development, resulting in the increased production of various myeloid cells. Previous studies in which mice models were used illustrated that the loss of TET2 function leads to characteristics similar to that of MPN patients. The aim of our study was to screen 25 PV, ET and PMF patients for mutations in the TET2 gene using high resolution melting (HRM) analysis and DNA sequencing analysis. In this study only two of the 24 primer sets could successfully be used to detect mutations using HRM analysis. The presence of non-specific amplification products, primer dimer, multiple melting domains and high melting background were suspected to be the causes of the unsuccessful HRM analysis. The DNA sequencing analysis of the TET2 gene was, however, successful. No novel variants were found in the current study. A total of 24 previously published TET2 variants were detected in the 25 Ph-negative MPN patients. The TET2 variants included missense single nucleotide polymorphisms (SNPs), synonymous SNPs, intronic SNPs, intronic deletions and intronic insertions. Unlike in previous studies, TET2 variants appeared to be more common in JAK2 V617F-negative MPN patients than in JAK2 V617F-positive MPN patients. It is speculated that most of the TET2 variants detected in the current study occur naturally in the TET2 gene and not exclusively in MPN patients. The prognostic value of TET2 variants in MPNs is as yet unknown. Since most of the SNPs in the current study appeared to be natural variants of the TET2 gene, it is possible that TET2 does not play a direct role in the development of MPNs. However, limited literature regarding the function of TET2 and how it contributes to abnormal haematopoiesis is available. Further investigation is therefore needed to establish the exact role of TET2 in the pathogenesis of MPNs and whether variants of this gene could predispose individuals to the development of MPNs.en_ZA
dc.description.abstractAfrikaans: Miëloproliferatiewe neoplasmas (MPNs) is klonale hematologiese stamselsiektes wat deur die oormatige produksie van een of meer van die miëloϊede bloedsellyne gekenmerk word. Mutasies in verskeie gene word met die patogenese van die Philadelphia (Ph) -negatiewe MPNs geassosieer. Die Ph-negatiewe MPNs bestaan uit polisitemie vera (PV), essensiële trombositemie (ET) en primêre miëlofibrose (PMF). Mutasies in die JAK2 en MPL gene is die algemeenste genetiese afwykings in Ph-negatiewe MPNs en vorm dus ‘n belangrike deel van die 2008 Wêreld Gesondheids Organisasie (WGO) diagnostiese kriteria vir PV, ET en PMF. JAK2 en MPL mutasies word egter nie altyd in PV, ET en PMF pasiënte aangetref nie. Die tien-elf-translokasie (TET) 2 tumor onderdrukker geen is onlangs met Ph-negatiewe MPNs geassosieer en word gespekuleer om tot die normale ontwikkeling van bloedselle by te dra, veral van die miëloϊede sellyn. Mutasies wat in die gekonserveerde domeine van TET2 geleë is, word gespekuleer om in te meng met die katalitiese aktiwiteit van die proteϊen wat ‘n onaktiewe tumor suppressor geen tot gevolg kan hê. Mutasies wat buite die gekonserveerde domeine van TET2 geleë is, het die potensiaal om die funksie van die proteϊen te verander. Vorige studies het getoon dat die gebrekkige uitdrukking van TET2 abnormale hematopoϊese, insluitende verhoogde produksie van sekere miëloϊede selle kan veroorsaak. Studies waarin muismodelle gebruik is, het getoon dat die verlies van die TET2 geen simptome soortgelyk aan wat in MPN pasiënte waargeneem word, kan veroorsaak. Die doel van hierdie studie was om hoë-resolusie-smeltkromme (HRS) analise en deoksiribonukleϊensuur (DNS) volgordebepaling te gebruik om vas te stel of mutasies in die TET2 geen van 25 PV, ET en PMF pasiënte voorkom. Slegs twee van die 24 priemstukke in die huidige studie kon suksesvol gebruik word met die HRS analise vir die identifisering van variante in die TET2 geen. Die oorsake vir die onsuksesvolle HRS analise was die teenwoordigheid van nie-spesifieke amplifiseringsprodukte, priemstuk dimere, veelvoudige smeltdomeine en hoë smeltingsagtergrond. Die DNS volgordebepaling van die TET2 geen was egter suksesvol. Geen nuut beskryfde TET2 variante is in die huidige studie geïdentifiseer nie. ‘n Totaal van 24 TET2 variante wat van te vore beskryf is, is in die PV, ET en PMF pasiënte gevind. Die TET2 variante sluit aminosuur veranderende enkelnukleotiedpolimorfismes (ENPs), sinonieme ENPs, introniese ENPs, introniese delesies en introniese invoegings in. Die geϊdentifiseerde TET2 variante in die huidige studie was meer algemeen in JAK2 V617F-negatiewe MPN pasiënte in vergelyking met JAK2 V617F-positiewe MPN pasiënte. Vorige studies het egter getoon dat TET2 mutasies meer algemeen is in JAK2 V617F-positiewe MPN pasiënte. Dit blyk of meeste van die geϊdentifiseerde variante in die huidige studie natuurlike variante van die TET2 geen is. Die prognostiese waarde van TET2 variante in MPN pasiënte is tans onbekend. Terwyl dit blyk of meeste van die geïdentifiseerde variante natuurlik voorkom in die TET2 geen, is dit moontlik dat TET2 nie ‘n direkte rol in die ontwikkeling van MPNs speel nie. Daar is tans beperkte wetenskaplike literatuur rakende die funksie van TET2 en die impak van mutasies in hierdie geen op hematopoiëse beskikbaar. Toekomstige studies moet dus uitgevoer word om te bepaal of TET2 variante moontlike risikofaktore kan wees vir die ontwikkeling van MPNs om sodoende die rol van TET2 in die patogenese van MPNs vas te stel.af
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectMyeloproliferative neoplasm (MPN)en_ZA
dc.subjectPolycythaemia vera (PV)en_ZA
dc.subjectEssential thrombocythaemia (ET)en_ZA
dc.subjectPrimary myelofibrosis (PMF)en_ZA
dc.subjectTET2en_ZA
dc.subjectTumor suppressor geneen_ZA
dc.subjectHigh resolution melting (HRM)en_ZA
dc.subjectDNA sequencingen_ZA
dc.subjectSingle nucleotide polymorphisms (SNP)en_ZA
dc.subjectBlood -- Diseasesen_ZA
dc.subjectMyeloproliferative disordersen_ZA
dc.subjectDissertation M.Med.Sc. (Human Molecular Biology))--University of the Free State, 2014en_ZA
dc.titleMutational analysis of the TET2 gene in Philadelphia negative myeloproliferative neoplasmsen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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