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Clinical Characterization and Prognostic Impact of SF3B1 and TP53 Gene Mutations in Myelodysplastic Syndromes

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Clinical Characterization and Prognostic Impact of SF3B1 and TP53 Gene Mutations in Myelodysplastic Syndromes Universitätsklinikum Ulm Zentrum für Innere Medizin Klinik für Innere Medizin III Hämatologie, Onkologie, Palliativmedizin, Rheumatologie und Infektionskrankheiten Ärztlicher Direktor: Prof. Dr. med. H. Döhner Clinical characterization and prognostic impact of SF3B1 and TP53 gene mutations in myelodysplastic syndromes Dissertation zur Erlangung des Doktorgrades der Medizin der Medizinischen Fakultät der Universität Ulm Vorgelegt von Till Wallrabenstein, geb. in Münster 2017 Amtierender Dekan: Prof. Dr. rer. nat. Thomas Wirth 1. Berichterstatter: Prof. Dr. med. Konstanze Döhner 2. Berichterstatter: Prof. Dr. med. Ramin Lotfi Tag der Promotion: 25. Oktober 2018 Table of contents Abbreviations ......................................................................................................... III 1 Introduction ...................................................................................................... 1 1.1 MDS .................................................................................................................................... 1 1.1.1 Pathogenesis .............................................................................................................. 1 1.1.2 Standards of diagnosis and classification .................................................................. 2 1.1.3 Prognostic scores ....................................................................................................... 6 1.1.4 Therapy options .......................................................................................................... 8 1.2 SF3B1 ................................................................................................................................. 8 1.3 TP53 ................................................................................................................................. 10 1.4 Study objectives ............................................................................................................... 11 2 Methods ......................................................................................................... 13 2.1 Reagents .......................................................................................................................... 13 2.2 Machines and devices ...................................................................................................... 14 2.3 Patients ............................................................................................................................. 15 2.3.1 Patient cohort ........................................................................................................... 15 2.4 Isolation of mononuclear cells from BM/PB ..................................................................... 18 2.5 DNA/RNA extraction ......................................................................................................... 18 2.5.1 Extraction kit and preparation ................................................................................... 19 2.5.2 Cell disruption, homogenization and separation of DNA from RNA ......................... 19 2.5.3 Extraction of RNA and DNA ..................................................................................... 20 2.5.4 Product measuring and quality control ..................................................................... 21 2.6 cDNA-synthesis ................................................................................................................ 23 2.7 PCR .................................................................................................................................. 23 2.7.1 SF3B1 DNA-based assay ........................................................................................ 24 2.7.2 SF3B1 cDNA-based assay ....................................................................................... 25 2.7.3 TP53 cDNA-based assay ......................................................................................... 28 2.7.4 TP53 DNA-based assay ........................................................................................... 28 2.7.5 Product control by gel-electrophoresis ..................................................................... 29 2.8 PCR-cloning ..................................................................................................................... 30 2.9 DHPLC-screening............................................................................................................. 32 2.10 Purification of PCR-products ............................................................................................ 34 2.11 Purification of gel-bound PCR-product ............................................................................. 34 2.12 Cycle sequencing reaction ............................................................................................... 35 2.13 Purification of CSR-products ............................................................................................ 36 2.14 Sequencing and sequence analysis ................................................................................. 36 2.15 Mutation nomenclature ..................................................................................................... 37 2.16 Statistical analysis ............................................................................................................ 38 I 3 Results ........................................................................................................... 40 3.1 Frequency and distribution of SF3B1 mutations .............................................................. 40 3.2 Frequency and distribution of TP53 mutations ................................................................. 41 3.3 Splicing mutations of TP53 ............................................................................................... 43 3.3.1 Mutation c.782_783ins64bp ..................................................................................... 43 3.3.2 Mutation c.560_672del113bp ................................................................................... 44 3.4 Associations of SF3B1 mutations with clinical characteristics ......................................... 47 3.5 Associations of TP53 mutations with clinical characteristics ........................................... 51 3.6 Association of SF3B1 with TP53 mutations ..................................................................... 54 3.7 Survival analysis according to SF3B1 mutation status .................................................... 54 3.8 Survival analysis according to TP53 mutation status ....................................................... 55 3.9 Associations of TP53 polymorphism c.639A>G ............................................................... 56 4 Discussion ..................................................................................................... 57 4.1 Discussion of results regarding SF3B1 mutations ........................................................... 57 4.1.1 Frequency of SF3B1 mutations ................................................................................ 57 4.1.2 Associations with SF3B1 mutations ......................................................................... 57 4.1.3 SF3B1 mutations and diagnosis/prognosis .............................................................. 58 4.1.4 SF3B1 mutations and therapy .................................................................................. 60 4.2 Discussion of results regarding TP53 mutations .............................................................. 61 4.2.1 Frequency of TP53 mutations .................................................................................. 61 4.2.2 Associations of TP53 mutations ............................................................................... 62 4.2.3 TP53 mutations and diagnosis/prognosis ................................................................ 63 4.2.4 TP53 mutations and therapy .................................................................................... 64 4.3 Molecular effects caused by SF3B1 and TP53 mutations ............................................... 65 4.3.1 Molecular effects of SF3B1 mutations ...................................................................... 65 4.3.2 Molecular effects of TP53 mutations ........................................................................ 66 5 Summary ....................................................................................................... 69 6 References .................................................................................................... 71 Acknowledgements .............................................................................................. 83 Curriculum vitae ................................................................................................... 84 II Abbreviations % per cent < less than = equals ≈ approximately > greater than °C degrees Celsius µl microliter 3’-5’ three prime to five prime (reading/repair direction) 5’-3’ five prime to three prime (elongation direction) ∞ infinite A Adenosine ABCB7 ATP-binding cassette sub-family B member 7 (human gene) ACC Adenoid cystic carcinoma aCML Atypical chronic myeloid leukemia, BCR-ABL1–negative AML Acute myeloid leukemia ANC Absolute neutrophil count ASXL1 Additional Sex Combs Like Transcriptional Regulator 1 (human gene) BCR-ABL1 Fusion gene between the breakpoint cluster region (BCR) on chromosome 22 and the ABL gene (Abelson murine leukemia viral
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