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Molecular Characterisation of Translocations Involving Chromosome Band 1P36 in Acute Myeloid Leukaemia

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Molecular Characterisation of Translocations Involving Chromosome Band 1P36 in Acute Myeloid Leukaemia MOLECULAR CHARACTERISATION OF TRANSLOCATIONS INVOLVING CHROMOSOME BAND 1P36 IN ACUTE MYELOID LEUKAEMIA Christopher Slape, B.Sc. (Hons) A thesis submitted in fulfilment of the requirements for the degree of Doctor of PhilosoPhY to The University of Adelaide Department of Medicine (The Queen Elizabeth HosPital) October 2002 Table of Contents 1l Table of Contents vi List of Tables vi List of Figures vlll Abstract X Declaration xi Acknowledgements xlv Abbreviations Chapter I Introduction pl pl 1.0 Overview p2 1.1 Leukaemia p3 Prevalence of Leukaemia 1.1.1 p3 HaematoPoiesis 1.1.2 p4 1 .1 .3 Classification of the Leukaemias 1.1.3.1 TYPes of Leukaemia p4 1.1.3.2 SubtYPes of AML p6 Translocations p8 1 .2 Chromosome p8 1.2.1 Translocations in Leukaemia and Other Malignancies p10 l.2.2EffecIs of Translocations on Gene Expression |.2.2.IDysregulationofExpressionofoncogenesbyTranslocation pl0 p 11 1.2.2.2 Formation of Fusion Genes by Translocation p72 1.2.3 Mechanisms of Translocation p14 1.3 Approaches to Breakpoint Identification p15 1.3.1 Candidate Gene APProach (FISH) p16 1 .3.2 Fluores çençe in slfø Hybridisation p16 1.3.3 Somatic Cell HYbridisation pl7 1.3.4 Positional Cloning p18 1.3.5 Somatic Cell HYbrid PCR p19 1.4 The Human Genomic Sequence p19 1.4.1 The GeneMaPg9 Genome MaP p20 l.4.2The Human Genome Project p22 1.4.3 Celerart Public Access Genome Database p22 1.5 Chromosome Band lP36 p24 1.6 Translocations Investigated in this Thesis p24 1.6.I t(l;3)(P36;q2t) p24 1 1.6.2 inv ins (1 ; I 2)(P2 1 -P36;P 3) p25 1.6.3 t(l;3XP36;P2I) p25 1.7 Aims Chapter 2 p26 Materials and Methods p26 2.1 Materials p26 2.1.1 Enzymes p27 2.1.2 Buffers and Solutions p28 2.1.3 Media p29 2.2 Methods p29 2,2.1Basic Nucleic Acid Isolation p29 2.2.1.1Mononuclear Cell Isolation from Peripheral Blood p30 2.2.1.2 Thawing of Frozen SamPles p30 2.2.1.3 DNA Isolation from Cell Suspension p3l 2.2.1.4 RNA Isolation from Cell Suspension p3l 2.2.1.5 Plasmid DNA Isolation p31 2.2.2 Basic Nucleic Acid Manipulation p31 2.2.2.1 DNA PreciPitation p32 2.2.2.2 DNA Sequencing p32 2.2.2.3 DNA/RNA Quantitation p32 2.2.2.4 DNA ElectroPhoresis p33 2.2.2.5 RNA ElectroPhoresis p33 2.2.2.6 Restriction Endonuclease Digestion of DNA p33 2.2.2.7 DNA Ligation p34 2.2.3 Polymerase Chain Reaction (PCR) p34 2.2,3.1Standard PCR p34 2.2.3.2 Long TemPlate PCR p35 2,2.3.3 Reverse Transcription (RT) using MMLV p35 2.2.3.4 Reverse Transcription (RT) using Superscript II p36 2.2.3.5 DNaseI Treatment of RNA p37 2.2.3.6 Procedure for 3'RACE p37 2.2.3.7 Inverse PCR p39 2.2.3.8 Direct Purification of PCR Products p39 2.2.3.g PCR Product Purification from Agarose Gels p39 2.2.3.10 PCR Product Cloning p40 2.2.3.11 PCR Primer Sequences p40 2.2.4 Northern AnalYsis p42 2.2.5 Cell Culture p42 2.2.5.1Thawing of Cells for Cell Culture p42 2.2.5.2 Maintenance of Cells in Culture p43 2.2.5.3 Freezing of Cells p43 2.2.5.4 Somatic Cell Hybridisation Chapter 3 p45 The (1 ;3)(p3 6;q2l\ Translocation p45 3.1 t(l;3Xp36;q2l) in AML and MDS p45 3.1.1Background p46 3.1.1.1 The MDSI-EVII Gene in Leukaemia p48 3.1.1.2 PRDM Genes in Oncogenesis p49 3 .1 .2 P atient Information p50 3.2 Somatic Cell HYbrid AnalYsis p51 3.2.1 Chromosome I Breakpoint Analysis p56 3.2.2 Chromosome 3 Breakpoint Analysis p56 3.3 Amplification and Sequencing of Breakpoints p59 3.4 Expression AnalYsis p60 3.4.1 MELI ExPression AnalYsis p62 3.4.1.1Identification of MELI Splice Variant p63 3.4.l.zldentification of Other Genes in the Breakpoint Region p63 3.4.2 ARPM2 ExPression AnalYsis p64 3.4.3 ARHGEFIó Expression Analysis p65 3.4.4 GATA2 ExPression AnalYsis p66 3.4.5 EYII ExPression AnalYsis p67 3.4.6 Expression AnalYsis Summary p68 3.5 Discussion p68 3.5.1 Position of Translocation Breakpoints in Patient 1 p69 3.5.2 MELI ExPression p69 3.5.2.1Identification of aNovel MELI Splice Variant p69 3.5.z.zExpression of MEL| in MDS and AML Patients without t(l;3Xp36;q2l) p73 3.5.2.3 Oncogenic Significance of MELI observations p74 3 .5.2.4 MELI Expression is Insufficient to Explain clinical p76 3.5.3 ARPM2 ExPression AnalYsis p77 3.5.4 ARHGEFIó Expression Analysis p78 3.5.5 GATA2 ExPression AnalYsis p78 3.5.5.1 Normal Function of GATA2 in Haematopoiesis p80 3.S.5.2GATA2ExpressioninPatients'\'ith3q2lRearangements 3.5.5.3 Co-expression of GATA2 with MELI or EVII p8l p81 3.5.5.4 Mechanism of Dysregulationof GATA2 Expression p82 3.5.5.5 GATA Family Member Overexpression Studies p83 3.5.5.6 FunctionalRedundancy of GATA Family Genes p87 3.5.6 Interactions Between GATA Family Members and PRDM Family Members p90 3.6 Conclusions lv Chapter 4 p93 The ins(l2;1)(p13;p36p21) Rearrangement p93 4.1 Rearrangements of 1p36, l2pl3 and lp21 in AML & MDS p93 4.1.1Background p96 4.1 .2 P atient Information p97 4.2 Somatic Cell HYbrid AnalYsis p97 4.2.1 Chromosome I AnalYsis p99 4.3 Identification of lp36-12p13 Breakpoint Sequence p 104 4.4 Investigation of 12p13-1p21 Breakpoint Sequence p 106 4.5 Investigation of lp36-1p21 Breakpoint Sequence p 111 4.6 TEL Northern AnalYsis p 113 4.7 Discussion p 113 4.7. 1 Identifrcation of the lp36'l2pl3 breakpoint Sequence p ll4 4.7 .2The lp36-lp2l and l2pl3-1p36 Breakpoints p 117 4.7.3 Expression of MELI in Patient 2 p 118 4.7.4The TEL-MDS2 Fusion Gene in t(1;12)(p36;p13) p 119 4.8 Conclusions Chapter 5 p l2l The (1 ;3Xp3 6;P2l) Translocation p l2l 5. I t( 1 ;3Xp3 6 ;p2l) as a Recurrent Translocation p 5.l.l Background l2l p 122 5.1.2 Patient Information p 123 5.2 Somatic Cell HYbrid AnalYsis p 123 5.2.1 Chromosome 1 AnalYsis p 124 5.2.2 Chromosome 3 AnalYsis p 128 5.3 Northern AnalYsis p 129 5.4 Investigation of a Fusion Between GOLGA4 and MELI p 131 5.4.1 GOLGA4 SPlice Variant p 132 5.5 3'RACE AnalYsis p 134 5.6 Inverse PCR p r37 5.7 Discussion p 137 5.7.1 Identification of the 3p2l Breakpoint within GOLGA4 in Patient 3 p 138 5.7 .2 Confirmation of the Presence of a Go LGA4 Fusion Gene p 140 5.7.3 Investigation of a Fusion Between GOLGA4 and MEL| p 5.7.43' RACEAnalysis of GOLGA4 Transcripts l4l p r43 5.7.5 Inverse PCR Analysis of 3p21 Breakpoint p 146 5.8 Conclusions Chapter 6 p 147 Conclusions p 147 6.1 Heterogeneity of lp36 Translocation Breakpoints p 149 6.2 Molecular Outcomes of 1p36 Rearrangements p 151 6.3 Molecular Mechanisms of Transformation p 157 6.4 Summary of Major Findings p 159 References v List of Tables Table 1.1. The FAB Subtypes of Acute Myeloid Leukemia' p6A Table l.2.Balanced Recurrent lp36 Translocations in Haematological Malignancy p23 p 404 T able 2.1. Primer Sequences. Table 3.1. Summary of Expression Analysis' p 671^ p 934 Table 4.1 . The fusion partners of the TEL oncogene. List of Figures Figure 1.1. A Scheme of Haematopoiesis. p4 Figure 1.2. Schematic Representation of the various Types of chromosomal Rearrangements. p8A Figure L3. Formation of Fusion Gene by chromosome Translocation. p llA Figure 1.4. Schematic Representation of Construction of Somatic Cell Hybrids' p t7A Figure 2.1. Schematic of Inverse PCRDesign. p 384 504 Figure 3.1. Patient I KaryotYPe. p 514 Figure 3.2. Chromosome I Marker Screen of PW Hybrid Cell Lines' p Figure 3.3.425F32 Screen of PW Hybrid Cell Lines' p 521^ 534 Figure 3.4 Orientation of RPll-425F18 and GA X2HTBKR3SEG p Figure 3.5. Markers used in chromosome 1 Breakpoint Analysis of PWN. p54 Figure 3.6. Alignment of MELI Protein Sequence with PRDMl6 and MDSl-EVIl Protein Sequences p 554 55B Figure 3.7. Orientation of RPl l-425F18 and GA x2HTBKR3SEG' p Figure 3.8. Markers used in chromosome 3 Breakpoint Analysis of PWN. p57 584 Figure 3.9. Primer combinations for breakpoint amplification' p Figure 3.10. Long Template PCR Analysis of (1;3)(p36;q2l) Breakpoint in Patient l. p 588 Figure 3.114. Sequence Alignment of the Patient I der 3 Breakpoint Sequence with Normal Chromosome I and 3 Sequences p 58C Figure 3,1lB. Sequence Alignment of the Patient I der I Breakpoint Sequence with Normal Chromosome I And 3 Sequences p 58D 594 Figure 3.12. Map of Patient I Breakpoint with respect to MELl and kPNl p Figure 3.13. Map of t(l;3Xp36;q2l) Breakpoints Reported in Mochizuki et al (2000). p 598 p 604 Figure 3.14 MELI Expression by RT-PCR Analysis' p 6lA Figure 3.15.
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