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Identification of Disease Gene Variants That Can Lead to Familial Myelodysplasia and Acute Myeloid Leukaemia

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Identification of Disease Gene Variants That Can Lead to Familial Myelodysplasia and Acute Myeloid Leukaemia Identification of disease gene variants that can lead to familial myelodysplasia and acute myeloid leukaemia A thesis submitted for the degree of PhD Shirleny Romualdo Cardoso Supervisors: Professor Inderjeet Dokal, and Professor Tom Vulliamy Centre for Genomics and Child Health, Blizard Institute Barts and The London School of Medicine & Dentistry, Queen Mary University of London In loving memory of my beloved sister Karla Romualdo Cardoso 2 I, Shirleny Romualdo Cardoso, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Shirleny Romualdo Cardoso 3 Abstract Myelodysplasia (MDS) is characterised by inefficient haematopoiesis with dysplastic features of blood and bone marrow, reduction of mature blood cells and continuous bone marrow failure (BMF). Acute myeloid leukaemia (AML) is characterised by the accumulation of immature myeloid blasts in the bone marrow. MDS and AML are mostly sporadic clonal disorders affecting older patients. Familial occurrence of MDS/AML is rare, and most of these cases occur in the setting of genetic syndromes. However, it has also been reported to be caused by germline heterozygous mutations in genes including RUNX1, CEBPA, TERC, TERT, GATA2, SRP72, and ANKRD26. Our group has collected 115 families that have two or more individuals with BMF with at least one of whom has MDS or AML. The aim of this project was to identify disease causing gene variants that can lead to familial MDS/AML. Identification of predisposing variants to familial MDS/AML is critical for effective management in these families. This will also provide new insights into the biology of MDS/AML in general. Herein, we have characterised a subset of families with MDS/AML as well as identified candidate disease genes using a range of genetic studies. Specifically, we have: i. Identified new genetic variants in some of the known disease genes such as RUNX1 and GATA2. ii. Our studies have substantiated the discovery of DDX41 as a disease gene as we have identified several families harbouring novel heterozygous loss of function (LoF) DDX41 variants. iii. Identified germline heterozygous LoF RTEL1 variants in a subset of families with myelodysplasia and liver disease. This defines a new disease group in this field, RTEL1 can now be added to the list of familial MDS/AML disease genes. iv. We have identified nine new candidate disease genes 4 which are involved in RNA splicing, transcription factor, DNA modification, cell signalling and intracellular transport. 5 Contents List of figures 12 List of tables 15 List of abbreviations 17 Publications and presentations arising from this thesis 27 Acknowledgments 29 Introduction 31 1.1 General introduction 32 1.2 Myelodysplasia syndromes 32 1.2.1 Patterns of acquired genetic variants in MDS 37 1.3 Acute myeloid leukaemia 41 1.3.1 Patterns of acquired genetic variants in AML 45 1.4 Familial myelodysplasia and/or acute myeloid leukaemia with germline predisposition syndromes 49 1.4.1 Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) 51 1.4.2 Thrombocytopenia 2 51 1.4.3 Thrombocytopenia 5 52 1.4.4 Familial AML with mutated CEBPA 53 1.4.5 Familial MDS/AML with mutated GATA2 53 1.4.6 Familial aplastic anaemia/MDS with SRP72 54 1.4.7 Bone marrow failure syndromes 54 1.4.7.1 Fanconi anaemia 55 1.4.7.2 Dyskeratosis congenita 56 1.5 Our cohort of families with familial MDS/AML 60 1.6 Aims of the project 63 Material and methods 64 6 2.1 Introduction 65 2.2 Materials 65 2.2.1 Patient samples 65 2.2.2 Primers 65 2.2.3 Chemicals and reagents 66 2.3 Methods 67 2.3.1 DNA quantification 67 2.3.2 Polymerase chain reaction – PCR 68 2.3.3 Agarose gel electrophoresis 68 2.3.4 DNA restriction digestion 69 2.3.5 Rolling circle amplification reaction 69 2.3.6 Alkaline gel electrophoresis 71 2.3.7 Southern blotting 71 2.3.8 Hybridisation and chemiluminescence detection 73 2.3.9 Gel extraction and purification 74 2.3.10 Sanger sequencing 74 2.3.11 Analysis of sequencing traces 76 2.3.12 RNA extraction 77 2.3.13 Reverse Transcriptase PCR 77 2.3.14 Monochrome multiplex quantitative PCR 78 2.3.15 Denaturing high-performance liquid chromatography 79 2.3.16 Whole exome sequencing 81 2.3.17 Nextera library preparation (enrichment of exonic fragments) 81 2.3.18 Tagmentation of genomic DNA and first PCR amplification 83 2.3.19 First hybridisation and first capture 84 2.3.20 Targeted resequencing – design of probes 86 2.3.21 Targeted resequencing library preparation 87 2.3.22 Sequencing of exome library 90 2.3.23 Variant calling 92 2.3.24 Mammalian cell culture 94 2.3.25 Polyacrylamide gel electrophoresis and Western blotting 95 2.3.26 In silico analyses 96 7 Variants identified in known disease genes associated with familial MDS/AML and related disorders 97 3.1 Introduction 98 3.1.1 Genetically uncharacterised familial MDS/AML patients 98 3.2 Results 99 3.2.1 Variants identified in previously known familial MDS/AML causing genes 100 3.2.1.1 Variants identified in RUNX1 100 3.2.2 Variants identified in genes associated with complex phenotypes 104 3.2.2.1 Variants identified in GATA2 104 3.2.2.2 Variant identified in TERT 109 3.2.2.3 TERT promoter region and ANKRD26 5’UTR screening in our cohort of patients by dHPLC 113 3.2.3 Variants identified in genes associated with inherited syndromes with predisposition to MDS/AML 118 3.2.3.1 Shwachman-Diamond syndrome 118 3.2.3.2 Fanconi anaemia 121 3.2.4 Variants identified in genes associated with other inherited syndromes 124 3.2.4.1 Wiskott-Aldrich syndrome 124 3.3 Discussion 127 Germline heterozygous DDX41 variants in a subset of familial myelodysplasia and acute myeloid leukaemia 131 4.1 Introduction 132 4.1.1 Familial myelodysplasia and acute myeloid leukaemia with germline predisposing variants in DDX41 132 4.1.2 DDX41 – DEAD-box helicase 41 structure and function 133 4.1.3 The role of DExD/H box helicases and DDX41 in the innate immunity response 136 8 4.1.4 Association of DDX41 variants with myeloid neoplasms and defects in mRNA splicing 141 4.1.5 The role of DDX41 in ribosome biogenesis and in post- transcriptional regulation of protein translation in cell growth 149 4.2 Results 153 4.2.1 DDX41 germline variants identified in our cohort 155 4.2.2 Telomere length analysis in patients harbouring DDX41 variants 158 4.3 Discussion 159 RTEL1 variants leading to myelodysplasia and liver disease 163 5.1 Introduction 164 5.1.1 RTEL1 protein structure and function 164 5.1.2 RTEL1 in homologous recombination 167 5.1.3 RTEL1 in telomere maintenance 170 5.1.4 RTEL1 in human diseases 177 5.1.4.1 RTEL1 germline biallelic variants leading to dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome 177 5.1.4.2 RTEL1 germline heterozygous variants leading to pulmonary fibrosis 179 5.2 Results 182 5.2.1 RTEL1 germline variants identified in our cohort 185 5.2.1.1 Patients with biallelic RTEL1 variants 185 5.2.1.2 Patients with heterozygous loss of function RTEL1 variants 189 5.2.1.3 Families with heterozygous variants of unknown significance and heterozygous likely benign variants 193 5.2.2 Short telomeres and T-circles in distinguishing the pathogenic status of RTEL1 variants 195 9 5.3 Discussion 198 Variants identified in familial MDS/AML candidate genes 202 6.1 Introduction 203 6.1.1 Genetically uncharacterised familial MDS/AML patients 203 6.2 Results 206 6.2.1 Data analysis 206 6.2.1.1 All MDS/AML families 208 6.2.1.2 Family FML012 217 6.2.1.3 Family FML003 223 6.2.1.4 All MDS/AML families – gene-level and variant-level metrics combined to assess potential pathogenicity of a variant 227 6.3 Discussion 235 Discussion 239 7.1 Variants identified in known disease genes associated with familial MDS/AML and related disorders 240 7.2 Germline heterozygous LoF DDX41 variants in a subset of familial myelodysplasia and acute myeloid leukaemia 244 7.3 RTEL1 LoF variants leading to myelodysplasia and liver disease 247 7.4 Variants identified in familial MDS/AML candidate genes 250 7.5 Future work 259 7.6 Concluding remarks 261 10 Appendices 263 Appendix 1 – Known familial MDS/AML causing genes – published mutations to date 264 Appendix 2 – Primer sequences 274 Appendix 3 – TSCA studies 277 Appendix 4 – Deletion of RUNX1 in family FML053 278 References 279 11 List of figures Figure 1.1.
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