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2016 Mian Syed Ethesis This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Unravelling the molecular pathogenesis of Myelodysplastic Syndrome Patients with Ringed Sideroblasts Mian, Syed Asif Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 06. Oct. 2021 Unravelling the molecular pathogenesis of Myelodysplastic Syndrome Patients with Ringed Sideroblasts Syed Asif Mian Thesis submitted for the Degree of Doctor of Philosophy King’s College London October 2015 1 Declaration of Authorship I hereby declare that I alone composed this thesis and that work presented here is my own, except where stated otherwise. October 2015 2 Abstract Myelodysplastic syndromes (MDS) are a collection of clonal hematopoietic stem cell disorders with diverse phenotypes, characterized mainly by ineffective haematopoiesis. Refractory anaemia with ringed sideroblasts (RARS) is an acquired form of MDS with accumulation of mitochondrial iron that gives rise to ringed sideroblasts. In this study, whole-exome sequencing performed in 12 patients all of whom had >25% ringed sideroblasts identified mutations in one particular gene, splicing factor 3b subunit 1 (SF3B1), a component of the major and minor spliceosomes, in 11/12 cases. No SF3B1 mutations were observed in a single case of congenital sideroblastic anaemia, however, a constitutional mutation in ALAS1 gene was confirmed in this patient. Subsequently, amplicon sequencing using a 22 myeloid-gene panel in 154 MDS patients revealed 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harbouring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (p<0.03). Moreover, patients with splicing factor mutations alone had a better overall survival than those with coexisting additional mutations. Next, combination of techniques including xenotransplantation and single cell clonogenic assays were used to study the characteristics of SF3B1 mutant cells. Mutational analysis of hematopoietic stem/progenitor cells showed that SF3B1 mutations arise in phenotypically defined CD34+CD38-CD45RA-CD90+CD49f+ stem cells, therefore revealing the existence of MDS-initiating stem cells that propagate mutations to myeloid progenitors only. A persistent engraftment restricted to myeloid lineage was observed which was initiated only via the xenotransplantation of HSCs. Genetically diverse evolving subclones of mutant SF3B1 were detected in mice, indicating a branching multi-clonal as well as ancestral evolutionary paradigm. Subclonal evolution in mice was also mirrored in the clinical evolution in patients. Sequential sample analysis uncovered a clonal evolution in hematopoietic progeny and selection of the malignant driving clone leading to AML transformation. 3 Acknowledgements Apart from one’s own efforts, the success of any project depends largely on the encouragement and guidelines of many others. I would like to take this opportunity to express my gratitude to the people who have been instrumental in the successful completion of my PhD. First of all I would like to show my greatest appreciation and extend my heartfelt gratitude to my supervisors Prof. Ghulam J Mufti and Dr. Alexander Smith for giving me the opportunity to carry out this work which was funded by Leukaemia and Lymphoma Research. I can’t say thank you enough for their tremendous support and help which I received whilst working on this project. Without their encouragement and guidance this project would not have been successful. I would also like to thank all the patients who have donated their bone marrow tissues to King’s College London tissue bank. Also, I would like to acknowledge and extend my heartfelt gratitude to the people who made the completion of my project successful including Prof. Dominique Bonnet and Dr. Kevin Rouault. I owe my most sincere gratitude to Dr. Aytug Kizilors not only for her scientific advice but also for her deep understanding and help during my difficult moments. I would like to thank my colleagues in the department of Haematological medicine for their help and support when it was needed, but most of all for making me feel at home amongst them. It was a pleasure to work with them all. I would also like to thank all my friends especially Selen Bozkurt for her support and encouragement over the years. Finally, I am forever indebted to my family especially my parents for their understanding, encouragement, and endless love ‘come rain or shine’. I owe them so much. 4 Table of Contents Title…………………………………………………………………….…………………….. 1 Declaration………………………………………………………………………………..… 2 Abstract………………………………………………………………………………..……. 3 Acknowledgements………………………………………………………………………… 4 List of Figures…………………………………………………………………………...….. 9 List of Tables…………………………………………………………………………..….. 11 List of Abbreviations……………………………………………………………………… 13 1 General Introduction ....................................................................................... 15 1.1 Myelodysplastic syndromes ....................................................................................... 16 1.2 Spectrum of cytogenetic abnormalities in MDS ...................................................... 20 1.2.1 Deletion 5q ................................................................................................................... 21 1.2.2 Chromosome 7 and 7q deletion ................................................................................ 22 1.2.3 Trisomy 8 ...................................................................................................................... 23 1.2.4 Other Cytogenic Lesions ............................................................................................ 23 1.3 SNP Karyotyping ......................................................................................................... 24 1.4 Gene Mutations in MDS ............................................................................................. 25 1.4.1 Epigenetic modifier gene mutations ......................................................................... 26 1.4.2 Transcription regulation/Cell signalling pathway gene mutations ........................ 37 1.5 Spliceosome ................................................................................................................ 40 1.5.1 Splicing factor-3 b1 (SF3B1) ..................................................................................... 46 1.6 Refractory anaemia with ring sideroblasts and Iron metabolism ......................... 49 1.7 Therapeutic strategies for RARS .............................................................................. 59 1.8 High throughput molecular technologies ................................................................. 60 5 1.8.1 Second Generation Sequencing ............................................................................... 60 1.8.2 Microarray analysis ..................................................................................................... 62 1.9 Aims .............................................................................................................................. 64 2 Materials and Methods ................................................................................... 65 2.1 Materials ....................................................................................................................... 66 2.1.1 Patient Samples .......................................................................................................... 66 2.1.2 Commercial Kits .......................................................................................................... 69 2.1.3 Buffers ........................................................................................................................... 70 2.2 Methods ........................................................................................................................ 72 2.2.1 Extraction of Genomic DNA ....................................................................................... 72 2.2.2 DNA Quantitation using Quant-iT picoGreen dsDNA Assay kit. .......................... 72 2.2.3 RNA Extraction ...........................................................................................................
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