The Genetics of Familial Leukaemia and Myelodysplasia __________________ Ahad Fahad H Al Seraihi A thesis submitted for the Degree of Doctor of Philosophy (PhD) at Queen Mary University of London January 2019 Centre for Haemato-Oncology Barts Cancer Institute Charterhouse Square London, UK EC1M 6BQ Statement of Originality Statement of Originality I, Ahad Fahad H Al Seraihi, 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. Signature: Date: 30th January 2019 2 Details of Collaborations and Publications Details of Collaborations: Targeted deep sequencing detailed in Chapter 3 was carried out at King’s College Hospital NHS Foundation Trust, London, UK, in the laboratory for Molecular Haemato- Oncology led by Dr Nicholas Lea and bioinformatics analysis was performed by Dr Steven Best. RNA sequencing in Chapter 3 was performed at Oxford Genomics, UK, and bioinformatics analyses were carried out by Dr Jun (Alex) Wang and Dr Ai Nagano at the Barts Cancer Institute, Queen Mary University of London, London, UK. Final steps of Sanger sequencing (capillary electrophoresis) were carried out at GATC Biotech (Eurofins) services based in Germany. Samples from 3 GATA2-mutated families with reduced penetrance were kindly provided by Dr Marcin Wlodarski at the Department of Pediatric Hematology and Oncology, University Children’s Hospital Freiburg, Freiburg, Germany. Section 1.1 of the Introduction (Chapter 1) was reproduced from a book chapter entitled: “Genomics and Diagnostics in Acute Myeloid Leukaemia” published by The Royal Society of Chemistry in which I was first author (see details below). Much of the work described in the Results sections (Chapter 3 and Chapter 4) was published in the journal Leukemia – Springer Nature (Appendix 1) in which I was first author jointly with Dr Ana Rio-Machin (see details below). I performed and analysed all the experiments while Ana contributed to the study design and analysis. _______________________________________________________________________ Details of Publications: Al Seraihi AF, Rio-Machin A, Tawana K, Charrot S, Di Bella D, Bödör C, Butler T, Farren T, Grantham M and Fitzgibbon J. Genomics and Diagnostics in Acute Myeloid Leukaemia. Book Chapter in: Whitehouse D and Rapley R (eds) Genomics and Clinical Diagnostics. The Royal Society of Chemistry, London. (Book is scheduled for release on 29 January 2019). Al Seraihi AF, Rio-Machin A, Tawana K, Bödör C, Wang J, Nagano A, Heward JA, Iqbal S, Best S, Lea N, McLornan D, Wlodarski MW, Kozyra E, Niemeyer CM, Scott H, Ellison A, Tummala H, Cardoso SR, Vulliamy T, Dokal I, Smith M, Cavenagh J and Fitzgibbon J. GATA2 monoallelic expression underlies reduced penetrance in inherited GATA2- mutated MDS/AML. Leukemia. 2018. 10.1038/s41375-018-0134-9. 3 Details of Collaborations and Publications Tummala H, Dokal AD, Walne AJ, Ellison A, Cardoso S, Kirwan M, Browne I, Sidhu JK, rajeeve V, Rio-Machin A, Al Seraihi A, Duncombe AS, Jenner M, Smith OP, Enright H, Norton A, Aksu T, Ozbek NY, Pontikos N, Cutillas PR, Dokal I and Vulliamy T. Genome instability is a consequence of transcription deficiency in bone marrow failure patients harboring biallelic ERCC6L2 variants. PNAS. 2018. Cardoso SR, Ellison A, Walne A, Cassiman D, Raghavan M, Kishore B, Ancliff P, Rodriguez- Vigil C, Dobbels B, Rio-Machin A, Al Seraihi A, Pontikos N, Tummala H, Vulliamy T and Dokal I. Myelodysplasia and liver disease extend the spectrum of RTEL1 related telomeropathies. Heamatologica. 2017; 8:e293-e296. Tawana K, Wang J, Király PA, Kállay K, Benyó G, Zombori M, Csomor J, Al Seraihi A, Rio- Machin A, Matolcsy A, Chelala C, Cavenagh J, Fitzgibbon J and Bödör C. Recurrent somatic JAK-STAT pathway variants within a novel RUNX1-mutated pedigree. Eur J Hum Genet. 2017; 8:1020-1024. Cardoso SR, Ryan G, Walne AJ, Ellison A, Lowe R, Tummala H, Rio-Machin A, Collopy L, Al Seraihi A, Wallis Y, Page P, Akiki S, Fitzgibbon J, Vulliamy T and Dokal I. Germline heterozygous DDX41 variants in a subset of familial myelodysplasia and acute myeloid leukemia. Leukemia. 2016; 10:2083-2086. Okosun J, Wolfson RL, Wang J, Araf S, Wilkins L, Castellano BM, Escudero-Ibarz L, Al Seraihi AF, Richter J, Bernhart SH, Efeyan A, Iqbal S, Matthews J, Clear A, Guerra- Assunção JA, Bödör C, Quentmeier H, Mansbridge C, Johnson P, Davies A, Strefford JC, Packham G, Barrans S, Jack A, Du MQ, Calaminici M, Lister TA, Auer R, Montoto S, Gribben JG, Siebert R, Chelala C, Zoncu R, Sabatini DM and Fitzgibbon J. Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma. Nature Genetics. 2016; 48:183–188. Manuscripts Pending Review or in Preparation: Heward JA, Konali L, D’Avola A-L, Yeomans A, Rahim T, Al Seraihi AF, Wang J, Korfi K, Araf S, Iqbal S, Calaminici M, Clear A, Okosun J, Johnson P, Gribben J, Packham G, Fitzgibbon J. KDM5 inhibition is a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas. (Submitted in December 2018 – in review). _____________________________________________________________________________ Conference Presentations Oral Communications: Al Seraihi A. Reduced Penetrance in GATA2-mutated Familial Leukaemia. Barts Cancer Institute Annual Postgraduate Day. London, UK. June 2018. Al Seraihi A. Variable Penetrance in GATA2 Deficiency. 3rd Saudi Hematology Research Day. Saudi Hematology Annual Congress. Riyadh, Saudi Arabia. March 2018. 4 Details of Collaborations and Publications Al Seraihi A. The Genetics of Familial MDS/AML: Challenges and Insights. Saudi Hematology Year Review 2018. Saudi Society of Blood & Marrow Transplantation. Riyadh, Saudi Arabia. February 2018. Al Seraihi A. Germline GATA2 P.T354M mutations: why so shy? Barts Cancer Institute Seminar Series. London, UK. July 2017. Al Seraihi A. Variable Penetrance in GATA2 Deficiency. Friday Scientific Workshop on Inherited Hematopoietic Malignancies. 58th American Society of Hematology (ASH) Annual Meeting and Exposition, San Diego, CA, USA. December 2016. Posters: Rio-Machin A, Cardoso S, Ellison A, Tawana K, Wang J, Chelala C, Plagnol V, Pontikos N, Page P, Wallis Y, Ryan G, Al Seraihi A, Walne A, Tummala H, Fitzgibbon J, Dokal I and Vulliamy T. Identifying new disease genes in familial myeolodisplasia/acute myeloid leukemia. 23rd Congress of the European Haematology Association (EHA), Stockholm, Sweden. June 2018. Al Seraihi A, Rio-Machin A, Tawana K, Bödör C, Heward J, Iqbal S, Ellison A, Tummala H, Cardoso SR, Cavenagh J, Vulliamy T, Dokal I and Fitzgibbon J. GATA2 Monoallelic Expression Underlies Reduced Penetrance in GATA2 Deficiency. 4th International Conference on Acute Myeloid Leukemia “Molecular and Translational”: Advances in Biology and Treatment, European Society of Hematology (ESH). Estoril, Portugal. October 2017. Al Seraihi A, Rio-Machin A, Wang J and Fitzgibbon J. GATA2 monoallelic expression underlies reduced penetrance in inherited GATA2-mutated MDS/AML. Barts Cancer Institute Annual Postgraduate Day. London, UK. June 2017. Al Seraihi A, Rio-Machin A, Tawana K, Bödör C, Araf S, Heward J, Smith M, Iqbal S, Best S, Lea N, McLornan D, Ellison A, Tummala H, Cardoso SR, Cavenagh J, Vulliamy T, Dokal I and Fitzgibbon J. Variable Penetrance is Linked with Monoallelic Gene Expression in Inherited GATA2-mutated MDS/AML. 58th American Society of Hematology (ASH) Annual Meeting and Exposition, San Diego, CA, USA. December 2016. Rio-Machin A, Cardoso SR, Tawana K, Wang J, Chelala C, Plagnol V, Wallis Y, Ryan G, Ellison A, Al Seraihi A, Walne A, Tummala H, Fitzgibbon J, Dokal I and Vulliamy T. Whole Exome sequencing reveals novel candidate genes in familial MDS/AML. 21st Congress of the European Haematology Association (EHA), Copenhagen, Denmark. June 2016. Cardoso SR, Ryan G, Walne A, Tummala H, Rio-Machin A, Ellison A, Collopy L, Al Seraihi A, Wallis Y, Page P, Akiki S, Fitzgibbon J, Vulliamy T and Dokal I. Germline heterozygous DDX41 variants account for a subset of familial myelodysplasia and acute myeloid leukaemia. 36th World Congress of the International Society of Hematology and the British Society of Haematology (ISH/BSH). Glasgow, Scotland. April 2016. _______________________________________________________________________ 5 Abstract Abstract Background & Aim: While the majority of myelodysplasia and acute myeloid leukaemia (MDS/AML) cases are sporadic, rare familial predisposition syndromes have been delineated and are regarded a separate disease entity in the 2016 WHO classification system. Germline mutations in 14 disease genes have been uncovered thus far, with GATA2 representing one of the key transcriptional regulators commonly mutated in inherited leukaemias. The rarity of these familial cases opens the door to fundamental questions in biology, one of which is the phenomenon of reduced penetrance posing a clinical challenge particularly when identifying “silent” mutation carriers for genetic screening and exclusion as potential stem cell transplant donors. We have noted that this is indeed a feature within certain GATA2-mutated families, especially those carrying germline missense mutations such as (p.Thr354Met). In our example, two first-degree cousins developed MDS/AML with monosomy 7 while a third cousin presented with significant monocytopenia and neutropenia. This contrasted with the parental generation mutation carriers who all remain symptom free into their mid-late 60s.