Determining the causes of recessive retinal dystrophy By Mohammed El-Sayed Mohammed El-Asrag BSc (hons), MSc (hons) Genetics and Molecular Biology Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Medicine September 2016 The candidate confirms that the work submitted is his own, except where work which has formed part of jointly- authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated overleaf. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Mohammed El-Sayed Mohammed El-Asrag to be identified as author of this work has been asserted by his in accordance with the Copyright, Designs and Patents Act 1988. © 2016 The University of Leeds and Mohammed El-Sayed Mohammed El-Asrag Jointly authored publications statement Chapter 3 (first results chapter) of this thesis is entirely the work of the author and appears in: Watson CM*, El-Asrag ME*, Parry DA, Morgan JE, Logan CV, Carr IM, Sheridan E, Charlton R, Johnson CA, Taylor G, Toomes C, McKibbin M, Inglehearn CF and Ali M (2014). Mutation screening of retinal dystrophy patients by targeted capture from tagged pooled DNAs and next generation sequencing. PLoS One 9(8): e104281. *Equal first- authors. Shevach E, Ali M, Mizrahi-Meissonnier L, McKibbin M, El-Asrag ME, Watson CM, Inglehearn CF, Ben-Yosef T, Blumenfeld A, Jalas C, Banin E and Sharon D (2015). Association between missense mutations in the BBS2 gene and nonsyndromic retinitis pigmentosa. JAMA Ophthalmology 133(3): 312-318. Chapter 4 (second results chapter) of this thesis is entirely the work of the author and appears in: Ravesh Z, El-Asrag ME*, Weisschuh N, McKibbin M, Reuter P, Watson CM, Baumann B, Poulter JA, Sajid S, Panagiotou ES, O'Sullivan J, Abdelhamed Z, Bonin M, Soltanifar M, Black GC, Amin-ud Din M, Toomes C, Ansar M, Inglehearn CF, Wissinger B and Ali M (2015). Novel C8orf37 mutations cause retinitis pigmentosa in consanguineous families of Pakistani origin. Molecular Vision 21: 236-243. *Equal first-authors. iii Bedoni N*, Haer-Wigman L*, Vaclavik V, Tran HV, Farinelli P, Balzano S, Royer- Bertrand B, El-Asrag ME, Bonny O, Ikonomidis C, Litzistorf Y, Nikopoulos K, Yioti G, Stefaniotou M, McKibbin M, Ellingford J, Booth AP, Black G, Toomes C, Inglehearn CF, Hoyng CB, Bax N, Klaver CCW, Thiadens AA, Murisier F, Schorderet DF, Ali M, Cremers FPM, Andréasson S, Munier FL and Rivolta C (2016). Mutations in the polyglutamylase gene TTLL5, expressed in photoreceptor cells and spermatozoa, are associated with cone-rod degeneration and reduced male fertility. Human Molecular Genetics doi:10.1093/hmg/ddw282. *Equal first-authors. Khan KN*, El-Asrag ME*, Ku CA, Holder GE, McKibbin M, Arno G, Poulter JA, Carss K, Bommireddy T, Bagheri S, NIHR BioResource-Rare Diseases, UK Inherited Retinal Disease Consortium, Bakall B, Scholl HP, Raymond FL, Toomes C, Inglehearn CF, Pennesi ME, Moore AT, Michaelides M, Ali M, and Webster AR (2016). Recessive mutations in MFSD8 are a cause of non-syndromic rod-cone dystrophy with severe macular involvement. Submitted to Investigative Ophthalmology and Visual Science. *Equal first-authors. Chapter 5 (third results chapter) of this thesis is entirely the work of the author and appears in: El-Asrag ME*, Sergouniotis PI*, McKibbin M, Plagnol V, Sheridan E, Waseem N, Abdelhamed Z, McKeefry D, Van Schil K, Poulter JA, Johnson CA, Carr IM, Leroy BP, De Baere E, Inglehearn CF, Webster AR, Toomes C and Ali M (2015). Biallelic mutations in the autophagy regulator DRAM2 cause retinal dystrophy with early macular involvement. American Journal of Human Genetics 96(6): 948-954. *Equal first- authors iv Sergouniotis PI*, McKibbin M*, Robson AG, Bolz HJ, De Baere E, Muller PL, Heller R, El-Asrag ME, Van Schil K, Plagnol V, Toomes C, Ali M, Holder GE, Charbel Issa P, Leroy BP, Inglehearn CF and Webster AR (2015). Disease expression in autosomal recessive retinal dystrophy associated with mutations in the DRAM2 gene. Investigative Ophthalmology and Visual Science 56(13): 8083-8090. *Equal first-authors Publication in preparation from this thesis El-Asrag ME, McKibbin M, Poulter JA, Abdelmottaleb DI, Black GC, Toomes C, Inglehearn CF and Ali M. Whole exome sequencing identifies LARGE as novel gene candidate for autosomal recessive retinal dystrophy. In preparation. Publications related to other work done during this study Al-Amri A, Saegh AA, Al-Mamari W, El-Asrag ME, Ivorra JL, Cardno AG, Inglehearn CF, Clapcote SJ and Ali M (2016). Homozygous single base deletion in TUSC3 causes intellectual disability with developmental delay in an Omani family. American Journal of Medical Genetics Part A 170(7): 1826-1831. El-Asrag ME, McKibbin M, Mohamed MD, Toomes C, Inglehearn CF and Ali M. Next generation sequencing identifies novel loss of function mutations in NRL cause autosomal recessive retinitis pigmentosa. In preparation. v Acknowledgements First and above all, I praise God Almighty for providing me this opportunity, for without His blessings, guidance and assistance, this study would not have been possible. I also offer my immeasurable appreciation and deepest gratitude to Dr Manir Ali for his expert guidance, profound interest, support, assistance, valuable remarks and continuous encouragement. I also my grateful thanks to Prof. Chris Inglehearn for accepting me as a doctoral student at Leeds University, for his friendly advice, thoughtful guidance and for imparting his knowledge and bringing his expertise to this study. Also many thanks to Dr Carmel Toomes for her unfailing advice and constructive criticism; she did not spare any efforts in guiding me towards the best. Sincere appreciation also goes to my colleague Dr James Poulter for his valuable help and friendship throughout this study. I would also like to thank all my laboratory colleagues, past and present, in Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical Sciences (LIBACS) who in one way or another have contributed to the successful completion of this study, particularly Ahmed, Zakia, Kamron, Claire, Evi, Carla, Layal, Tom, Denisa, Emma, Ekram, Clare, Suba and Kasia. Many thanks also to Dr David Parry and Dr Ian Carr for their help with bioinformatics analysis and to Prof. Colin Johnson and Dr Jacquelyn Bond for their friendly advice. I would also like to thank Mr Martin McKibbin for providing the clinical data and images of the patients. I would also like to acknowledge the financial support that I received from the Egyptian Ministry of Higher Education. I would like to warmly thank my parents and my brother Ahmed for their spiritual support in all aspects of my life. To my dearest daughters, Maram and Maryam, as you grow older, I want you to know that just watching you smile makes me realize how beautiful my life is, and that I love you, and have done this for you. Hopefully, I have made you proud. Finally, my lovely wife, dear Dina, although I know that you did not want to be named, you put your own career on hold to come with me to the United Kingdom. Without your constant support and encouragement, I would never have finished this work. You became the bedrock of our family, and I know how difficult it was for you, so all I can say is: Thank you for everything and may Allah reward you for your generosity of spirit. vi Abstract Inherited retinal dystrophies (RDs) are a clinically heterogeneous group of eye diseases that result from mutations in more than 250 genes. Genetic diagnosis of these diseases has, until recently, been hampered by the lack of suitable technologies to perform high throughput screening. This thesis describes two different strategies for using next generation sequencing (NGS) in RD patients to find the pathogenic mutation(s) involved. In the first results chapter, a customised capture reagent (called Retinome) designed against the known retinal dystrophy genes (RetNet, June 2010) was used in NGS analysis of 20 RD families. The disease-causing mutations were identified in 12 of 20 cases (60%). These included previously reported mutations in ABCA4 (c.6088C>T, p.R2030*; c.5882G>A, p.G1961E), RDH12 (c.601T>C, p.C201R; c.506G>A, p.R169Q), PROM1 (c.1117C>T, p.R373C), GUCY2D (c.2512C>T, p.R838C), RPGRIP1 (c.3565C>T, p.R1189*), BBS2 (c.1895G>C, p.R632P) and SPATA7 (c.253C>T, p.R85*) and new mutations in CRB1 (c.2832_2842+23del), USH2A (c.12874A>G, p.N4292D), RP2 (c.884-1G>T) and ABCA4 (c.3328+1G>C). In eight cases the causative mutation could not be unambiguously identified. In the second results chapter, whole-exome NGS was performed on five RD families that had been pre-screened with the Retinome reagent. This identified mutations in three known RD genes, MFSD8 (c.1006G>C, p.E336Q; c.1394G>A, p.R465Q), C8orf37 (c.555G>A, p.W185*) and TTLL5 (c.1627G>A, p.E543K), and mutations in two potentially new RD genes, LARGE (c.2089G>T, p.V697L) and FDFT1 (c.930C>G, p.F310L). In the third results chapter, whole-exome NGS was performed, without pre- screening of known genes, in a family with atypical adult-onset RD with early macular involvement. NGS identified a mutation in a novel RD gene, DRAM2 (c.140delG, p.G47Vfs*3). Further DRAM2 screening in DNA panels identified a compound heterozygote case (c.494G>A, p.W165*; c.131G>A, p.S44N).