INVESTIGATING SINGLE-GENE DISORDERS OF CHILDHOOD INFECTIOUS DISEASE Bayarchimeg Mashbat A thesis submitted for the degree of Doctor of Philosophy Section of Paediatrics, Division of Infectious Diseases Department of Medicine, Imperial College London United Kingdom October 2017 Abstract A common feature of infectious diseases, including an infection with Neisseria meningitidis (Nm), is that only a small proportion of the individuals exposed to the same strain of the bacteria suffer from the clinical disease. Host genetics has long been considered to be an important determinant of both predisposition to and severity of outcome from invasive meningococcal disease (IMD). The human complement system is central to protection against IMD. It is well established that individuals with terminal or alternative complement deficiencies are predisposed to invasive, often recurrent meningococcal infections. However, the occurrence of these putative genetic deficiencies is rare, such that complement deficiencies account for less than 3 % of the disease cases. The current study sought to uncover novel genetic aetiologies of IMD, by employing WES and GWAS, in conjunction with molecular functional characterisation assays. Firstly, genetic analysis of six familial IMD exomes revealed a novel mutation in the SPLUNC1 gene. The encoding protein has been shown to play an important role in innate immune defence against a number of Gram-negative bacterial infections. The characterisation assays undertaken in this work suggest that the protein encoded by SPLUNC1 is also implicated with host innate immune defence against Nm infection, by providing protection against nasopharyngeal colonisation of a pathogenic Nm strain. The results further suggest that harbouring rare pathogenic mutations that impact the function of the encoding protein is associated with reduced host defence activities in the resulting protein, which in turn may possibly lead to increased susceptibility to IMD in the carriers. Furthermore, a large-scale GWAS was performed to define common polymorphisms underlying host susceptibility and severity of IMD, using 1236 individuals with confirmed disease and over 5000 controls. In this work, efforts were made to understand the biological plausibility of the genetic associations identified through the GWAS analysis. i ii Declaration of Originality This thesis is submitted for the degree of Doctor of Philosophy to Imperial College London. The work presented here was undertaken in the Section of Paediatrics, Department of Medicine at Imperial College London. The research was co- supervised by Dr.Vanessa Sancho-Shimizu and Professor Michael Levin. I declare that none of the research results offered in this thesis has been previously submitted by me for any other degree or qualifications at Imperial College London or any other university. I certify that all the material included in this thesis is my own and those from contributors have been appropriately acknowledged and referenced. iii iv Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work v vi Acknowledgements I would like to thank my supervisors, Dr. Vanessa Sancho-Shimizu and Prof. Michael Levin for the opportunity they have given me to undertake this exciting project at Imperial College London. I would like to thank Dr. Sancho-Shimizu for her excellent knowledge, trust in me and endless encouragements through the ups and downs of my PhD. I would also like to thank Prof. Levin for his continuous support and providing me with the opportunity to develop my research skills through international collaborations. I am truly inspired by both these wonderful mentors for their unwavering passion and dedication to their daily work and their ambitious visions to help alleviate suffering from some of the most fatal diseases in the world. I am also very grateful for the patients and their families who provided samples for this study. I would also like to thank the EUCLIDS Consortium members for their huge contributing to my development as a scientist through many scientific meetings, where I was given the opportunities to present my research and obtain valuable feedback. I would like to extend my thanks to Irene and Elli from EUCLIDS for offering me their friendship, sharing so many good times during our socials and being there for me when I needed their support. I would like to extend my thanks to my assessors, Dr. Simon Nadel and Dr. Anna Puel for taking diligent care to review this thesis. I would like to thank everyone in Paediatrics and especially to those who provided support, guidance and mentorship throughout the years including Prof. P.Langford, Dr. M.Li, Dr .F.Bidmos, Dr. V.Wright, Dr. Y.Li, Dr. A.Cunnington, Dr. J.Herberg and Dr. C.Hoggart. I would also like to thank my work friends Liyana, Roberto, Shea, Stephanie, Beth, Tom, Myrsini, Evan and Rahmeen for their consolidation over coffee breaks, shared laughs, and attempted running clubs. I am truly blessed to have the most wonderful friends Simon, Kate, Kasia, Pawel, Enkhjargal and David, whom I was always able to depend on in every way possible. Most importantly, I would like to thank my parents and my siblings, Saruul, Badamtsetseg and Amartaivan for their love and support from afar. vii Special thanks go to my aunty, Mashbileg who has always been my inspiration and taught me to excel in everything I do. Finally, I thank my partner, Karim for his encouragement for me to embark on this challenge and always helping me to live life to the full. viii To my love and my parents, ix x Table of Contents Abstract ................................................................................................................................ i Declaration of Originality .................................................................................................. iii Copyright Declaration ......................................................................................................... v Acknowledgements .......................................................................................................... vii Table of Contents ............................................................................................................... xi Abbreviations .................................................................................................................. xvii List of Figures .................................................................................................................. xxi List of Tables ................................................................................................................... xxv Chapter 1 ............................................................................................................................. 1 Introduction ......................................................................................................................... 2 1.1. Genetic basis of infectious diseases ...................................................................... 2 1.2. Genetic evidence from twin and adoptee studies ................................................... 3 1.3. Single gene defects underlying host susceptibility to infection ............................... 3 1.4. Genomic approaches for identifying genetic cause of infectious diseases ............. 5 1.5. Whole exome sequencing for uncovering genetic basis of Mendelian disease ...... 6 1.6. Genome-wide association studies uncovering polymorphisms .............................. 9 1.7. Neisseria meningitidis .......................................................................................... 11 1.8. Nm colonisation and nasopharyngeal carriage .................................................... 12 1.9. Experimental models to study Nm colonisation and host-pathogen interactive biology 13 1.10. Invasive meningococcal disease ......................................................................... 14 1.11. Antibiotic treatments and vaccination ................................................................... 15 1.12. Host immunity to Nm ........................................................................................... 17 1.12.1. Innate immune response to Nm ................................................................... 18 xi 1.12.1.1. Mucosal barrier ............................................................................................ 18 1.12.1.2. Recognition of Nm by the innate immune system......................................... 20 1.12.1.3. Complement cascade .................................................................................. 21 1.12.2. Humoral immune response against Nm ....................................................... 23 1.13. Host genetics underlying susceptibility and severity of IMD ................................. 24 1.13.1. Mendelian defects in IMD ............................................................................. 25 1.13.1.1. Complement deficiencies ............................................................................. 25 1.13.1.2. Properdin and MBL .....................................................................................