University College London Identification of novel genetic mutations leading to rare monogenic inflammatory diseases Ciara Maria Mulhern Thesis submitted for PhD Infection, Immunity and Inflammation Research and Teaching Department 1 UCL, Great Ormond Street, Institute of Child Health I, Ciara Maria Mulhern, state that all experimental and analytical work presented in this thesis has been carried out by myself. Where others have contributed to this work, this has been indicated in the thesis. 2 Acknowledgements Firstly, I would like to say a huge thank you to my supervisors; Dr Despina Eleftheriou, Dr Ying Hong and Professor Paul Brogan. I cannot say enough words of thanks to Despina and Ying, two incredible women. Despina has always been so supportive and encouraging. Thank you for always being consistently present throughout my PhD, even when you were on holidays, you still answered my emails. No problem is ever too small for you. Ying, Thank you for always advising me, pushing me and giving me confidence in my work. Thank you for showing me how to work as a proper scientist, how to carry out assays, plan experiments and most importantly, how to get assays to work. You have been a constant support throughout my time at ICH, not just to me but to everyone around you. Thank you as well to Paul; your constant optimism and positivity was always appreciated. Thank you for your enthusiasm and words of encouragement. I would also like to thank Ebun and Dara two mighty postdocs. Ebun, you are a constant delight, always smiling and happy. Thank you for your expertise while gene hunting; you are an absolute pleasure to work with. Dara, thank you for the all the Fr. Ted jokes, and always making me laugh with the constant Irish humour that you provide in the lab . I would like to say thank you to my parents, without whom this PhD would not have been possible. Thank you for letting me escape London, and sponge off you for 3 months, in Ireland, while I tackled the writing. Thank you especially to Mum for joining me for walks in the countryside and to Dad for always being inquisitive about my work. Thanks also to my siblings; Colin, Sean, Niamh and Patrick, to my grandparents, and to all my friends who put up with my PhD moaning. You can all rejoice now; it’s over! 3 Abstract Over the past 20 years, an increasing number of monogenic inflammatory diseases have been described. In particular, monogenic autoinflammatory disorders are characterized by episodic and unprovoked inflammation, without the existence of high-titre autoantibodies or antigen specific T cells. When such diseases occur early in infancy, the cause is often suspected to be genetic. The emergence of nextgeneration genetic sequencing technologies has provided a fast and reliable means upon which to investigate causative genes, leading to the discovery of novel monogenetic autoinflammatory diseases. This technology also aids our understanding of the molecular pathways underpinning these disorders. The aims of this PhD project were to discover novel genetic causes of inflammatory diseases through the use of next generation sequencing and to explore the functional relevance of identified variants in candidate genes. In this thesis, a cohort of children with suspected monogenic inflammation were subject to next- generation sequencing. Several discoveries were made, and various families are discussed herein. For the first family, the index case was an 8 year old girl suffering from severe neuroinflammation manifesting as left sided focal seizures, left sided hemiplegia, granulomatous cerebral inflammation and chorioretinitis. I identified a rare missense p.T647P mutation in the TNFAIP3 gene as the cause of her predominantly neurological presentation. I was then able to show that this variant induces activation of NF-ƙB pathway and enhances NLRP3 inflammasome activity, but also results in significant upregulation of type I interferon signaling pathway. Targeted therapeutic intervention through administration of a Janus kinase inhibitor, resulted in a complete resolution of neuroinflammation and improvement in her clinical presentation. 4 The second family was a consanguineous Pakistani family, where 3 affected individuals were suffering from a systemic inflammatory disorder characterized by fevers, susceptibility to viral infection, rashes, and haemophagocytosis. I identified a homozygous p.M228K mutation in CCR7 in all affected individuals. Functional experiments showed decreased expression of the CCR7 protein in all affected individuals and defective migration of immune cells following chemokine stimulation. Immunophenotyping also revealed complete absence of central memory T cells and defective IFNγ production in response to viral and bacterial stimuli. Lastly, a homozygous p.D118N mutation in BTNL2 was discovered in the third family I studied, a consanguineous family from Somalia wherein affected individuals were suffering from a familial leukocytoclastic vasculitis. Therefore, through the use of next generation sequencing, coupled synergistically with pertinent functional readouts, I have identified and characterised three novel monogenic immunological diseases, each associated with varying degrees of autoinflammation and immune deficiency. 5 Impact Statement This PhD project explored the potential genetic causes of a broad spectrum of children presenting with varying features of immune dysregulation from early in life. The primary beneficiaries of this project, are the patients and families with these rare diseases. Commonly, these diseases present in infancy and segregate within families, so a genetic cause is often suspected, but not always identified by the current routinely available genetic tests in the UK National Health Service (NHS). Due to the overlapping phenotypes of these disorders, broad (i.e. exome wide), genetic sequencing is required for timely and accurate diagnosis, and (where possible) targeted clinical intervention. The major way in which patients with this disease benefit from this study are summarised below: 1. The patients involved in this project benefitted from genotype specific targeted therapy. In the case of A-III-1, this patient had been sick for years with what was suspected to be an infection or malignant process. Many different treatments had already been administered, having little to no effect. The identification of the p.T647P mutation in TNFAIP3, enabled a deeper understanding the pathophysiology of her clinical condition and an ability to provide the correct targeted therapy, which resulted in a complete clinical and radiological resolution of her unusual neuroinflammatory condition. I have elucidated the mechanism by which aberrations in this gene resulted in the inflammatory manifestations observed in this patient. My findings, therefore, have the following impact: (i) Patients with heterozygous TNFAIP3 mutations and autoinflammation should be investigated for enhanced type I interferon activity, and targeting the IFN pathway should be considered when deciding upon therapeutic interventions for future 6 patients with mutations in this gene. (ii) Patients with heterozygous TNFAIP3 mutations should also be screened for underlying cerebral inflammation, even in the absence of overt clinical symptoms. 2. I am the first to describe an immunodysregulatory disorder associated with homozygous p.M228K in CCR7. Through this work, I have provided key insights into the cellular and molecular mechanisms underpinning this rare disease associated with immunodeficiency and autoinflammation. My findings also had therapeutic implications for this family since the affected individuals were fast- tracked to allogeneic haematopoietic stem cell transplantation. It is likely that future studies will expand the phenotype and report more patients with different genotypes associated with CCR7 deficiency. 3. Patients with autoinflammation, neuroinflammation and immunodeficiency are now systematically screened for mutations in TNFAIP3 and CCR7 as these genetic targets are now included in two routinely used in clinical practice genetic panels: the Vasculitis Inflammation Panel (VIP); and Neuroinflammation Panel (NIP). Some of the data generated in this PhD has been published in open access peer- reviewed journals; the remainder is soon to be submitted for peer reviewed publication, hence ultimately contributing to significant scientific knowledge advancement about the genetic causes of rare inflammatory disease for academic beneficiaries in the field. This project also contributes to worldwide academic 7 advancement to address issues of importance such as the pathogenesis of autoinflammatory and immunodysregulatory syndromes. Lastly, my PhD enabled my training as a highly skilled researcher and facilitated my academic career progress. I have developed expertise and knowledge in this multidisciplinary and collaborative environment. My project also contributes towards the health of the rheumatology/neurology academic discipline, with publications and national/international presentations. 8 Table of Contents Table of Contents 9 Table of Figures 24 Table of Tables 32 Abbreviations 35 1 Introduction 36 1.1 Monogenic inflammatory Diseases 36 1.1.1 Familial Mediterranean Fever (FMF) 41 1.1.2 TNF receptor-associated periodic syndrome (TRAPS) 43 1.1.3 Cryopyrin associated periodic syndrome (CAPS) 44 1.1.4 Deficiency of Adenosine Deaminase 2 (DADA2) 46 1.1.5 Deficiency of IL-1 Receptor Antagonist (DIRA) 47 1.1.6 Blau Syndrome 49 1.1.7 A20 Haploinsufficiency 50 1.1.8