Novel genes and mechanisms in monogenic autoinflammatory disorders

Abstract

© 2018 Dr. Fiona Eyesun MoghaddasMonogenic autoinflammatory disorders are a heterogeneous group of rare conditions characterised by innate immune dysregulation. Patients often present early in life with recurrent fevers and features of systemic inflammation without high titres of autoantibodies or self-reactive T cells. Since the introduction of whole exome sequencing in the diagnostic evaluation of patients with suspected monogenic autoinflammatory disorders, the number of genetically defined conditions has greatly increased as has the phenotypic diversity. This study aimed to optimise methods of validating the pathogenicity of previously undescribed variants in vitro and to establish a national registry for patients with suspected or confirmed monogenic autoinflammatory disorders. Two variants in inflammasome forming proteins were evaluated. An inflammasome is a large multiprotein complex that forms in response to danger or pathogens. It serves as a platform for caspase-1 activation, resulting in cleavage of pro-IL-1 beta and pro-IL-18 to their active forms, as well as inflammatory cell death, pyroptosis. The first variant investigated was found through Sanger sequencing of MEFV in a family with a dominantly inherited suppurative dermatological condition. The novel variant p.Glu244Lys pyrin segregated with disease and was associated with increased inflammasome activation in vitro. This residue was shown using immunoprecipitation to be important for the binding of the regulatory proteins 14-3-3 and the substitution to lysine resulted in the auto-activation of pyrin. The second novel variant was found in two unrelated children with autoinflammation and macrophage activation syndrome. Although different genetic sequencing techniques were used, both children were found to harbour heterozygous p.Trp655Cys NLRC4. In vitro modelling revealed that this variant caused a caspase-1-dependent increase in IL-1 beta and IL-18 release with priming alone. Through the evaluation of the potential mechanisms of auto-activation, a previously unknown leucine rich repeat interface was revealed to exist between two NLRC4 monomers in the oligomeric state. Furthermore, an additional distinct interface was shown to exist between p.Trp655Cys NLRC4 and residues of the adjacent leucine rich repeat domain. Finally, the Australian Autoinflammatory Diseases Registry was established with fifteen tertiary hospitals across six Australian states currently involved. Thirty-seven patients with suspected autoinflammatory disorders have been recruited to date along with seventy-seven family members. The first twenty participants in whom no pathogenic mutation had been detected using National Association of Testing Authorities approved diagnostic testing underwent whole exome sequencing alongside their biological parents to determine variants that may be causing disease. The results of this analysis are presented here, including the identification of a novel variant in SHARPIN, encoding a component of the linear ubiquitin chain assembly complex involved in both the NF-kB and NLRP3 inflammasome pathways. This is the subject of ongoing investigation. The work described in this thesis has led to the first ethically approved national Australian registry for patients with monogenic autoinflammatory disorders. Furthermore, the in vitro validations of several AIDs within this thesis provide exquisite examples of some of the techniques that can be utilised in the future evaluation of variants of interest generated through the recruitment and sequencing of patients through the Australian Autoinflammatory Diseases Registry