Godinho Lopes De Melo Gomes, Sónia; (2023) Somatic Mosaicism and Novel Gene Discovery in Autoinflammation: NLRP3 Mutation Negative CAPS and Beyond. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Since its inception over 20 years ago, the knowledge on autoinflammation and autoinflammatory diseases has grown exponentially, in part due to the development of next generation sequencing (NGS) technologies. However, a significant proportion of patients with autoinflammatory phenotypes and suspicion of a genetic monogenic disorder, still do not have a molecular diagnosis. In this thesis, I have used NGS techniques, mostly whole exome sequencing (WES), to identify a genetic cause for several families in this predicament. Firstly, I studied a cohort of ten patients with a clinical diagnosis of cryopyrin associated periodic syndrome (CAPS), who were deemed NLRP3 mutation negative, by assessing the possibility of low-level somatic mosaicism as well as the possibility of the contribution of other genes to their disease. These studies revealed the presence of somatic NLRP3 mosaicism in 40% of this cohort, in varying degrees of allelic frequency. The remainder, studied by WES, had a possibly pathogenic variant in a different exon of the NLRP3 gene (not included in the initial routine clinical Sanger approach), other known genes related to autoinflammation (NOD2, IL36RN) and a gene not previously associated with autoinflammation (MTOR). In two patients a tentative genetic cause has not yet been identified, despite thorough analysis of the WES data. In relation to the mosaicism cases, I assessed the mosaicism distribution by analysing the frequency of the identified NLRP3 mutation in different tissues and assessed the stability of the mosaicism overtime by repeating the analysis at different timepoints. These studies have shown that mosaicism distribution may support an earlier occurrence of the mutational event in the paediatric cohort than in an adult cohort of patients with late onset CAPS, and that in certain patients, mosaicism frequency can indeed vary overtime, with significant implications in mosaicism detection as well as treatment. Secondly, I have applied the same concept and methodology to a cohort of patients with varied autoinflammatory phenotypes. These included: a patient with inflammatory polyarthritis and syndromic features shown to have a truncating variant in the ARID1B gene, which prompted my observation that inflammatory arthritis could in fact be a feature of Coffin-Siris/NicolaidesBaraitser syndromes; a patient with intestinal inflammation and corneal changes harbouring compound heterozygous variants in the ADAM17 gene with possible functional implications in desmosome integrity; two unrelated patients with malignant atrophic papulosis/ Degos disease who had interesting variants in the IFIH1 and RNASET2 genes respectively, perhaps supporting existing concepts in the literature that Degos disease can be an end product of different physiopathological mechanisms on the one hand; and that it presents features of an interferonopathy on the other. Lastly, in this thesis I described two novel syndromes: - one in a patient with a very severe type I interferonopathy phenotype which I linked to a dominant negative variant in TRIM28 with incomplete penetrance. The functional data supported the central hypothesis of loss of a specific inhibitor of IRF7 resulting in a strong type I interferon signature at gene expression and cytokine level and conditional knock-out transfection studies in control cells showed similar readouts as those seen in the affected patient. - the other in siblings with a clinical diagnosis of early onset sarcoidosis, for which I proposed a digenic model caused by heterozygous variants in the TNFRSF25 and IL27RA genes, both previously implicated in the pathogenesis of sarcoidosis and/or granuloma formation. Whilst still requiring confirmation by transfection studies, this model was shown to segregate with disease within the family and the functional work performed thus far supported this hypothesis by showing increased protein expression of TNFRSF25 and increased P65 phosphorylation; and in relation to IL27RA, by showing increased STAT1/STAT3 phosphorylation and a consistent type I interferon signature at both gene expression and cytokine level. In summary, the studies described in this thesis underscore the importance of NGS combined with functional studies to characterise the molecular pathogenesis of rare autoinflammatory diseases, with important therapeutic implications.

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