FRI0001 NEXT GENERATION SEQUENCING ANALYSIS OF AN NLRP3 MUTATION NEGATIVE CAPS COHORT

Abstract

Background: Cryopyrin Associated Periodic Syndromes (CAPS) are caused by autosomal dominant gain of function mutations in the NLRP3 gene. However, a subset of these patients with typical clinical features and good response to anti-IL-1 treatment, have no mutation in NLRP3 detected by conventional Sanger DNA sequencing. Somatic mosaicism may account for between 19 to 69% of these, from previous reports. Genetic heterogeneity has also been implicated in patients with CAPS-like phenotypes, with the identification of pathogenic mutations in the NLRP12, PLCG2 and NLRC4 genes (Familial Cold Autoinflammatory Syndrome subsets 2, 3 and 4, respectively). Objectives: To explore the genetic cause(s) in a paediatric cohort of mutation negative CAPS patients using Next Generation Sequencing (NGS) techniques, by assessing: the rate of somatic NLRP3 mosaicism, and possible contribution of other/novel genes Methods: Patients from a single paediatric centre who met the CAPS diagnostic criteria and had no NLRP3 mutations identified by partial (exons 3, 4 and 6) or total Sanger sequencing, were identified. The NGS approach used in this study was Amplicon-based Deep Sequencing (ADS) for identification of NLRP3 mosaicism; and Whole Exome Sequencing (WES) for exploration genetic heterogeneity (i.e. other causative mutations). ADS was performed on an Ion Torrent platform using the Ion Torrent PGM HiQ sequencing kit. Resulting sequences were analysed using Torrent Server and in reporter softwares.WES was performed using the Nextera Exome Capture sequencing kit and Hiseq sequencing platforms. Exome data was analysed in the Galaxy web-based suit. The wANNOVER web-based software was used for variant annotation. Results: We identified a total of 8 patients who met the CAPS clinical diagnostic criteria and had no mutation identified by conventional Sanger sequencing. ASD identified the presence of a somatic mutation in 3 of these patients (37.5%), with an allelic frequency varying from 3.1 to 14.5%. The remaining patients were studied by WES. In Patient 4 a novel NLRP3 mutation was identified in exon 5, which had been previously missed due to a restricted Sanger approach. Patient 5 had a novel NOD2 mutation, thus redefining the diagnosis as Blau syndrome. WES findings in the other patients are summarized in the Table. Conclusion: In this study, somatic NLRP3 mutations were identified in 3/8 (37.5%) of our cohort, and 3/8 had variants in 3 other genes, potentially of relevance to the phenotype. In particular, the identification of a NOD2 mutation and subsequent reclassification of that patient as Blau syndrome, illustrates the limitations of current CAPS diagnostic criteria as well as potential clinical overlap between different autoinflammatory syndromes, reiterating the use of a wider NGS approach. At present it is unclear how the variants found in patients 6 and 7 could modulate the phenotype, therefore in 3 patients a clear genetic cause is yet to be found. In conclusion, this study suggests that NGS approaches are superior to conventional sequencing for the molecular diagnosis of CAPS and CAPS like phenotypes since they can detect somatic mosaicism or alternative molecular diagnoses mimicking CAPS. Disclosure of Interests: Sonia Melo Gomes: None declared, Ebun Omoyinmi: None declared, Juan Arostegui: None declared, Ying Hong: None declared, Nigel Klein: None declared, Paul Brogan Grant/research support from: SOBI, Novartis, Roche, Novimmune, Chemocentryx, Consultant for: Roche, SOBI, Speakers bureau: SOBI, Roche, Novartis, UCB