BackgroundMonogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the “Vasculitis and Inflammation Panel” (VIP) for AID and vasculitis.MethodsThe Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic.ResultsVIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%).ConclusionsThe high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID.

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BackgroundMonogenic autoinflammatory diseases (AID) are severe lifelong systemic inflammatory disorders with dysregulated innate immunity, causing significant morbidity, mortality, and economic burden. The number of AID is rapidly expanding. Securing a molecular diagnosis is of major importance for treatment, prognosis, and genetic counselling. Routine genetic screening is time-consuming, costly, and lacks sensitivity since only common disease harbouring exons of a minority of the known AID genes are currently tested using Sanger sequencing. Next-generation sequencing (NGS) offers the ability to rapidly and cost-effectively screen all exons of a gene panel containing hundreds of genes. This approach has not yet been routinely introduced in the UK for AID.ObjectivesTo develop and evaluate the performance of a NGS gene panel for AID.MethodsThe Agilent SureDesign tool was used to design an NGS panel targeting 113 genes, grouped into 9 broad clinical phenotypes: AID; monogenic vasculitis/vasculopathy; complement defects; monogenic lupus; hemophagocytic lymphohistiocytosis (HLH); early-onset inflammatory bowel disease; autoimmune lymphoproliferative syndromes; monogenic stroke; and hereditary amyloidosis. The targeted region includes coding exons, conserved non-coding exons, upstream promoter regions, and splice sites. Captured and indexed libraries (QXT Target Enrichment System) were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in paired-end mode. Positive controls for panel validation comprised 19 DNA samples from patients with confirmed mutations in a variety of genes on the panel. We then applied the panel to test 29 prospective samples with suspected but unconfirmed monogenic inflammation. Read alignment, variant calling, and annotation were performed using Agilent SureCall v3.0 software.ResultsIn the validation stage, our targeted panel detected all known mutations in the 19 positive control samples with an average read depth of 244X (+/− 29X) across the captured genes. The panel was effective at detecting different types of variants, including rare and common single nucleotide variants (SNVs), insertion/deletions, splice-junction variants, upstream promoter region variants, and somatic mosaicism. Prospective testing of the panel in 29 patients revealed potential pathogenic variants in 14 of the 29 patients giving a detection rate of 48%. These included atypical patients with novel mutations in specific exons/genes, not currently routinely available for testing by Sanger sequencing in the NHS.ConclusionsThis study demonstrates the clinical utility of a comprehensive NGS-based targeted gene panel which was both highly sensitive and specific in detecting different sequence variants of clinical significance. We are currently integrating this panel into a routine diagnostic laboratory testing strategy for monogenic inflammation. This will facilitate accurate and timely molecular diagnosis, more targeted treatment, and ultimately better outcomes for our patients.AcknowledgementThis work was su...

ObjectiveTo report a series of patients with cerebral arteriopathy associated with heterozygous variants in the casitas B-lineage lymphoma (CBL) gene and examine the functional role of the identified mutant Cbl protein. We hypothesized that mutated Cbl fails to act as a negative regulator of the RAS-mitogen-activated protein kinases (MAPK) signaling pathway, resulting in enhanced vascular fibroblast proliferation and migration and enhanced angiogenesis and collateral vessel formation.MethodsWe performed whole-exome sequencing in 11 separate families referred to Great Ormond Street Hospital, London, with suspected genetic cause for clinical presentation with severe progressive cerebral arteriopathy.ResultsWe identified heterozygous variants in the CBL gene in 5 affected cases from 3 families. We show that impaired CBL-mediated degradation of cell surface tyrosine kinase receptors and dysregulated intracellular signaling through the RAS-MAPK pathway contribute to the pathogenesis of the observed arteriopathy. Mutated CBL failed to control the angiogenic signal relay of vascular endothelial growth factor receptor 2, leading to prolonged tyrosine kinase signaling, thus driving angiogenesis and collateral vessel formation. Mutant Cbl promoted myofibroblast migration and proliferation contributing to vascular occlusive disease; these effects were abrogated following treatment with a RAF-RAS-MAPK pathway inhibitor.ConclusionsWe provide a possible mechanism for the arteriopathy associated with heterozygous CBL variants. Identification of the key role for the RAS-MAPK pathway in CBL-mediated cerebral arteriopathy could facilitate identification of novel or repurposed druggable targets for treating these patients and may also provide therapeutic clues for other cerebral arteriopathies.