Novel UNC13D intronic variant disrupting a NF κB enhancer in a patient with recurrent macrophage activation syndrome and systemic juvenile idiopathic arthritis

Abstract

Abstract Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (SJIA), and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH-3 (FHL3), but the role of non-coding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS. A custom enrichment library was constructed to sequence approximately 1 MB flanking UNC13D in 24 patients with SJIA, recurrent MAS, and negative prior genetic (exon/coding) testing. Functional consequences of intronic variants were assessed using quantitative PCR on patient derived peripheral blood mononuclear cells (PBMC), electrophoretic mobility shift assay (EMSA), in vitro transcriptional enhancer assays, and in vitro NK cell degranulation assays. We report a patient with SJIA and recurrent episodes of MAS, found to have a novel functional intronic variant in UNC13D, c.117+143A>G. This variant occurs in a proposed regulatory region that drives an alternative, lymphocyte specific UNC13D transcript, and is associated with reduced transcript levels in patient PBMC. In support of this, the patient’s variant disrupts NFκB binding to a functional transcriptional enhancer, leading to reduced enhancer activity and NK cell degranulation in vitro. Another patient was identified with a previously described UNC13D intronic variant, for a total non-coding variant hit rate of 8.3% (2/24). These findings highlight that intronic variants in key regulatory regions may be associated with MAS in patients with SJIA, and support deep sequencing approaches when causative coding variants are not identified.