Splice site and de novo mutations can cause mixed gain of function/dominant negative PLCG2-associated immune dysregulation with cold urticaria (CU-PLAID)

BackgroundPhospholipase C{gamma}2 (PLC{gamma}2) is an important signaling molecule that receives and transmits signals from various cell surface receptors in most hematopoietic lineages. Variants of PLCG2 cause PLC{gamma}2-associated immune dysregulation (PLAID), a family of conditions that are classified by mutational effect. PLAID with cold urticaria (CU-PLAID) is caused by in-frame deletions of PLCG2 that are dominant negative at physiologic temperatures but become spontaneously active at sub-physiologic temperatures. ObjectiveTo identify genetic lesions that cause PLAID by combining RNA sequencing of full-length PLCG2 with whole genome sequencing. MethodsWe studied nine probands with antibody deficiency and a positive evaporative cooling test, together with two known CU-PLAID patients and three healthy subjects. Illumina sequencing was performed on full-length PLCG2 cDNA synthesized from peripheral blood mononuclear cell RNA and whole genome sequencing was used to identify genetic lesions. Novel alternate transcripts were overexpressed in the Plcg2-deficient DT40 cell overexpression system. ERK phosphorylation was quantified by flow cytometry with and without BCR crosslinking. ResultsTwo probands expressed novel alternative transcripts of PLCG2 with in-frame deletions. The first, expressing PLCG2 without exons 18-19, carried a splice site mutation in intron 19. The second, expressing PLCG2 without exons 19-22, carried a 14kb de novo deletion of PLCG2. DT40 cells overexpressing the exon 18-19 or exon 19-22 deletions failed to phosphorylate ERK in response to BCR crosslinking. ConclusionIn addition to autosomal dominant genomic deletions, de novo deletions and splice site mutations of PLCG2 can also cause CU-PLAID. All of these can be identified by cDNA-based sequencing. Capsule SummaryBy identifying both the first de novo and splice site variants to cause PLCG2-associated immune dysregulation with cold urticaria (CU-PLAID), we demonstrate the diagnostic utility of PLCG2-specific RNA-sequencing.