Abstract
AbstractBackgroundPhospholipase Cγ2 (PLCγ2) is an important signaling molecule that receives and transmits signals from various cell surface receptors in most hematopoietic lineages. Variants ofPLCG2cause PLCγ2-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 ofPLCG2that 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-lengthPLCG2with 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-lengthPLCG2cDNA synthesized from peripheral blood mononuclear cell RNA and whole genome sequencing was used to identify genetic lesions. Novel alternate transcripts were overexpressed in thePlcg2-deficient DT40 cell overexpression system. ERK phosphorylation was quantified by flow cytometry with and without BCR crosslinking.ResultsTwo probands expressed novel alternative transcripts ofPLCG2with in-frame deletions. The first, expressingPLCG2without exons 18-19, carried a splice site mutation in intron 19. The second, expressingPLCG2without exons 19-22, carried a 14kbde novodeletion ofPLCG2. 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 novodeletions and splice site mutations ofPLCG2can also cause CU-PLAID. All of these can be identified by cDNA-based sequencing.Capsule SummaryBy identifying both the firstde novoand splice site variants to causePLCG2-associated immune dysregulation with cold urticaria (CU-PLAID), we demonstrate the diagnostic utility ofPLCG2-specific RNA-sequencing.
Publisher
Cold Spring Harbor Laboratory