Splice site and de novo variants can cause PLCG2-associated immune dysregulation with cold urticaria

Phospholipase Cγ2 (PLCγ2) is an important signaling molecule that receives and transmits signals from various cell surface receptors in most hematopoietic lineages. Variants of PLCG2 cause PLCγ2-associated immune dysregulation (PLAID), a family of conditions classified by mutational effect. PLAID with cold urticaria (PLAID-CU) is caused by in-frame deletions of PLCG2 that are dominant negative at physiologic temperatures but become spontaneously active at subphysiologic temperatures. We identified genetic lesions that cause PLAID by combining RNA sequencing of full-length PLCG2 with whole genome sequencing. We studied 9 probands with antibody deficiency and a positive evaporative cooling test, along with 2 known PLAID-CU patients and 3 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. Extracellular signal-regulated kinase (ERK) phosphorylation was quantified by flow cytometry with and without B-cell receptor crosslinking. Two probands expressed novel alternative transcripts of PLCG2 with in-frame deletions. Proband 1, expressing PLCG2 without exons 18-19, carried a splice site mutation in intron 19. Proband 2, expressing PLCG2 without exons 19-22, carried a 14 kb de novo deletion of PLCG2. DT40 cells overexpressing the exon 18-19 or exon 19-22 deletions failed to phosphorylate ERK in response to B-cell receptor crosslinking. In addition to autosomal dominant genomic deletions, de novo deletions and splice site mutations of PLCG2 can also cause PLAID-CU. All of these can be identified by cDNA-based sequencing.