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

Background: Phospholipase Cγ2 (PLCγ2) is an important signaling molecule that receives and transmits signals from various cell surface receptors in most hematopoietic lineages. Variants of cause 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 of that are dominant negative at physiologic temperatures but become spontaneously active at sub-physiologic temperatures.

Objective: To identify genetic lesions that cause PLAID by combining RNA sequencing of full-length with whole genome sequencing.

Methods: We 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 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 -deficient DT40 cell overexpression system. ERK phosphorylation was quantified by flow cytometry with and without BCR crosslinking.

Results: Two probands expressed novel alternative transcripts of with in-frame deletions. The first, expressing without exons 18-19, carried a splice site mutation in intron 19. The second, expressing without exons 19-22, carried a 14kb deletion of . DT40 cells overexpressing the exon 18-19 or exon 19-22 deletions failed to phosphorylate ERK in response to BCR crosslinking.

Conclusion: In addition to autosomal dominant genomic deletions, deletions and splice site mutations of can also cause CU-PLAID. All of these can be identified by cDNA-based sequencing.