Splice site and de novo mutations can cause mixed dominant negative/gain of function PLCG2-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 PLCG2 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 PLCG2 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 PLCG2 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 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. Results Two 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. Conclusion In 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 Summary By 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (Z01-AR041198). Additional support was provided by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Z01-AI001098). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Board of the National Institutes of Health gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors