The 2020 Pediatric Rheumatology Symposium, originally scheduled for April 29 – May 2, was postponed due to COVID-19; therefore, abstracts were not presented as scheduled.
Session Type: Poster Breakout Session
Session Time: 5:10PM-5:40PM
Background/Purpose: PLCG2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammatory PLAID (APLAID) are autosomal dominant diseases caused by mutations of PLCG2. APLAID is clinically characterized by episodic fever, severe interstitial lung disease (ILD), antibody deficiency and inflammation of the eyes, skin and gut. To date, 3 mutations have been reported to cause APLAID (S707Y, L848P, M1141K), each leading to increased PLCg2-dependent signaling following stimulation. Since our initial descriptions of PLAID/APLAID, we have received >100 referrals of patients with severe forms of immune dysregulation and mutations in PLCG2. Here, we leverage this cohort to expand our understanding of PLCG2 variants that cause autoinflammatory phenotypes.
Methods: Subjects with immune dysregulation and mutations in PLCG2 were referred to our group for evaluation; those with autoinflammatory features (episodic fever or ILD) were identified and their mutations were investigated. Characteristics of PLCG2 variants, including allele frequencies, metrics of evolutionary conservation and predicted functional consequences, were assessed using Annovar. Mutant forms of PLCG2 were generated for in vitro studies by site-directed mutagenesis of a GFP-tagged PLCg2 construct. Constructs were overexpressed in Plcg2-deficient DT40 B cells. Cells were assayed for BCR-induced calcium flux and ERK phosphorylation. Cells were stained with the calcium-binding fluorophore, Indo-1, and intracellular calcium levels were observed by flow cytometry at baseline and following BCR cross-linking with anti-IgM antibody. Similarly, ERK phosphorylation was measured by flow cytometry before and after BCR-stimulation.
Results: We identified 16 subjects with autoinflammatory features bearing 16 ultra-rare/novel missense mutations of PLCG2. The most common autoinflammatory features were episodic fever (81%), cutaneous inflammation, including blistering, granulomatous or nodular skin lesions (63%) and ILD (38%). Arthritis and gastrointestinal inflammation were each present in 25% of patients, while half of patients had antibody deficiency. All mutations were located at evolutionarily conserved residues, each was predicted to alter protein function and 14 were previously unreported. Overexpression studies identified 3 groups of variants that produced distinct responses to BCR cross-linking: 1) 5 variants whose response was indistinguishable from wild type PLCG2; 2) 5 variants with hypermorphic responses consistent with S707Y; and 3) 6 variants with hypomorphic responses, 1 of which was amorphic. In all, we identified signaling alterations in 11/16 PLCG2 mutations found in these patients.
Conclusion: This study describes 14 new mutations of PLCG2 found in 16 APLAID patients with clinical features consistent with published cases. A third of the mutations produced a typical hypermorphic pattern in our in vitro system of B cell activation, but surprisingly, another third of variants reproducibly produced hypomorphic or amorphic responses. Future investigations should focus on the interrogation of primary patient cells and specific examination of differential effects in different PLCG2-expressing cells.
To cite this abstract in AMA style:Baysac K, Fisher C, Nakano H, Sun G, Milner J, Ombrello M. Functional Characterization of PLCG2 Mutations Found in Subjects with Autoinflammation and PLCG2-Associated Antibody Deficiency and Immune Dysregulation (APLAID) Reveals Both Hypermorphic and Hypomorphic Mutants [abstract]. Arthritis Rheumatol. 2020; 72 (suppl 4). https://acrabstracts.org/abstract/functional-characterization-of-plcg2-mutations-found-in-subjects-with-autoinflammation-and-plcg2-associated-antibody-deficiency-and-immune-dysregulation-aplaid-reveals-both-hypermorphic-and-hypomorp/. Accessed November 29, 2021.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functional-characterization-of-plcg2-mutations-found-in-subjects-with-autoinflammation-and-plcg2-associated-antibody-deficiency-and-immune-dysregulation-aplaid-reveals-both-hypermorphic-and-hypomorp/