PLCG2-associated immune dysregulation (PLAID) comprises broad and distinct clinical presentations related to functional classes of genetic variants

Abstract

Background: Pathogenic variants of phospholipase C gamma 2 (PLCG2) cause 2 related forms of autosomal-dominant immune dysregulation (ID), PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammatory PLAID (APLAID). Since describing these conditions, many PLCG2 variants of uncertain significance have been identified by clinical sequencing of patients with diverse features of ID.

Objective: We sought to functionally classify PLCG2 variants and explore known and novel genotype-function-phenotype relationships.

Methods: Clinical data from patients with PLCG2 variants were obtained via standardized questionnaire. PLCG2 variants were generated by mutagenesis of enhanced green fluorescent protein (EGFP)-PLCG2 plasmid, which was overexpressed in Plcg2-deficient DT-40 B cells. B-cell receptor-induced calcium flux and extracellular signal-regulated kinase phosphorylation were assayed by flow cytometry. In some cases, stimulation-induced calcium flux was also measured in primary patient cells.

Results: Three-fourths of PLCG2 variants produced functional alteration of B-cell activation, in vitro. Thirteen variants led to gain of function (GOF); however, most functional variants defined a new class of PLCG2 mutation, monoallelic loss of function (LOF). Susceptibility to infection and autoinflammation were common with both GOF and LOF variants, whereas a new phenotypic cluster consisting of humoral immune deficiency, autoinflammation, susceptibility to herpesvirus infection, and natural killer cell dysfunction was observed in association with multiple heterozygous LOF variants detected in both familial and sporadic cases. In some cases, PLCG2 variants produced greater effects in natural killer cells than in B cells.

Conclusions: This work expands the genotypic and phenotypic associations with functional variation in PLCG2, including a novel form of ID in carriers of heterozygous loss of PLCG2 function. It also demonstrates the need for more diverse assays for assessing the impact of PLCG2 variants on human disease.

Keywords: Phospholipase C gamma 2; antibody deficiency; autoinflammation; immune dysregulation; primary immune deficiency; variants of uncertain significance.

Figure 1.. Summary of PLCG2 variants identified in subjects with immune dysregulation.

A linear schematic of PLCγ2 displays the domain localization of PLCG2 variants identified in subjects with immune dysregulation in this study (A). PLAID-causing deletions (grey bars) and gain-of-function hotspots (asterisks) are noted above the schematic; variants that alter in vitro activation in this study are shown in bold; previously reported variants are italicized. In panels B-D, pie charts illustrate distributions of variant characteristics, including variant allele frequencies from the Genome Aggregation Database (gnomAD) v2.1.1 population (B), degree of evolutionary conservation as indicated by SiPhy scores (C), and damage prediction as indicated by phred-scaled CADD scores (D) for the 60 PLCG2 variants examined in the study.

Figure 2.. Loss of function variation is the largest functional class of PLCG2 variation in subjects with immune dysregulation.

A linear schematic of PLCγ2 displays the domain localization of the 29 hypomorphic variants identified in subjects with immune dysregulation in this study (A). In Panel B, bar graphs display relative fold-change (FC) of ERK phosphorylation by flow cytometry in Plcg2-deficienct DT-40 cells overexpressing either wild type or mutant PLCG2 constructs 5 minutes after BCR cross-linking (B). The bars indicate the median of 3 or more independent experiments, error bars indicate interquartile range, and asterisks indicate statistical significance (p < 0.05) by Mann-Whitney nonparametric test. Variant labels (A) and bar plots (B) are colored to indicate the degree of hypomorphism (FC>0.75, green; FC 0.75–0.51, orange; FC 0.49–0.25, yellow; FC<0.25, red).

Figure 3.. Autosomal dominant familial immune dysregulation co-segregates with variants of uncertain significance of PLCG2.

Pedigrees are displayed for 4 families in whom PLCG2 variants co-segregated with multi-generational immune dysregulation. The PLCG2 variant status is indicated beneath each subject. The table displays a summary of clinical features of immune dysregulation observed in the affected members of the 4 families. wt, wild type; IBD, inflammatory bowel disease; NK, natural killer; ILD, interstitial lung disease.

Figure 4.. Deficient natural killer cell activity in 2 families with PLCG2 variants, autoinflammation and recurrent herpesvirus infections.

NK cytotoxic killing, expressed as % specific lysis of K562 cells in ⁵¹CR release ex vivo assay, is plotted in 2 affected subjects from Family 2 and an unrelated control subject (A) and 2 affected subjects and an unaffected family member from Family 3 (B). Representative tracings of in vitro stimulated calcium flux in Plcg2^−/− DT-40 cells expressing N1097del (C) and L170F (D) variants are paired with tracings of ex vivo stimulated calcium flux in primary B-cells from Family 2–2 and 2 unrelated control subjects (E) and 1 unaffected and 2 affected members of Family 3 (F). In vitro tracings are representative of 3 or more independent experiments. IL-2, interleukin-2.

Figure 5.. A subset of PLCG2 variants from subjects with immune dysregulation are hypermorphic following stimulation and/or constitutively active.

A linear schematic of PLCg2 displays the domain localization of reported hypermorphic variants of PLCG2 (above), together with the hypermorphic variation identified in subjects with immune dysregulation in this study (bottom, A). In Panels B and C, wild-type (WT) or mutagenized PLCG2 plasmids were overexpressed in a Plcg2-deficienct DT-40 B-cell line and transfected cells were stimulated with IgM. Cytoplasmic calcium was measured in resting cells by flow cytometry for 5 seconds and displayed as fold change of the area under the curve (AUC) for that interval, relative to WT PLCG2 (B). Cytoplasmic calcium was also measured by flow cytometry for 240 seconds following stimulation with IgM and ERK phosphorylation was measured by flow cytometry 10 minutes after stimulation (C). Calcium data are displayed as fold change of the area under the curve (AUC) for the interval, relative to WT PLCG2. ERK phosphorylation is displayed as fold-change of mean fluorescence intensity (MFI), relative to WT PLCG2. Bars indicate the median of 3 or more independent experiments, error bars display the interquartile range. Statistical comparisons were performed using the Mann-Whitney U Test. *, p < 0.05. EV, empty vector. WT, wild type.