A Germline Mutation in the C2 Domain of PLCγ2 Associated with Gain-of-Function Expands the Phenotype for PLCG2-Related Diseases

Abstract

We report three new cases of a germline heterozygous gain-of-function missense (p.(Met1141Lys)) mutation in the C2 domain of phospholipase C gamma 2 (PLCG2) associated with symptoms consistent with previously described auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome and pediatric common variable immunodeficiency (CVID). Functional evaluation showed platelet hyper-reactivity, increased B cell receptor-triggered calcium influx and ERK phosphorylation. Expression of the altered p.(Met1141Lys) variant in a PLCγ2-knockout DT40 cell line showed clearly enhanced BCR-triggered influx of external calcium when compared to control-transfected cells. Our results further expand the molecular basis of pediatric CVID and phenotypic spectrum of PLCγ2-related defects.

Keywords: Germline PLCγ2 mutations; PLCγ2 C2 domain.

Fig. 1

Cutaneous manifestations of PLCG2 mutation. Various polymorphic cutaneous manifestations including a vesiculobullous (case 1) b urticarial (case 1) and c, d nonspecific erythematous, polymorphic eruptions (case 2)

Fig. 2

Structure of PLCγ2 and multiple sequence alignment. a Schematic representation of the protein domains of PLCγ2. Annotated are previous cases of APLAID, PLAID, and somatic cancer-associated mutations linked to the Met1141 site. The yellow star refers to the mutation identified in the current report. b The region surrounding the p.Met1141Lys substitution site was aligned to the corresponding regions in a variety of organisms. Indicated in red is the substitution site. Asterisks indicate perfect conservation. PLCγ2, phospholipase C gamma 2; PH, pleckstrin homology domain; EF, EF hand; SH2, Src Homology 2 domain; SH3, Src Homology 3 domain; C2, C2 domain; APLAID, autoinflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency, and immune dysregulation (APLAID) syndrome; PLAID, PLCG2-associated antibody deficiency, and immune dysregulation syndrome

Fig. 3

B cell subset immunophenotyping of CD19+ B cells from patient (PLCγ2^M1141K) and healthy pediatric donor. a CD21^+/lowCD10+ transitional B cells. b CD38^highCD10+ transitional B cells. c CD27+IgD+IgM+ marginal zone B cells, CD27+IgD-IgM-switched memory B cells and CD27-IgD+IgM-naïve B cells. d IgG+IgM+, IgG-IgM-, and IgG-IgM+ B cells. e CD24^highCD38^high transitional B cells, CD24^highCD38^low memory B cells and CD24^lowCD38^low naïve B cells. 7AAD was used to exclude dead cells. Each of these plots represents three different experiments

Fig. 4

Evidence of altered BCR signaling. a Calcium flux in primary CD19+ B cells after BCR stimulation. Fresh PBMCs from patient (PLCγ2^M1141K) and healthy control were incubated in Ca²⁺-free media and stimulated via BCR followed by addition of exogenous Ca²⁺. Representative of three independent blood collections including one prior to initiation of IVIG therapy with three different sex-matched pediatric controls. b, c ERK1/2 phosphorylation after BCR stimulation. Primary CD19⁺ B cells from patient (PLCγ2^M1141K) and healthy control were stimulated for the indicated times and the phosphorylation of PLCγ2 (b) and ERK1/2 (c) was measured by intracellular flow cytometry. These experiments were repeated with three blood draws from the patient. Each data point is presented as a ratio of MFI to baseline MFI at start of incubation. Values represent means ± SEM. d Collagen-induced platelet alpha granule release. Fresh blood was collected without use of a tourniquet from the patient (PLCγ2^M1141K) and healthy controls into sodium citrate tubes with a discard tube first and stimulated with collagen. Alpha granule release from platelets was assessed by measuring the surface expression of CD62P on CD61+ platelets by flow cytometry. Each column represents data from 3 individual samples. e Equal amounts of PLCγ2 protein in about 1 million isolated CD19+ B cells was confirmed by immunoblotting. Representative of two independent collections

Fig. 5

BCR stimulation of PLCγ2 KO DT40 cells transfected with PLCγ2 constructs. a PLCγ2 KO DT40 cells were transiently transfected with empty plasmid, normal, and mutant PLCγ2^M1141K construct corresponding to our patients’ genetic variant. After overnight incubation, the transfected DT40 cells were incubated in Ca²⁺-free media and stimulated via BCR followed by addition of exogenous Ca²⁺. Each data point is representative of three independent experiments with measurements shown in 15-s intervals. Viable cells were analyzed for 60 s prior to BCR stimulation in order to establish an average baseline by which the relative fold increase in fluorescence is measured. b Western blot showing equal transfection expression of PLCγ2 constructs