Severe Autoinflammatory Manifestations and Antibody Deficiency Due to Novel Hypermorphic PLCG2 Mutations

Abstract

Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demonstrated by both ex vivo calcium responses of the patient's B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation.

Keywords: APLAID; Autoinflammatory diseases; PLCγ2; agammaglobulinemia; caspase-1; inflammasome; interleukin-1.

Fig. 1

Familial pedigrees and features of PLCG2 variants. Panel a Pedigrees of enrolled families. Black filled symbols represent affected individuals, open symbols unaffected individuals, squares male individuals and circles female individuals. Panels b, c Schemes of filtering of the next-generation sequencing strategies used in the enrolled families. Panel d Sanger sense chromatograms of the PLCG2 gene from patients (upper boxes) and from wild-type healthy subjects (bottom boxes). Gray arrows indicate the PLCG2 variants detected in the patients. Panel e Scheme of structural domains of phospholipase Cγ2 protein. The already known APLAID-associated PLCG2 mutations are showed in red fonts, while the PLCG2 variants described in the present work are displayed in blue fonts. Panel f Multiple sequence alignment of human phospholipase Cγ2 and sixteen orthologues. The single asterisk represents the amino acid residue 708 of human phospholipase Cγ2, while two asterisks indicate the amino acid residues 845–848

Fig. 2

Description of cutaneous, pulmonary, and ocular inflammatory manifestations in patients. Panel a Multiple papules and serosal and hemorrhagic vesicles on the hands and palms detected in patient 1 at the age of 4 years. Panel b Large areas of cutis laxa in the abdominal region detected in patient 1 at the age of 7 years. Panels c, d Ocular inflammatory lesions including intense bilateral conjunctivitis, keratitis, episcleritis, and nodules in the sclera detected over the course of the disease in patient 1. Panel e Bronchiectasis detected in patient 1. Panel f Blistering inflammatory cutaneous lesions in the leg detected in patient 2 at the age of 6 months. Panel g Areas of cutis laxa detected in patient 2 at the age of 6 years. Panel h Ocular inflammatory lesions including conjunctivitis and corneal limbitis detected in patient 2 at the age of 7 years

Fig. 3

PLC activity analyses. Panel a The effect of p.Ala708Pro and p.Leu845_Leu848del variants on PLC activity was measured in transfected COS-7 cells under basal conditions (basal) or after stimulation by EGF (stimulated). Each data point represents the mean of triplicates and error bars indicate standard error of the mean. Expression levels of PLCγ2, corresponding to increasing concentrations of plasmids used for transfection, were measured using WES (top, inset). Further evaluation of the differences in PLC activities between the WT and variants was performed for the points with an equal protein expression. Panel b Position of p.Ala708 and p.Leu845-Leu848 segment (red arrows) is mapped on the structure of cSH2 and spPH domain, respectively. Positions of other mutations, reported for the main PLCγ1- and PLCγ2-linked pathologies that map to the same domains, are labeled using single letters and numbers corresponding to PLCγ2 sequence. Residues so far found to be mutated only in PLCγ1 are shown in gray. Other residues mutated in APLAID (p.Ser707 and p.Leu848) and Ali14 mice (p.Tyr495) are indicated by orange arrows

Fig. 4

Involvement of NLRP3-inflammasome activation in sterile inflammation. Panel a Heat map of cytokine analysis from peripheral blood mononuclear cell (PBMCs) supernatants after LPS stimulation as indicated (1 μg/mL, 2 h) isolated from healthy controls (n = 5), patients with CAPS (heterozygous for p.Arg260Trp NLRP3 mutation; n = 2) or APLAID carrying the heterozygous p.A708P (n = 1) or p.Leu845_Leu848del (n = 1) PLCG2 variants. Representative of relative values of minimum and maximum concentrations measured per cytokine. Panel b Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) speck forming monocytes by flow cytometry and active caspase-1 by YVAD-Fluorochrome Inhibitor of Caspases (FLICA) staining on monocytes after LPS stimulation as indicated (1 μg/mL, 2 h) isolated from healthy controls, patients with CAPS, or patients with APLAID. Showed results are representative of duplicate experiments. Panel c PBMCs IL-1β and IL-18 cytokine production at baseline and after LPS stimulation as indicated (1 μg/mL, 2 h) in healthy controls, patients with CAPS, and patients with APLAID. Showed results are representative of duplicate experiments. Panel d PBMCs IL-1β and percentage of monocytes stained for active caspase-1 by FLICA at baseline and after LPS stimulation (1 μg/mL, 2 h) in the presence or absence of BAPTA-AM (20 μM) or U73122 (2.5 μM) as indicated in APLAID patient 1 (p.Leu845_Leu848del PLCG2 variant) and her healthy mother. Showed results are representative of experiments performed only once due to limited availability of samples. Panel e PBMCs IL-1β and ASC speck forming monocytes upon canonical NLRP3 activation by LPS priming (1 μg/mL, 2 h) followed by 30-min treatment with ATP (3 mM) or nigericin (10 μM) in healthy controls, patients with CAPS, and patients with APLAID. Showed results are representative of duplicate experiments. ND, not detected