A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency

Abstract

Whole-exome sequencing was performed in a family affected by dominantly inherited inflammatory disease characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency. Exome data from three samples, including the affected father and daughter and unaffected mother, were filtered for the exclusion of reported variants, along with benign variants, as determined by PolyPhen-2. A total of eight transcripts were identified as possible candidate genes. We confirmed a variant, c.2120C>A (p.Ser707Tyr), within PLCG2 as the only de novo variant that was present in two affected family members and not present in four unaffected members. PLCG2 encodes phospholipase Cγ2 (PLCγ2), an enzyme with a critical regulatory role in various immune and inflammatory pathways. The p.Ser707Tyr substitution is located in an autoinhibitory SH2 domain that is crucial for PLCγ2 activation. Overexpression of the altered p.Ser707Tyr protein and ex vivo experiments using affected individuals' leukocytes showed clearly enhanced PLCγ2 activity, suggesting increased intracellular signaling in the PLCγ2-mediated pathway. Recently, our laboratory identified in individuals with cold-induced urticaria and immune dysregulation PLCG2 exon-skipping mutations resulting in protein products with constitutive phospholipase activity but with reduced intracellular signaling at physiological temperatures. In contrast, the p.Ser707Tyr substitution in PLCγ2 causes a distinct inflammatory phenotype that is not provoked by cold temperatures and that has different end-organ involvement and increased intracellular signaling at physiological temperatures. Our results highlight the utility of exome-sequencing technology in finding causal mutations in nuclear families with dominantly inherited traits otherwise intractable by linkage analysis.

Figure 1

Clinical Manifestations in Two Individuals with the p.Ser707Tyr Substitution in PLCγ2 The inflammatory skin lesions observed in these affected individuals include recurrent eruptions of erythematous plaques (A) and vesiculopustular lesions (B) and the frequent evolution of skin lesions into cellulitis (C). (D) A dense interstitial and perivascular inflammatory infiltrate in the dermis is composed of lymphocytes, histiocytes, eosinophils, and much karyorrhectic nuclear debris. Karyorrhectic nuclear debris is typically seen in neutrophilic dermatosis.

Figure 2

Exome Sequencing Identifies a De Novo PLCG2 c.2120C>A Mutation in a Family Affected by Systemic Inflammatory Disease (A) Schematic representation of the exome-data-filtering approach under the assumption of dominant inheritance in the family. The following abbreviations are used: MS, missense variant; SS, splice-site variant; stop, stop-codon variant; FS, frameshift indel; and NFS, nonframeshift indel. (B) A pedigree of the family shows the unaffected grandparents and the affected father and daughter and indicates that the disease-causative mutation (c.2120C>A) arose as a de novo event in the affected father (II-1) and was dominantly inherited in the third generation. Sanger sequencing confirmed complete cosegregation of p.Ser707Tyr in PLCγ2 within the family. (C) The evolutionary conservation of the p.Ser707Tyr substitution of PLCγ2. Sequence alignment of PLCγ2 among various species shows that Ser707 (red indicated with an arrow) is a highly conserved residue. (D) Structure of C-terminal SH2 (cSH2) and SH3 domains of PLCγ2. The top panel shows a schematic representation of PLCγ2 and the location of the p.Ser707Tyr substitution. The p.Ser707Tyr substitution is not located within the catalytic domain of PLCγ2 (X-box = 316–458; Y-box = 926–1,029) but is located in the cSH2 domain. The lower panel shows a three-dimensional structural model of the PLCγ2 cSH2 domain (green) and SH3 domain (orange) with the use of homology modeling by SWISS-Model. The substitution of Ser707 (blue) with Tyr (red) lies in the cSH2 domain. The three known phosphorylation sites are depicted in magenta; Tyr733 is located in the cSH2 domain, whereas Tyr753 and Tyr759 are located in the linker region (blue gray) between the cSH2 domain and the SH3 domain.

Figure 3

IFN-γ Production in Stimulated PBMCs and Immunophenotyping of Expanded Individual B Cells (A and B) Peripheral-blood mononuclear cells (PBMCs) were isolated from heparinized blood and cultured in the presence of the indicated agonists for 48 hr. The harvested supernatant was analyzed for the IFN-γ production by multiplex cytokine analysis. The indicated control was representative of multiple different healthy controls. (C) B cell expansion in response to CpG and SAC stimulation of the affected individual, control, and PLAID PBMCs for 4 days is shown here as the fold increase from baseline after expansion in the percentage of B cells. The percentage of B cells among input or cultured PBMC was measured by flow cytometry. (D) IgG- or IgA-positive B cells from the cultures in (C) are measured by ELISPOT in the control, affected individual, and PLAID PBMCs. They are represented as Ig-positive cells per million B cells in the initial PBMC culture.

Figure 4

The p.Ser707Tyr Substitution Enhances PLCγ2 Activity and Intracellular Ca²⁺ Flux via the IP₃ Signaling Pathway (A) PLCγ2 activity was assayed in COS-7 cells transfected with wild-type (WT) or p.Ser707Tyr altered PLCγ2 constructs. [³H]inositol phosphate production was measured after stimulation with 10 ng/μl epidermal growth factor (EGF). PLCγ2 levels were analyzed by immunoblot assay (insets). (B) The WT PLCγ2 construct (#RC200442, Origene, Rockville, MD) and the p.Ser707Tyr altered PLCγ2 construct (Site-Directed Mutagenesis Kit, QuikChange II, Stratagene-Agilent Technologies, Santa Clara, CA) were transiently introduced into 293T cells with Lipofectamine 2000 according to the manufacturers’ protocols. The transfected 293T cells were treated for 1 hr with EGF (150 ng/ml, #CN02, Cytoskeleton, Denver, CO) for Rac-mediated PLCγ2 activation and were subjected to the IP-one assay. The IP₁ (surrogate for IP₃) level in the cell lysate was measured by IP-One ELISA (#72IP1PEA, Cisbio, Bedford, MA) according to the manufacturer’s instructions. (C) Increasing concentrations of PLCγ2 WT and p.Ser707Tyr altered constructs (0, 1.5, or 3.0 μg/10⁶ cells) were transiently introduced into 293T cells for an assay of the intracellular Ca²⁺ level. The transfected 293T cells were plated onto 96-well plates (70,000 cells per well) and treated with EGF (150 ng/ml) and the calcium-indicating dye (Calcium Assay Kit 640176, BD Biosciences, San Diego, CA). The plates were cooled down to room temperature for 15 min, and the relative fluorescence was measured every 10 s for the duration of 6 min with the use of Victor (PerkinElmer, Waltham, MA) according the manufacturers’ manuals. (D) The protein levels of PLCγ2 WT and p.Ser707Tyr altered constructs in 293T cells were confirmed by immunoblot assay. All graphs represent three or four independent experiments, and error bard indicate the mean ± the standard error of the mean (SEM).

Figure 5

Affected Individuals’ PBMCs Exhibit Significantly Higher PLCγ2 Activity PBMCs of two healthy controls—the unaffected mother (II-2) and another healthy control—and two affected individuals (III-2 and II-1) were isolated and subjected to IP-one (A) and intracellular Ca²⁺ (B) assays. PBMCs were coated with IgE anti-Dinitrophenyl (DNP) mAb (#D8406, 10 μg/ml, Sigma-Aldrich, St. Louis, MO) for 2 hr and then stimulated with DNP-albumin (#A6661, 100 ng/ml, Sigma-Aldrich). The graphs represent the mean ± SEM from three or four independent experiments. Error bars indicate the SEM. (C) Affected individuals’ cells show increased ERK phosphorylation. CD19 B cells from individuals III-2 and II-1, a healthy control sample, and a PLAID-affected individual were stimulated by surface anti-IgM crosslinking and analyzed for expression of phosphorylated ERK. Lines indicate phosphostaining in unstimulated cells (red) and anti-IgM stimulated cells (blue). The data are representative of three independent experiments.