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. 2023 Nov;152(5):1292-1302.
doi: 10.1016/j.jaci.2023.06.020. Epub 2023 Jul 6.

A gain-of-function variation in PLCG1 causes a new immune dysregulation disease

Panfeng Tao  1 , Xu Han  2 , Qintao Wang  3 , Shihao Wang  2 , Jiahui Zhang  2 , Lin Liu  4 , Xiaorui Fan  2 , Chenlu Liu  2 , Meng Liu  5 , Li Guo  6 , Pui Y Lee  7 , Ivona Aksentijevich  8 , Qing Zhou  9
Affiliations

A gain-of-function variation in PLCG1 causes a new immune dysregulation disease

Panfeng Tao et al. J Allergy Clin Immunol. 2023 Nov.

Abstract

Background: Phospholipase C (PLC) γ1 is a critical enzyme regulating nuclear factor-κB (NF-κB), extracellular signal-related kinase, mitogen-activated protein kinase, and nuclear factor of activated T cells signaling pathways, yet germline PLCG1 mutation in human disease has not been reported.

Objective: We aimed to investigate the molecular pathogenesis of a PLCG1 activating variant in a patient with immune dysregulation.

Methods: Whole exome sequencing was used to identify the patient's pathogenic variants. Bulk RNA sequencing, single-cell RNA sequencing, quantitative PCR, cytometry by time of flight, immunoblotting, flow cytometry, luciferase assay, IP-One ELISA, calcium flux assay, and cytokine measurements in patient PBMCs and T cells and COS-7 and Jurkat cell lines were used to define inflammatory signatures and assess the impact of the PLCG1 variant on protein function and immune signaling.

Results: We identified a novel and de novo heterozygous PLCG1 variant, p.S1021F, in a patient presenting with early-onset immune dysregulation disease. We demonstrated that the S1021F variant is a gain-of-function variant, leading to increased inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and increased phosphorylation of extracellular signal-related kinase, p65, and p38. The transcriptome and protein expression at the single-cell level revealed exacerbated inflammatory responses in the patient's T cells and monocytes. The PLCG1 activating variant resulted in enhanced NF-κB and type II interferon pathways in T cells, and hyperactivated NF-κB and type I interferon pathways in monocytes. Treatment with either PLCγ1 inhibitor or Janus kinase inhibitor reversed the upregulated gene expression profile in vitro.

Conclusions: Our study highlights the critical role of PLCγ1 in maintaining immune homeostasis. We illustrate immune dysregulation as a consequence of PLCγ1 activation and provide insight into therapeutic targeting of PLCγ1.

Keywords: PLCG1; autoinflammation; gain-of-function variant; immune dysregulation.

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Conflict of interest statement

Conflict of Interest Disclosure: The authors declare no conflict of interest relevant to the study.

Figures

Figure 1.
Figure 1.. Identification of a de novo PLCG1 variant in a patient with early-onset immune dysregulation disease.
(A) Schematic of data-filtering criteria identifying de novo exonic variant. SNV: single-nucleotide variants; INDEL: insertion & deletion variants; MS: missense; SS: splice-site. (B) Sanger sequencing verification of de novo variant in PLCG1. (C) Pedigree of the patient with de novo heterozygous variant S1021F in PLCG1. (D) The evolutionary conservation of the serine at PLCγ1 1021 site across molecules in the PLC family (left) and species (right). (E) Domain diagram of PLCγ1 drawn to scale. Position of S1021F is marked. (F) PLCγ1 structure (PDB: 6pbc) with domains coloured as in (E) and expanded view of wild type and S1021F mutation on PLCγ1 auto-inhibition interface. Protein structural analysis was executed with Chimera. (G) Protein expression levels of PLCγ1 in the patient and three healthy control T cells.
Figure 2.
Figure 2.. PLCγ1 gain-of-function variation causes strong activation of inflammatory signalling in the patient.
(A) Enrichment of differential expressed genes between patient and three healthy controls analyzed by gene set enrichment analysis (GSEA). NES, normalized enrichment score. (B) Transcription level of type I IFN, NF-κB and type II IFN pathways in PBMCs from the patient and three healthy controls. Analysis of each sample was performed in duplicate. For gene names, see Figure S1. (C) Type I and type II interferon score of the patient and three healthy controls (HCs). (D) qPCR analysis of cytokine and chemokine-related genes in PBMCs from the patient (sampling three times) compared with seven healthy controls (HCs). P values determined by unpaired two-tailed t-test. (E) Serum Levels of cytokines IL-6, IL-1β, and chemokines IL-8, and IP-10 in the patient (sampling three times) and fourteen healthy controls (HCs) were detected by cytometric bead array (CBA). P values determined by unpaired two-tailed t-test.
Figure 3.
Figure 3.. PLCγ1 gain-of-function variant S1021F enhances PLC activity and promotes downstream signalling pathways activation.
(A) IP1 level of PLCG1-knockout Jurkat cells complemented with empty vector (EV), wild-type (WT) PLCγ1 or S1021F mutant, treated with anti-human CD3 (5 μg/mL) for 1 hour. P values were determined by unpaired two-tailed t-test. (B) Intracellular calcium level of PLCG1-knockout Jurkat cells complemented with empty vector (EV), wild-type (WT) PLCγ1 or S1021F mutant, treated with anti-human CD3 (2.5 μg/mL). The fluorescence was measured every 6 s. (C) Western blots of PLCG1-knockout Jurkat cells complemented with empty vector (EV), wild-type (WT) PLCγ1 or S1021F mutant, treated as indicated. (D) Western blots of COS-7 cells over-expressed with empty vector (EV), wild-type (WT) PLCγ1 or S1021F mutant, treated as indicated. (E) NF-κB (top) and NFAT (bottom) luciferase assays in transiently transfected COS-7 cells. EV, empty vector; WT, wild-type PLCγ1. P values were determined by unpaired two-tailed t-test. (F) Flow cytometry analysis of phosphorylation of ERK in PLCG1-knockout Jurkat cells complemented with empty vector (EV), wild-type (WT) PLCγ1 or S1021F mutant, treated as indicated. (G) qPCR analysis of cytokines transcription level in PLCG1-knockout Jurkat cells complemented with empty vector (EV), wild-type (WT) PLCγ1 or S1021F mutant, treated as indicated. P values were determined by unpaired two-tailed t-test. (H) IP1 level of purified T cells from the patient and three healthy controls (HCs) treated as indicated for 1 hour. (I) Intracellular calcium level of purified T cells from the patient and three healthy controls treated with EGF (left) and anti-human CD3 (right) respectively. The fluorescence was measured every 10 s. (J) qPCR analysis of IFN and NF-κB pathway genes in PBMCs from the patient and healthy controls, with or without PLC inhibitor U73122 treatment.
Figure 4.
Figure 4.. Single-cell analysis of immunophenotype and activated inflammatory signalling in the patient.
(A) Single-cell RNA sequencing analysis. Annotation of clusters based on different cell type identities (upper), unique cluster in the patient (bottom). Arrow indicates the LDG cluster. Eryth, erythrocyte; MK, megakaryocyte; Treg, T regulatory cell; LDG, low-density granulocyte. (B) Expression levels of genes in NF-κB (TNFAIP3, IL8) and interferon pathway (ISG15, IFI44L) in T cells, CD14+ monocytes and CD16+ monocytes among patient and controls. (C) GSEA plot of differential expressed genes between patient and healthy controls in monocytes with NF-κB (NES=1.857, P<0.01) and IFN-α response (NES=2.272, P<0.0001) gene sets and T cells with NF-κB (NES=1.653, P<0.05) and IFN-γ response (NES=1.549, P<0.05) gene sets. (D) Expression levels of IFN-γ receptors in cells express them simultaneously. LDG, low-density granulocyte; DC, dendritic cell. (E) Proportion of immune cell subsets of the patient (sampling two times) represented as percent of agranulocytes (CD45+CD66b−) compared with two healthy controls. (F) Mean intensity of inflammatory markers relative to the average of two healthy controls. (G) qPCR analysis of IFN and NF-κB pathway genes in PBMCs from the patient and healthy controls, with or without JAK inhibitor baricitinib (Bari) treatment.
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