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. 2022 Jun;74(6):1083-1090.
doi: 10.1002/art.42070. Epub 2022 Apr 23.

Haploinsufficiency of PSMD12 Causes Proteasome Dysfunction and Subclinical Autoinflammation

Kai Yan  1 , Jiahui Zhang  1 , Pui Y Lee  2 , Panfeng Tao  3 , Jun Wang  1 , Shihao Wang  1 , Qing Zhou  1 , Minyue Dong  1
Affiliations

Haploinsufficiency of PSMD12 Causes Proteasome Dysfunction and Subclinical Autoinflammation

Kai Yan et al. Arthritis Rheumatol. 2022 Jun.

Erratum in

  • Erratum.
    [No authors listed] [No authors listed] Arthritis Rheumatol. 2023 Jul;75(7):1138. doi: 10.1002/art.42563. Arthritis Rheumatol. 2023. PMID: 37377006 Free PMC article. No abstract available.

Abstract

Objective: Proteasome-associated autoinflammatory syndrome (PRAAS) is caused by mutations affecting components of the proteasome and activation of the type I interferon (IFN) pathway. This study was undertaken to investigate the pathogenic mechanisms of a newly recognized type of PRAAS caused by PSMD12 haploinsufficiency.

Methods: Whole-exome sequencing was performed in members of a family with skin rash, congenital uveitis, and developmental delay. We performed functional studies to assess proteasome dysfunction and inflammatory signatures in patients, and single-cell RNA sequencing to further explore the spectrum of immune cell activation.

Results: A novel truncated variant in PSMD12 (c.865C>T, p.Arg289*) was identified in 2 family members. The impairment of proteasome function was found in peripheral blood mononuclear cells (PBMCs), as well as in PSMD12-knockdown HEK 293T cell lines. Moreover, we defined the inflammatory signatures in patient PBMCs and found elevated IFN signals, especially in monocytes, by single-cell RNA sequencing.

Conclusion: These findings indicate that PSMD12 haploinsufficiency causes a set of inflammation signatures in addition to neurodevelopmental disorders. Our work expands the genotype and phenotype spectrum of PRAAS and suggests a bridge between the almost exclusively inflammatory phenotypes in the majority of PRAAS patients and the almost exclusively neurodevelopmental phenotypes in the previously reported Stankiewicz-Isidor syndrome.

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Figures

Figure 1
Figure 1
Identification of the PSMD12 variant and proteasome dysfunction in patients with the variant. A, Pedigree of the family carrying the heterozygous novel PSMD12 truncated variant p.R289*. Family members with the variant are patient 1 (P1) (proband [arrow]), an 8‐year‐old girl with urticarial skin rashes, severe intellectual disability, and developmental delay, and her father, patient 2 (P2), with congenital uveitis and mild intellectual disability. B, Schematic representation of the whole‐exome sequencing analysis and variant filtering approach used to identify the pathogenic variant in PSMD12. Single‐nucleotide polymorphism (SNP) includes missense (MS), splice site (SS), and stop codon variants and indel, frameshift, and non‐frameshift insertions and deletions. C, Confirmation of the nonsense variant by Sanger sequencing. D and E, Wild‐type (WT) and truncated PSMD12 expression in transfected HEK 293T cells (D) and in patient and control (C) peripheral blood mononuclear cells (PBMCs) (E). Experiments were performed at least 3 times for each sample. F, Significant differences in 3 types of proteasome proteolytic activity between patient and healthy control (HC) PBMCs. Symbols represent individual samples; for healthy controls, bars show the mean ± SEM. *** = P < 0.001; **** = P < 0.0001. G, Native gel analysis of the proteasome assembly in PBMCs from patients and controls. The assembly of different parts of the proteasome was illustrated using antibodies to the corresponding subunits. The loading amount of each sample is presented relative to the level of β‐actin determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. H, K48 ubiquitin (Ub) levels in PBMCs from the patients and 2 controls. FL = full‐length; RLU = relative luminescence units; CP = core particle; REG = regulatory particle.
Figure 2
Figure 2
Elevated inflammatory signals in PBMCs from patients with the PSMD12 variant. A, Western blots of interferon (IFN) signaling pathway–involved proteins in poly(I‐C)–stimulated PBMCs from patients 1 and 2 and control subjects. Results shown are representative of 2 independent experiments. B, Levels of the cytokines interleukin‐6 (IL‐6), tumor necrosis factor (TNF), and IL‐1β in cultured supernatants of PBMCs from patient 2 and 6 control subjects, measured by enzyme‐linked immunosorbent assay. C and D, Expression levels of IFN‐stimulated genes in PBMCs from patient 2 and 3 control subjects. PBMCs were treated with lipopolysaccharide (LPS) (C) or IFNβ (D) or were left untreated. ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001. E, Concentrations of the proinflammatory cytokines IL‐8, IFNγ, and IL‐1β and the chemokine IFNγ‐inducible protein 10 (IP‐10) in serum from patients 1 and 2 and from control subjects, determined by cytometric bead array analysis. Two samples from each subject, obtained at different times, were assessed. F, Expression patterns of genes involved in the IFN and NF‐κB signaling pathways in PBMCs from patients 1 and 2, healthy control subjects, and disease controls, determined by RNA sequencing. G, IFN response gene scores (28‐gene, 25‐gene, and 3‐gene) and NF‐κB score (11‐gene) in the 2 patients (PA), healthy controls, and disease controls (DC), determined using bulk RNA sequencing data. H, Expression levels of IFN pathway genes in LPS‐stimulated PBMCs from patients 1 and 2 and healthy controls, with and without baricitinib (Bari) treatment. In B–E, G, and H, symbols represent individual samples; for healthy controls (and for studies in which >1 sample from each of the 2 patients was assessed), bars show the mean ± SEM. MDA5 = myeloma differentiation–associated protein 5; RIG‐I = retinoic acid–inducible gene I; IRF3 = IFN regulatory factor 3; IFIT3 = IFN‐induced protein with tetratricopeptide repeats 3; SLE = systemic lupus erythematosus; CRIA = cleavage‐resistant receptor‐interacting protein kinase 1–induced autoinflammatory; DADA2 = deficiency of adenosine deaminase 2; (see Figure 1 for other definitions).
Figure 3
Figure 3
Enrichment of inflammatory pathways in monocytes from patients with the PSMD12 variant. A, Single‐cell RNA sequencing analysis of cell type distributions in PBMCs from the 2 patients and 2 healthy controls (left), and different cell clusters in the patients and controls (right). Control 1 was age‐ and sex‐matched to patient 1, and control 2 was age‐ and sex‐matched to patient 2. B, FeaturePlots of interferon (IFN)–related genes, created using single‐cell RNA sequencing data. Colored dots indicate single cells in the UMAP plot; red color indicates a higher level of gene expression. C, Violin plots showing the expression of IFN‐stimulated genes in CD14+ and CD16+ monocytes (Mono) from the 2 patients and 2 healthy control subjects. Light blue dots show the median expression level of all cells. D, Enriched hallmark gene set pathways of pathways in CD14+ monocytes analyzed by gene set enrichment analysis (GSEA). E, GSEA plot of inflammatory response, as well as IFNα, tumor necrosis factor (TNF), and IFNγ responses in CD14+ monocytes from the patients and controls. F, Comparison of type I IFN signaling pathways in CD14+ and CD16+ monocyte subsets from patients and controls, determined by single‐cell RNA sequencing. NK = natural killer; pDCs = plasmacytoid dendritic cells; mTORC1 = mechanistic target of rapamycin complex 1; PI3K = phosphatidylinositol 3‐kinase; UPR = unfolded protein response; ROS = reactive oxygen species; NES = normalized enrichment score (see Figure 1 for other definitions).
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