Mutations in proteasome subunit β type 8 cause chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature with evidence of genetic and phenotypic heterogeneity

Abstract

Objective: Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE syndrome) is an autoinflammatory syndrome recently described in children. We undertook this study to investigate the clinical phenotype, genetic cause, and immune dysregulation in 9 CANDLE syndrome patients.

Methods: Genomic DNA from all patients was screened for mutations in PSMB8 (proteasome subunit β type 8). Cytokine levels were measured in sera from 3 patients. Skin biopsy samples were evaluated by immunohistochemistry, and blood microarray profile and STAT-1 phosphorylation were assessed in 4 patients and 3 patients, respectively.

Results: One patient was homozygous for a novel nonsense mutation in PSMB8 (c.405C>A), suggesting a protein truncation; 4 patients were homozygous and 2 were heterozygous for a previously reported missense mutation (c.224C>T); and 1 patient showed no mutation. None of these sequence changes was observed in chromosomes from 750 healthy controls. Of the 4 patients with the same mutation, only 2 shared the same haplotype, indicating a mutational hot spot. PSMB8 mutation-positive and -negative patients expressed high levels of interferon-γ (IFNγ)-inducible protein 10. Levels of monocyte chemotactic protein 1, interleukin-6 (IL-6), and IL-1 receptor antagonist were moderately elevated. Microarray profiles and monocyte STAT-1 activation suggested a unique IFN signaling signature, unlike in other autoinflammatory disorders.

Conclusion: CANDLE syndrome is caused by mutations in PSMB8, a gene recently reported to cause "JMP" syndrome (joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced childhood-onset lipodystrophy) in adults. We extend the clinical and pathogenic description of this novel autoinflammatory syndrome, thereby expanding the clinical and genetic disease spectrum of PSMB8-associated disorders. IFN may be a key mediator of the inflammatory response and may present a therapeutic target.

Conflict of interest statement

Conflict of interest: None.

Copyright © 2012 by the American College of Rheumatology.

Figures

Figure 1

Clinical features of CANDLE syndrome. A–D: Facial features and eruption in patients with CANDLE. Note the violaceous periorbital discoloration and edema, perinasal erythema, and significant fat loss most prominent in B. E, F: Finger swelling and violaceous lesions on the heel and toes. G: Discrete erythematous nodules and post-inflammatory hyperpigmentation. H, I: T2-weighted MRI of thighs (H) in one patient suggested loss of subcutaneous fat, particularly on the posterior aspect of both thighs; STIR image (I) showed increased signal intensity in the subcutaneous fat, suggesting panniculitis (asterisks). J: Synovial enhancement with rice body formation extending to the suprapatellar pouch.

Figure 2

Histopathology of lesional skin. A: H&E staining showed a dense perivascular and interstitial mononuclear dermal infiltrate. The overlying epidermis appears unremarkable. (H&E, 100× original magnification). B: Areas of karyorrhexis are shown. (H&E, 400× original magnification); C: Leder stain (Naphthol AS-D chloracetate esterase stain or specific esterase) specifically identifies cells of the granulocyte lineage, from the early promyelocyte stage to mature neutrophils. At high magnification (C-1: right upper panel), some of the mononuclear cells are Leder stain-positive, suggestive of immature neutrophils, and rare mature neutrophils. Another high magnification view (C-2: right lower panel) also shows karyorrhectic nuclear debris surrounded by a rim of Leder stain positive cytoplasm. (Leder stain, 400× original magnification). D–F: Staining for the macrophage markers KP-1 (CD68) (D) and CD163 (E), and myeloperoxidase positive cells (F). (Immunohistochemical stains, 200× original magnification).

Figure 3

Genetic analyses. A: Genome-wide homozygosity peaks discovered by HomozygosityMapper analysis. Black arrow indicates the chromosomal region of homozygosity shared by four patients (patients 1, 2, 4 and 5). B: Sequence analysis of PSMB8. a) wild type; b) heterozygous c.405C>A mutation; and c) homozygous c.405C>A mutation in patient 5. C: Sequence analysis of PSMB8. a) wild type; b) heterozygous c.224C>T mutation; and c) homozygous c.224C>T mutation in patients 1, 2, 4, 7, 8 and 9. D: Overview of PSMB8 coverage by exome sequencing demonstrating the identification of a homozygous mutation in PSMB8.

Figure 4

Functional assessment of PSMB8 mutations. A: Structural model of 20S proteasome. The model on the left shows a ring of β subunits in a proteasome, the inset on the upper right corner shows the position of the ring in 20S proteasome. The intact residues of the β5i subunit in the patient with the C135X mutation are shown in red and the deleted residues in gray. The T75 amino acid is represented by a purple ball. The model on the right shows two β4 subunits interacting with two β5i subunits on two adjacent rings, the inset on the upper right corner shows their position in a 20S proteasome. B: Cytokine expression in CANDLE patients 6, 7, 8, healthy controls, and NOMID patients. Data are shown on a log10 scale with error bars indicating standard errors of the mean (SEM). C: Whole blood microarray analysis was done on 4 CANDLE patients and 4 healthy age/gender matched controls. A color-coded heat map was generated on 42 IFN regulated genes from a transcript list that were two fold differentially expressed (p<0.05). Red indicates increased expression levels and blue indicates decreased expression levels compared to the mean of healthy controls.

Figure 5

Stat-1 -phosphorylation in CANDLE patients’ monocytes in response to IFN-γ stimulation, and inhibition by a JAK kinase inhibitor (tofacitinib). A: Stat-1 phosphorylation in response to 15 min IFN-γ stimulation in monocytes from CANDLE patients, healthy controls and a NOMID patient. The dashed line indicates an isotype control, the solid gray graph indicates the response of a healthy control and the solid line of the CANDLE patient. In the lower panels the bolded dashed line indicates the response of a NOMID patient. B: PBMCs from a healthy control and from pt7 were stimulated with 10IU/ml IFN-γ for 15 min in the absence (solid line) and presence of in 0.1 and 0.5μmol of tofacitinib (dashed and dotted lines respectively). A dose depended inhibition in stat-1 phosphorylation is seen in patient and control.