Second Case of HOIP Deficiency Expands Clinical Features and Defines Inflammatory Transcriptome Regulated by LUBAC

Abstract

Background: HOIP is the catalytic subunit of the linear ubiquitination chain assembly complex (LUBAC) that is essential for NF-κB signaling and thus proper innate and adaptive immunity. To date only one patient with HOIP deficiency has been reported with clinical characteristics that include autoinflammation, immunodeficiency, amylopectinosis, and systemic lymphangiectasia. Case: We sought to identify a genetic cause of a disease for an 8 year-old girl who presented with early-onset immune deficiency and autoinflammation. Methods: Targeted next generation sequencing of 352 immune-related genes was performed. Functional studies included transcriptome analysis, cytokine profiling, and protein analysis in patients' primary cells. Results: We identified biallelic variants in close proximity to splice sites (c.1197G>C and c.1737+3A>G) in the RNF31 gene. RNA extracted from patient cells showed alternatively spliced transcripts not present in control cells. Protein expression of HOIP and LUBAC was reduced in primary cells as shown by western blotting. Patient-derived fibroblasts demonstrated attenuated IL-6 production, while PBMCs showed higher TNF production after stimulation with proinflammatory cytokines. RNA sequencing of whole blood RNA and PBMCs demonstrated a marked transcriptome wide change including differential expression of type I interferon regulated genes. Conclusion: We report the second case of HOIP deficiency with novel compound heterozygous mutations in RNF31 and distinct clinical and molecular features. Our results expand on the clinical spectrum of HOIP deficiency and molecular signatures associated with LUBAC deficiency.

Keywords: CVID; HOIL1; HOIP; LUBAC; SHARPIN; autoinflammation; primary immunodeficiency.

Figure 1

Clinical characteristics of the HOIP deficient patient. (A) Eczematous dermatitis. (B) Histopathology of the skin biopsy sample. (C) Information about the pedigree and the detected variants. (D) Genomic architecture of the RNF31 gene through exons 6 to 10. ZF: zinc finger; NZF: Npl4-zinc finger; UBA: ubiquitin-associated. (E) Subcloning of the RT-PCR product demonstrated multiple forms of aberrant splicing. The table demonstrates the number of clones.

Figure 2

Effects of the identified RNF31 mutations on LUBAC assembly. (A) The expression of LUBAC subunits in PBMCs. HOIP (long) indicates long exposure. C, unrelated age-matched control; Pt, the HOIP deficient patient; Mo, the mother. (B) Overexpression of HA-tagged mutant HOIP plasmids with SHARPIN and HOIL-1 in HEK293T cells. To mimic compound heterozygosity, two mutant plasmids were co-transfected. e7_del and e9_del indicate skipping of exon 7 and exon 9, respectively. (C) Immunoprecipitation of HA-tagged HOIP to assess the LUBAC assembly, in the presence of SHARPIN and HOIL-1 overexpression.

Figure 3

Molecular consequences of the HOIP deficiency. (A) PBMCs from the HOIP deficient patient showed decreased levels of phosphorylated IκBα and delayed phosphorylation of IKKα/β after TNF stimulation (20 ng/ml) compared with an unrelated healthy control. (B) Cytokine responsiveness of CD14⁺ monocyte subsets in the HOIP deficient patient and unrelated healthy controls (N = 2). Representative FACS plots are shown. Analysis was performed in triplicates. Two-way factorial ANOVA with Bonferroni adjustment was performed for the statistical analysis. ^*adjusted p < 0.05.

Figure 4

Inflammatory signatures of the HOIP deficient patient. (A,B) Heatmaps showing genome-wide expression change in the patient's whole blood (A) and PBMCs (B) obtained during symptom-free periods. C1-3, M, and P indicate healthy unrelated controls, the mother and the patient, respectively. (C,D) Heatmaps showing the expression patterns of differentially expressed genes regulated by IFNα2 in whole blood (C) and PBMCs (D). (E) M-A plot analysis showing differentially expressed genes between PBMCs of the HOIP deficient patient and unrelated controls. Red dots indicate genes differentially expressed with statistical significance (edgeR, p < 0.001). (F) Heatmap showing the expression of 28 genes in PBMCs that are known to be upregulated in monogenic type I interferonopathies. (G) Heatmap showing the expression pattern of differentially expressed genes regulated by TNF.

Figure 5

Flow cytometry analysis of STAT1 phosphorylation. PBMCs were stimulated with IFNα2 (20 ng/ml) for 20 min and CD4⁺, CD8⁺, and CD14⁺ cells were assessed for STAT1 phosphorylation.