PMC6431612_01
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.
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F1
On examination at the age of 7 years old she was noted to have eczematous dermatitis (Figure 1A), splenomegaly, and clubbing of her toes and fingers. Skin biopsy demonstrated superficial perivascular chronic inflammation with dense infiltration of CD4+ and focal MPO+ cells (Figure 1B and Figure S1).
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.
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F2
Consistent with the previous report of HOIP deficiency, the patient's B cell proliferation and CD80 expression were impaired after CD40 ligand (CD40L) stimulation and preserved after B cell receptor stimulation (Figures S2A,B).
Molecular consequences of the HOIP deficiency. (A) PBMCs from the HOIP deficient patient showed decreased levels of phosphorylated IkappaBalpha and delayed phosphorylation of IKKalpha/beta 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.
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F3
T lymphocyte proliferation following various stimuli were normal (Figure S3).