Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency - PubMed (original) (raw)
. 2018 Mar;141(3):1060-1073.e3.
doi: 10.1016/j.jaci.2017.05.030. Epub 2017 Jun 17.
Michel J Massaad 1, Saetbyul Lee 1, Alessia Scarselli 2, Caterina Cancrini 2, Kunihiko Moriya 3, Yoji Sasahara 3, Arjan C Lankester 4, Morna Dorsey 5, Daniela Di Giovanni 6, Liliana Bezrodnik 6, Hidenori Ohnishi 7, Ryuta Nishikomori 8, Kay Tanita 9, Hirokazu Kanegane 9, Tomohiro Morio 9, Erwin W Gelfand 10, Ashish Jain 11, Elizabeth Secord 12, Capucine Picard 13, Jean-Laurent Casanova 14, Michael H Albert 15, Troy R Torgerson 16, Raif S Geha 17
Affiliations
- PMID: 28629746
- PMCID: PMC5733640
- DOI: 10.1016/j.jaci.2017.05.030
Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency
Daniel Petersheim et al. J Allergy Clin Immunol. 2018 Mar.
Abstract
Background: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in IκBα that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in IκBα have impaired noncanonical NF-κB activity and defective lymphorganogenesis.
Objective: We sought to establish genotype-phenotype correlation in patients with AD EDA-ID.
Methods: A disease severity scoring system was devised. Stability of IκBα mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-κB signaling in skin-derived fibroblasts.
Results: Disease severity was greater in patients with IκBα point mutations than in those with truncation mutations. IκBα point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-κB-dependent IL-6 secretion and upregulation of the NF-κB subunit 2/p100 and RELB proto-oncogene, NF-κB subunit (RelB) components of the noncanonical NF-κB pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-κB-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus α-lymphotoxin β receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations.
Conclusions: IκBα point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-κB activity in patients with AD EDA-ID.
Keywords: Ectodermal dysplasia with immune deficiency; NF-κB inhibitor α; canonical nuclear factor κB pathway; hematopoietic stem cell transplantation; lymphorganogenesis; noncanonical nuclear factor κB pathway.
Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Conflict of interest statement
The authors have declared that no conflict of interest exists.
Figures
Figure 1. Relative disease severity score in AD EDA-ID patients
A. Cumulative disease severity score expressed as a percentage of the maximal score based on the evaluable disease criteria listed in Table 2. B. Individual disease category scores for patients with IκBα point mutations and patients with IκBα truncations. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant; na, not applicable.
Figure 2. Expression of IκBα in fibroblasts and transfectants
A. Representative immunoblot of IκBα expression in cultured skin-derived fibroblasts from healthy controls (HC) and AD EDA-ID patients (P). P1 S32I and P2 S32I are patients 1 and 3 in Table I. B, C. Representative immunoblot (B) and quantitative analysis (C) of the expression of Flag-tagged WT and mutant IκBα in transfected HEK293T cells. Results in the left panel of C are expressed as the mean ratio of IκBα to tubulin of each individual mutant analyzed, relative to the mean of WT IκBα set at 1.0. Results in the right panel of C are expressed as the mean ratio of IκBα to tubulin of pooled IκBα truncation mutants and pooled IκBα point mutants relative to the mean of WT IκBα set at 1.0. D. RT-PCR analysis of mRNA encoding FLAG-tagged WT and mutant IκBα. The amounts of cDNA used were in the linear range determined for mRNA encoding FLAG-tagged WT IκBα. Data is representative of three independent experiments. Columns and bars represent the mean±SD of at least three independent experiments. **, p< 0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.
Figure 3. IκBα point mutants exert a stronger dominant negative effect on IL-6 secretion and accumulate to a greater extent following LPS stimulation than IκBα truncation mutants
A. LPS-driven IL-6 secretion by fibroblasts. Left panel: mean of three determinations on each of two healthy controls (HC), two patients with S32I, and individual patients with the other IκBα mutations. Right panel: Pooled results showing the mean IL-6 secretion by fibroblasts from HC (n=2), patients with IκBα truncation mutations (n=2) and patients with IκBα point mutations (n=4). B, C. Effect of LPS stimulation on IκBα levels: Representative (B) and quantitative (C) immunoblot analysis of IκBα expression in fibroblasts from two HC and AD EDA-ID patients before and after stimulation with LPS. Results from only one patient with S32I mutation are shown in B because of the limited number of lanes in the gel, but results from both patients with S32I mutation studied are included in the quantitative analysis, which includes 2 controls, 2 patients with IκBα truncation mutations and 4 patients with IκBα point mutations. Results in C are expressed as the mean ratio of IκBα to GAPDH relative to the mean of unstimulated HC set at 1.0. D. Mean ratio of mutant to WT IκBα in unstimulated and LPS-stimulated fibroblasts from AD EDA-ID patients with truncating mutations in IκBα. Columns and bars represent the mean±SD of three independent experiments. **, p<0.01; ***, p<0.001; ****, p<0.0001.
Figure 4. IκBα point mutants impair more severely LPS-driven expression of NF-κB2/p100 and RelB, and activation of the non-canonical NF-κB pathway than IκBα truncation mutants
A. Representative immunoblot analysis of p100 and RelB levels in fibroblasts from two healthy controls (HC) and AD EDA-ID patients (P) before and after stimulation with LPS for 48 hours. B, C. Quantitative analysis of expression of p100 (B) and RelB (C) in LPS-stimulated fibroblasts from two HC and individual patients (left panels), and pooled results representing the mean p100 and RelB expression levels in fibroblasts from patients with IκBα truncation mutations (n=2) and IκBα point mutations (n=4) (right panels). Results are expressed as the mean ratio of p100 and RelB to GAPDH relative to the mean of LPS-stimulated healthy controls set at 1.0. D. Representative immunoblot analysis of p100 and p52 levels in fibroblasts from two HC and AD EDA-ID patients before and after stimulation with LPS+anti-LTβR mAb for 48 hours. E, F. Quantitative analysis of expression of p52 (E) and p100 (F) in LPS+anti-LTβR mAb stimulated fibroblasts from healthy controls (HC) and individual patients (left panels) and pooled results representing the mean p52 and p100 expression levels in fibroblasts from HC (n=2), patients with IκBα truncation mutations (n=2) and IκBα point mutations (n=4) (right panels). Results are expressed as the ratio of p52 and p100 to GAPDH relative to the mean of LPS+anti-LTβR mAb stimulated (E) or unstimulated (F) HC set at 1.0. Columns and bars represent the mean±SD of at least three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.
Figure 5. IκBα point mutants impair the induction of genes important for lymphorganogenesis more severely than IκBα truncation mutants
A. Number of genes significantly (p<0.05) upregulated or downregulated by two folds or more in LPS+anti-LTβR mAb stimulated fibroblasts from healthy controls (n=3) and patients with IκBα truncation (n=2) and point mutations (n=2). B. Heat map analysis of NF-κB regulated genes the expression of which was significantly altered by LPS+anti-LTβR stimulation of normal fibroblasts. C. RNA-Seq analysis of the expression of CCL20, ICAM1 and VCAM1 mRNA in unstimulated and LPS+anti-LTβR mAb stimulated fibroblasts from healthy controls (n=3) and patients with IκBα truncation (n=2) and point mutations (n=2). Results are expressed as Fragments per Kilobase of Exon per Million (FPKM). Columns and bars represent the mean±SD for each of the three groups. D. q-PCR analysis of the expression of CCL20, ICAM1 and VCAM1 mRNA in unstimulated and LPS+anti-LTβR mAb stimulated fibroblasts from healthy controls (n=3) and patients with IκBα truncation (n=2) and point mutations (n=3). Results for ICAM1 and VCAM1 are expressed as the ratio of the gene of interest to GAPDH as calculated by the 2−ΔCt method relative to that of unstimulated healthy controls set at 1.0. Because CCL20 was undetectable in unstimulated fibroblasts, results are expressed as the ratio of CCL20 to GAPDH as calculated by the 2−ΔCt method. Columns and bars in D represent the mean±SD of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant.
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