Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency - 07/03/18

Doi : 10.1016/j.jaci.2017.05.030

Daniel Petersheim, MD ^a,^{^{‡
Daniel Petersheim, MD, is currently affiliated with the Department of Pediatrics, Dr von Hauner Children's Hospital, Ludwig-Maximilians-Universität, Munich, Germany.},}^{^{∗
These authors contributed equally to this work.}}, Michel J. Massaad, PhD ^a,^{^{∗
These authors contributed equally to this work.}}, Saetbyul Lee, PhD ^a, Alessia Scarselli, MD ^b, Caterina Cancrini, MD ^b, Kunihiko Moriya, MD, PhD ^c, Yoji Sasahara, MD, PhD ^c, Arjan C. Lankester, MD, PhD ^d, Morna Dorsey, MD ^e, Daniela Di Giovanni, MD ^f, Liliana Bezrodnik, MD ^f, Hidenori Ohnishi, MD, PhD ^g, Ryuta Nishikomori, MD ^h, Kay Tanita, MD ⁱ, Hirokazu Kanegane, MD, PhD ⁱ, Tomohiro Morio, MD ⁱ, Erwin W. Gelfand, MD ^j, Ashish Jain, MD ^k, Elizabeth Secord, MD, PhD ^l, Capucine Picard, MD, PhD ^m, Jean-Laurent Casanova, MD, PhD ^m,ⁿ, Michael H. Albert, MD ^o, Troy R. Torgerson, MD, PhD ^p, Raif S. Geha, MD ^a,^⁎
^a Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, Mass
^b Division of Immunology and Infectious Diseases, Department of Pediatrics, Bambino Gesù Children's Hospital, Rome, and University of Rome Tor Vergata, Rome, Italy
^c Department of Pediatrics, Tohoku University, Tohoku, Japan
^d Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands
^e Division of Allergy, Immunology, and Bone Marrow Transplantation, University of California, San Francisco, Calif
^f Immunology Service, Ricardo Gutiérrez Children's Hospital, Buenos Aires, Argentina
^g Department of Pediatrics, Gifu University, Gifu, Japan
^h Department of Pediatrics, Kyoto University, Kyoto, Japan
ⁱ Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan
^j Immunodeficiency Diagnosis and Treatment Program, Department of Pediatrics, National Jewish Health, Denver, Colo
^k Merck Research Laboratories Boston, Boston, Mass
^l Division of Allergy, Asthma, and Immunology, Children's Hospital of Michigan, Detroit, Mich
^m Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital, Paris Descartes University, Paris, France
ⁿ St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY
^o Department of Pediatric Hematology and Oncology, Dr von Hauner University Children's Hospital, Ludwig-Maximilians-Universität Munich, Munich, Germany
^p Department of Pediatrics, University of Washington School of Medicine, Seattle, Wash

^∗Corresponding author: Raif S. Geha, MD, One Blackfan Circle, Boston, MA 02115.One Blackfan CircleBostonMA02115

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.

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Graphical abstract

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Key words : Ectodermal dysplasia with immune deficiency, NF-κB inhibitor α, canonical nuclear factor κB pathway, noncanonical nuclear factor κB pathway, lymphorganogenesis, hematopoietic stem cell transplantation

Abbreviations used : AD EDA-ID, DN, FKPM, GAPDH, HSCT, IκBα, ICAM-1, IKK, LN, LTo, LTβR, NF-κB, NF-κB1, NF-κB2, NIK, PID, qPCR, RelB, RNA-Seq, S32, S36, SLO, TLR, VCAM-1, WT

Plan

Methods

Cell lines

Plasmids and targeted mutagenesis

Transient transfection of HEK293T cells

Semiquantitative RT-PCR from transfectants

Fibroblast stimulation

Immunoblotting

RNA sequencing analysis of LPS plus α-LTβR–stimulated fibroblasts

Quantitative PCR analysis of CCL20, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 mRNA levels

Statistical analysis

Results

Patients with AD EDA-ID with point mutations in IκBα have more severe disease than patients with truncation mutations and a poor outcome after HSCT

IκBα point mutants are expressed at higher levels compared with IκBα N-terminally truncated mutants

IκBα point mutants impair LPS-driven IL-6 secretion and accumulate after LPS stimulation significantly more than IκBα truncation mutants

IκBα point mutants impair noncanonical NF-κB activation to a greater extent than do IκBα truncation mutants

Induction of genes encoding chemokines and adhesion molecules important for lymphorganogenesis is impaired more severely in patients with AD EDA-ID with IκBα point mutations than IκBα truncation mutations

Discussion

Supported by a scholarship from the DAAD Thematic Network “Research for Rare Diseases and Personalized Medicine” and a Carl Duisberg scholarship from Bayer Foundations (to D.P.) and USPHS grant 1R21AI124101-01 (to R.S.G).

Disclosure of potential conflict of interest: D. Petersheim receives grant support from the DAAD and Bayer Foundations. M. Dorsey receives travel support from CSL Behring and support from the National Institutes of Health (NIH). J.-L. Casanova serves on the board for ADMA; serves as a consultant for Pfizer, Novartis, Sanofi, Genetech, and Neovacs; and receives grant support from Merck Sharpe & Dohme and Biogen Idec. M. H. Albert provides expert testimony for Biotest; receives grant support from GlaxoSmithKline; received payment for lectures from MSD and Jazz; holds stock with Amgen, BMS, Biotest, and Juno; and received travel expenses from Octapharma, Medac, and Neovii. T. R. Torgerson serves as a consultant for Shire Biosciences, UCB Biopharma, CSL Behring, and ADMA Biosciences and receives grant support from Baxalta BioSciences and CSL Behring. The rest of the authors declare that they have no relevant conflicts of interest.

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Vol 141 - N° 3

P. 1060 - mars 2018 Retour au numéro

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Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency - 07/03/18

Abstract

Background

Objective

Methods

Results

Conclusions

Graphical abstract

Plan

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