Elodie Bal1, Emmanuel Laplantine2, Yamina Hamel1, Virginie Dubosclard3, Bertrand Boisson4, Alessandra Pescatore5, Capucine Picard6, Smaïl Hadj-Rabia7, Ghislaine Royer1, Julie Steffann1, Jean-Paul Bonnefont1, Valeria M Ursini5, Pierre Vabres8, Arnold Munnich1, Jean-Laurent Casanova9, Christine Bodemer7, Robert Weil2, Fabrice Agou3, Asma Smahi10. 1. INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France. 2. Laboratory of Signaling and Pathogenesis, CNRS UMR 3691, Pasteur Institute, Paris, France. 3. Departments of Cell Biology and Infection and of Structural Biology and Chemistry, URA 2185, Pasteur Institute, Paris, France. 4. Rockefeller Branch, St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York, NY; Necker Branch, Laboratory of Human Genetics of Infectious Diseases, UMR 1163, Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France. 5. Institute of Genetics and Biophysics "Adriano Buzzati-Traverso" (CNR), Naples, Italy. 6. Rockefeller Branch, St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York, NY; Necker Branch, Laboratory of Human Genetics of Infectious Diseases, UMR 1163, Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France; Immunodeficiency Study Center, Necker Children's Hospital, Paris, France. 7. INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France; Department of Dermatology, Referral Center for Genodermatoses (MAGEC), Imagine Institute, Necker-Enfants Malades Hospital (AP-HP), Paris, France. 8. Department of Dermatology, Dijon CHU, Medicine Faculty and Bourgogne University, EA427 Genetic of Development Abonomalies, Bocage Hospital, Dijon, France. 9. Rockefeller Branch, St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York, NY; Necker Branch, Laboratory of Human Genetics of Infectious Diseases, UMR 1163, Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France; Pediatric Hematology, Immunology & Rheumatology Unit, Necker Children's Hospital, Paris, France. 10. INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France. Electronic address: asma.smahi@inserm.fr.
Abstract
BACKGROUND: Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [IKK] complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here we describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO. OBJECTIVES: We sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway. METHODS: We sequenced the IKBKG gene in patients with IP and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting, and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-β, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination using immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1β. RESULTS: We identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1β-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1-/- cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination. CONCLUSION: We identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.
BACKGROUND:Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [IKK] complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here we describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO. OBJECTIVES: We sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway. METHODS: We sequenced the IKBKG gene in patients with IP and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting, and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-β, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination using immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1β. RESULTS: We identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1β-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1-/- cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination. CONCLUSION: We identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.