Rescue of recurrent deep intronic mutation underlying cell type–dependent quantitative NEMO deficiency
Bertrand Boisson, … , Masatoshi Hagiwara, Takahiro Yasumi
Bertrand Boisson, … , Masatoshi Hagiwara, Takahiro Yasumi
Published November 13, 2018
Citation Information: J Clin Invest. 2019;129(2):583-597. https://doi.org/10.1172/JCI124011.
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Research Article Genetics Infectious disease

Rescue of recurrent deep intronic mutation underlying cell type–dependent quantitative NEMO deficiency

Abstract

X-linked dominant incontinentia pigmenti (IP) and X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) are caused by loss-of-function and hypomorphic IKBKG (also known as NEMO) mutations, respectively. We describe a European mother with mild IP and a Japanese mother without IP, whose 3 boys with EDA-ID died from ID. We identify the same private variant in an intron of IKBKG, IVS4+866 C>T, which was inherited from and occurred de novo in the European mother and Japanese mother, respectively. This mutation creates a new splicing donor site, giving rise to a 44-nucleotide pseudoexon (PE) generating a frameshift. Its leakiness accounts for NF-κB activation being impaired but not abolished in the boys’ cells. However, aberrant splicing rates differ between cell types, with WT NEMO mRNA and protein levels ranging from barely detectable in leukocytes to residual amounts in induced pluripotent stem cell–derived (iPSC-derived) macrophages, and higher levels in fibroblasts and iPSC-derived neuronal precursor cells. Finally, SRSF6 binds to the PE, facilitating its inclusion. Moreover, SRSF6 knockdown or CLK inhibition restores WT NEMO expression and function in mutant cells. A recurrent deep intronic splicing mutation in IKBKG underlies a purely quantitative NEMO defect in males that is most severe in leukocytes and can be rescued by the inhibition of SRSF6 or CLK.

Authors

Bertrand Boisson, Yoshitaka Honda, Masahiko Ajiro, Jacinta Bustamante, Matthieu Bendavid, Andrew R. Gennery, Yuri Kawasaki, Jose Ichishima, Mitsujiro Osawa, Hiroshi Nihira, Takeshi Shiba, Takayuki Tanaka, Maya Chrabieh, Benedetta Bigio, Hong Hur, Yuval Itan, Yupu Liang, Satoshi Okada, Kazushi Izawa, Ryuta Nishikomori, Osamu Ohara, Toshio Heike, Laurent Abel, Anne Puel, Megumu K. Saito, Jean-Laurent Casanova, Masatoshi Hagiwara, Takahiro Yasumi

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Figure 7

The CLK inhibitor TG003 suppresses IVS4+866 C>T–induced PE inclusion to restore the production of a functional NEMO protein.

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The CLK inhibitor TG003 suppresses IVS4+866 C>T–induced PE inclusion ...
(A) Genomic sequence surrounding the 44-bp PE of IKBKG. Black bars indicate the RNA sequences used for the pulldown assays (PE-5′ and PE-3′, WT, and IVS4+866 C>T). (B) Western blot of RNA-pulldown products (WT and IVS4+866 C>T) for U1snRNP subunits (U1-70k, U1-A, U1-C, and SmB/B′). (C) Western blot of RNA-pulldown products (PE-5′, PE-3′, WT, and IVS4+866 C>T) for phosphorylated SR proteins. (D) Western blot of P3-derived iPSC-Mϕ transfected with nonspecific siRNA or SRSF1-specific or SRSF6-specific siRNA. β-Actin served as the internal control. (E) TNF-α production by P3-derived iPSC-Mϕ transfected with nonspecific, SRSF1-specific, or SRSF6-specific siRNA for 72 hours was evaluated 4 hours after stimulation with LPS and IFN-γ (n = 3). (F) Diagram of the SPREADD reporter for IKBKG exons 4–5 with the IVS4+866 C>T mutation. The GFP signal indicates IKBKG exon 4^5 splicing, whereas the RFP signal indicates inclusion of the 44-nt PE. (G) The intensities of green and red fluorescence were quantified in HeLa cells transfected with the IKBKG (IVS4+866 C>T) SPREADD vector and treated with small-molecule compounds (TG003, TG009, and SRPIN340) or not (0.1% DMSO) for 24 hours. Dot plots represent the GFP/RFP ratio of 6 random fields (80–100 fluorescence-positive cells/field) from a single experiment. **P < 0.001. (H) Representative fluorescence micrographs of cells treated with 10 μM TG003 or 0.1% DMSO following transfection with the IKBKG (IVS4+866 C>T) SPREADD vector. Nuclei were counterstained with Hoechst 33342. Scale bars: 200 μm. (I) Western blot of NEMO and β-actin for protein extracts from P3-derived or control iPSC-Mϕ. (J) TNF-α production by P3-derived iPSC-Mϕ stimulated with LPS and IFN-γ for 4 hours, with or without TG003 pretreatment. Data are presented as the mean ± SD of 3 independent experiments using a representative clone. *P < 0.05, by 1-way ANOVA followed by Dunnett’s test.