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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 February 1, 2019; First published November 13, 2018
Citation Information: J Clin Invest. 2019;129(2):583-597. https://doi.org/10.1172/JCI124011.
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Categories: Research Article Genetics Infectious disease

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

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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 5

The level of aberrant IKBKG splicing caused by IVS4+866 C>T mutation is cell type dependent.

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The level of aberrant IKBKG splicing caused by IVS4+866 C>T mutation ...
(A) RT-qPCR analysis of total cDNA from iPSCs, iPSC-MLs, and iPSC-NPs obtained from a control and P3. Data were obtained in triplicate and are expressed as ΔΔCt normalized against GAPDH. Representative results of 2 independent experiments are shown. (B) RT-qPCR amplification of full-length IKBKG from RNA extracted from control and P3-derived iPSCs, iPSC-MLs, and iPSC-NPs. (C) Percentage of IKBKG transcripts obtained after TA cloning experiments on P3-derived iPSCs, iPSC-MLs, and iPSC-NPs from B. (D–F) NEMO protein levels in total cell extracts from (D) SV40-immortalized fibroblasts established from controls, P2, a patient with a hypomorphic IKBKG mutation (NEMOX420W/Y), and a patient with complete NEMO deficiency (NEMOΔ4–10/Y), (E) frozen PBMCs from a control and P3, and (F) P3-derived iPSCs, iPSC-MLs, or iPSC-NPs with 3 independent clones. Numbers indicate the expression levels of NEMO relative to GAPDH (D and E) or β-actin (F).
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