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.

Keywords: Genetics; Infectious disease; Molecular diagnosis; NF-kappaB.

Figure 1. Pedigrees.

(A) Schematic representation of IKBKG and the surrounding region between positions X: 153,700,000 and X: 154,000,000. IKBKG and IKBKGP are indicated in red, the 35-kb duplication in green, and the region masked for the reanalysis in black. The variants reported in the 1kG and dbSNP 134 databases are shown in blue/red and gray, respectively. (B) Pedigrees of kindreds A and B. Patients with EDA-ID are indicated in black, and the mother with mild IP is indicated in gray.

Figure 2. Genomic strategies to identify IKBKG variants.

(A) Comparison of WGS mapping results between the classical and alternative (masking of the IKBKGP locus) strategies. The red/blue reads are the mapped sequences that can be used for variant calls (mappy quality score [MQ] >20), and the gray/white reads are the mapped sequences for which no variant could be called (MQ = 0). The duplicated region is indicated with a green bar and the masked region with a black bar. (B) Specific amplification by PCR of the full-length (top) or partial (bottom) IKBKG locus. gDNA from 2 controls (CTL), a patient (P2) and his mother, and from 2 NEMO^Δ4–10 (from 1 male and 1 female patient) SV40 immortalized fibroblast lines was used as the template. (C) IKBKG and IKBKGP DNA sequence electropherograms for controls, SV40-immortalized fibroblasts from a patient (P2) and his mother and a healthy brother (A.II.3), and for the leukocytes from a patient (P3) and his mother.

Figure 3. Reanalysis of the IKBKG locus with 1kG Project data.

(A) Correlation between the MAF of each variant in this study and the MAF reported in the 1kG Project. Plots show all the variants except for the IKBKG locus and the correlation for variants of FAM3 and GAB3, respectively. (B) Magnification of the IKBKG locus, for a comparison of the variants identified in this study (blue circles) with those reported in the various databases (red, orange, and green circles correspond to 1kG, ExAC, and ESP6500 databases, respectively). IKBKG exons are depicted by red bars and G6PD by gray bars.

Figure 4. The IVS4+866 C>T intronic mutation causes aberrant IKBKG splicing.

(A) Description of the impact of the mutation on the IKBKG transcript, focusing on the junction of exons 4 and 5. The PE (blue rectangle) was found in P2 and P3 but not in the controls. White rectangles indicate exons 4 and 5, and black rectangles indicate the alternative donor site (exon 4A) or the alternative acceptor site (exon 5A) found in the patients and controls. The IVS4+866 C>T mutation is indicated by a red arrow. (B) Northern blot analysis of purified mRNA from a control, P2 and his mother, and NEMO^Δ4–10/Y SV40-immortalized fibroblasts. The mRNAs were hybridized with a full-length ³²P-labeled IKBKG or ACTB cDNA. (C) RT-qPCR analysis of total cDNA from controls, P2 and his mother, and NEMO^Δ4–10/Y SV40-immortalized fibroblasts, and of whole-blood cells from a control and P3. Data are expressed as ΔΔCt normalized against GAPDH. The mean ± SD of the data obtained from 8 independent experiments is shown for the fibroblasts. The mean of triplicate experiments is shown for whole-blood cells and is representative of 2 independent experiments. (D) Full-length IKBKG amplification of purified mRNA from control and P2 SV40-immortalized fibroblasts as well as from whole-blood cells from a control and P3. (E) Schematic representation of IKBKG transcripts obtained after TA cloning experiments on controls, P2, and P3 from D. Approximately 100 clones were sequenced, and the results are expressed as percentages. (F) Sashimi plot of the RNA-Seq data from SV40-immortalized control (CTL1 and CTL2) and P2 fibroblasts. The red arrow indicates the position of IVS4+866 C>T and the new exon. Black and red numbers indicates the number of reads overlapping 2 consecutive exons. E1, E2, etc., exon 1, exon 2, etc.; M, molecular weight ladder; MW, molecular weight.

Figure 5. The level of aberrant IKBKG splicing caused by IVS4+866 C>T mutation is cell type dependent.

(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 (NEMO^X420W/Y), and a patient with complete NEMO deficiency (NEMO^Δ4–10/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).

Figure 6. NEMO insufficiency impairs NF‑κB activation in patients’ fibroblasts and iPSC-derived Mϕ.

(A) Impaired NF-κB activation in response to TNF-α. Time course for TNF-α–stimulated SV40‑immortalized fibroblasts, showing impaired IKKα/β phosphorylation (p-IKKα/β) but no impairment of IκBα degradation in P2 relative to the control. (B) Impaired NF-κB and ERK activation in response to LPS in P3-derived iPSC-Mϕ. Time course for LPS-stimulated iPSC-Mϕ, showing impaired phosphorylation of p65 and ERK1/2, but not of p38 or JNK, which are 2 proteins activated independently of NEMO. NF‑κB and ERK activation was rescued by reexpression of WT NEMO (P3 + NEMO). (C and D) Impaired NF-κB p65 nuclear translocation (C) in P2-derived SV40-immortalized fibroblasts and (D) P3-derived iPSC-Mϕ. NF-κB p65 nuclear translocation was rescued by reexpression of WT NEMO (P3 + NEMO). Data are shown as the mean ± SD of cells from more than 4 random fields taken from 1 well per condition, and the number of cells analyzed in each condition is shown in parentheses. Representative results of 3 independent experiments are shown. **P < 0.001, by 1-way ANOVA followed by Turkey’s test for multiple comparisons. (E) Impaired IL-6 production in response to TNF-α, TNF-β, IL-1β, poly (I:C), IL-17, and PMA in SV40-immortalized fibroblasts from P2 and 2 reported NEMO-deficient patients (NEMO^X420W/Y and NEMO^Δ4–10/Y). n = 3 or 4. (F) Impaired TNF-α production in P3-derived iPSC-Mϕ. Control and P3-derived iPSC-Mϕ were stimulated with LPS and IFN-γ for 4 hours before TNF-α determination by ELISA. TNF-α production of P3-derived iPSC-Mϕ was restored by reexpression of WT NEMO. Data are shown as the mean ± SD of 3 independent clones and are representative of 2 independent experiments. *P < 0.05, by unpaired, 2-tailed Student’s t test.

Figure 7. The CLK inhibitor TG003 suppresses IVS4+866 C>T–induced PE inclusion to restore the production of a functional NEMO protein.

(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.