Human RELA dominant-negative mutations underlie type I interferonopathy with autoinflammation and autoimmunity

Abstract

Inborn errors of the NF-κB pathways underlie various clinical phenotypes in humans. Heterozygous germline loss-of-expression and loss-of-function mutations in RELA underlie RELA haploinsufficiency, which results in TNF-dependent chronic mucocutaneous ulceration and autoimmune hematological disorders. We here report six patients from five families with additional autoinflammatory and autoimmune manifestations. These patients are heterozygous for RELA mutations, all of which are in the 3' segment of the gene and create a premature stop codon. Truncated and loss-of-function RelA proteins are expressed in the patients' cells and exert a dominant-negative effect. Enhanced expression of TLR7 and MYD88 mRNA in plasmacytoid dendritic cells (pDCs) and non-pDC myeloid cells results in enhanced TLR7-driven secretion of type I/III interferons (IFNs) and interferon-stimulated gene expression in patient-derived leukocytes. Dominant-negative mutations in RELA thus underlie a novel form of type I interferonopathy with systemic autoinflammatory and autoimmune manifestations due to excessive IFN production, probably triggered by otherwise non-pathogenic TLR ligands.

Figure 1.

AD RELA deficiency in five families. (A) Pedigree of the five unrelated families showing familial segregation of the different RELA alleles. Generations are indicated by Roman numerals (I–II), and each individual is indicated by an Arabic numeral (1–12). Male and female individuals are represented by squares and circles, respectively. Affected patients are represented by closed black symbols, and asymptomatic carriers are indicated by a black vertical line. Individuals of unknown genotype are indicated by “E?” (B) Schematic representation of the RELA gene. Coding exons are numbered (1–11). The positions of the variants observed in the patients are indicated by arrows. Novel mutations in this study are shown in bold. DN mutations are underlined.

Figure S1.

Analysis of the RELA variants found in the patients. (A) A dot plot was created with PopViz-2 (Zhang et al., 2018). MAF: minor allele frequency. c.1416dupC is not shown because the CADD score was not available. (B) Total mRNA extracted from the whole blood of three controls and patients (P1/2/4/5) was subjected to RT-qPCR for the assessment of total RELA expression. Data are displayed as 2−ΔCt after normalization according to endogenous β-actin control gene expression. Mean ± SEM. N = 3. (C) RT-PCR products of control and P5 samples were separated by 3.0% agarose gel electrophoresis. We sequenced the three bands indicated by arrowheads. The normal size band in the P5 lane contained two sequences, and the extra band at ∼1,500 bp was retained by intron 10. The largest band was considered a heteroduplex. The PCR primers were 5′-CCT​ACT​GTG​TGA​CAA​GGT​GCA​GA-3′ and 5′-CTC​CTG​AAA​GGA​GGC​CAT​TG-3′. (D) The mutant sequence contained in the normal size band. Sequencing was performed by using the pCR2.1-TOPO vector (Thermo Fisher Scientific) with the purified band introduced. (E and F) Immunoblot analysis of the cytoplasmic (E) and nuclear (F) fractions primary fibroblasts stimulated with TNF for 30 min, including primary fibroblasts from two healthy controls (C1 and C2). P1 and P3 were subjected to immunoblot analysis with an anti-RELA antibody following induction with TNF for 30 min. Tubulin and lamin A/C were used as loading controls for the cytoplasmic and nuclear fractions, respectively. The results shown are representative of three independent experiments. Molecular weight units are given in kilodaltons. Source data are available for this figure: SourceData FS1.

Figure 2.

Effect of the pathogenic RELA variants on the expression and dimerization of RelA protein. (A) Immunoblot analysis of RelA protein levels in lymphoblastoid cell line and primary fibroblasts from controls and patients with AD RELA deficiency. The results shown are representative of three independent experiments. Molecular weight units were given in kilodaltons. C1 and C2 mean healthy controls 1 and 2. (B) Immunoblot analysis of RelA protein levels in total protein extracts of HEK293 cells transfected with anti–c-Myc, anti-Flag, or anti–β-actin antibody. (C) Whole-cell lysate immunoprecipitated with anti-HA antibody was used as an experimental negative control. Whole cell lysates were immunoprecipitated with anti–c-Myc antibody and then immunoblotted with anti-Flag or anti-Myc antibody. The p.R246* mutant, which causes RELA haploinsufficiency, impaired binding to WT RelA, whereas the other mutants exhibited normal binding to WT RelA. Two independent experiments were performed to confirm the results. Source data are available for this figure: SourceData F2.

Figure 3.

NF-κB reporter assay. (A) 25 ng plasmids encoding WT or mutant RelA protein was used. The p.E473Rfs*18 and p.H487Tfs*7 RELA variants were hypomorphic, having ∼8.0 and 32.3% residual activity, respectively, whereas the other five mutants, including p.R246*, were classified as LOF. (B) The total amount of expression vector, containing WT (12.5 or 25 ng) and mutant (12.5, 25, or 37.5 ng) RELA, was adjusted to 50 ng by supplementation with EV. All of the mutants, except for p.R246* mutants, which cause RELA haploinsufficiency, showed a dose-dependent negative effect against WT RelA. The mean ± SEM of three independent experiments is shown.

Figure S2.

Functional characterization of the RelA mutants in an overexpression system. (A and B) NF-κB reporter assay. 25 ng of WT or mutant RELA was used. The p.Y349Lfs* RELA was considered LOF. (B) The total amount of expression vector containing WT (12.5 or 25 ng) and mutant (12.5, 25, or 37.5 ng) RELA was adjusted to 50 ng by supplementation with EV. The p.Y349Lfs* RELA, as well as the p.Y349* mutant, showed dose-dependent negative effects against WT RELA. (C–J) Optimization of co-immunoprecipitation conditions. (C, E, G, and I) Immunoblot analysis to assess expression levels of Flag-RELA and Myc-tagged protein (RELA, p50, STAT1 or mock) in total protein extracts of HEK293 cell transfectants. (D, F, H, and J) Whole-cell lysates were immunoprecipitated with anti–c-Myc antibody and then immunoblotted with anti-Flag, anti-Myc, or anti–β-actin antibody. WT and mutant Flag-RELA did not bind to Myc-mock (D and H) or Myc-STAT1 (F and J). Under the same conditions, the p.R246* mutant which caused RELA haploinsufficiency impaired binding to WT RELA, whereas WT and the p.R329* mutant presented normal binding to WT RELA (D and F). Similarly, the p.R246* mutant impaired binding to WT p50, whereas WT and the p.R329* mutant presented normal binding to WT p50 (H and J). To confirm the result, two independent experiments were performed. (K and L) Dimerization between RELA and p50. (K) Immunoblot analysis to assess the expression levels of Flag-RELA and Myc-p50 protein in total protein extracts of HEK293 cells transfectants. (L) Extracts immunoprecipitated with anti-HA antibody are shown as experimental negative controls. Whole-cell lysates were co-immunoprecipitated with anti–c-Myc antibody and immunoblotted with anti-Flag, anti-Myc, or anti–β-actin antibody. The p.R246* mutant, which causes RELA haploinsufficiency, exhibited impaired binding to WT p50, whereas other mutants bound to WT p50. Two independent experiments were performed to confirm the result. Source data are available for this figure: SourceData FS2.

Figure 4.

RELA DN mutations underlie type I interferonopathy. (A and B) Relative expression of (A) six ISGs and (B) IFN scores of 11 healthy controls, five patients with DN RELA mutations, and three patients with AGS were evaluated by RT-qPCR. The relative abundance of each transcript was normalized to the expression level of β-actin, and the results are shown relative to a single calibrator. The experiment was performed in triplicate. The median of the relative quantification of the six ISGs was used to calculate the IFN score for each patient. IFN scores greater than +2 SD of the average of 11 healthy controls (5.04) were designated as positive. For each DN RELA patient except P3, multiple blood draws were taken at regular follow-up appointments in the absence of obvious signs of infection or fever. For P3, only one sample, taken just before bone marrow transplant (under conditioning), was available. For disease controls, data from three AGS patients (two genetically diagnosed: IFIH1 [p.R779H/WT] and IFIH1 [p.R720Q/WT], and one clinically diagnosed) was used. (C and D) PBMC stimulation assay. PBMCs of P2/3/5, patients with STAT1 GOF or STAT3 DN mutations, and healthy donors were stimulated with TLR7, TLR8, TLR7/8 agonists, or LPS for 24 h. (C) Secreted cytokines as measured by a LEGENDplex assay. NS, not stimulated. (D) Gene expression as determined by qPCR. GUSB was used as an internal control. In C and D, results from two experiments were compiled. Statistical significance of the difference between RELA DN patients and healthy controls was determined by two-tailed Wilcoxon’s rank sum test with FDR adjustment. Two independent experiments were performed to confirm the results. Bars represent the mean and SEM.

Figure S3.

Enhanced expression of type I and III IFNs and ISGs. (A and B) The concentration of (A) IFN-α2 and (B) TNF in the sera of five patients and five healthy controls. With the exception of P1, P5, and healthy control #5, the IFN-α2 concentration was below the limit of quantification (<4 pg/ml). (C–E) PBMC stimulation assay with neutralizing antibodies. PBMCs from P2/3/5 (biological duplicates for P3) and healthy donors were stimulated with TLR7, TLR8, TLR7/8 agonists, or LPS for 24 h, together with monoclonal antibodies neutralizing TNF, IFN-α, IFN-β, or IL-29, or corresponding isotype control antibodies. (C and D) Secreted cytokines were measured by a LEGENDplex assay. (E) ISG mRNA levels were determined by qPCR. GUSB was used as an internal control. Relative suppression was defined as RQ_{T, Neut} × RQ_{NS, Isotype}/RQ_{NS, Neut} × RQ_{T, Isotype}, where RQ is a GUSB-normalized relative expression level, T is a TLR7/8 agonist, NS is a nonstimulated condition, Neut denotes a neutralizing antibody, and Isotype a corresponding isotype control antibody. Values below 1 indicate suppression of a given ISG by addition of a given neutralizing antibody. In C–E, bars represent the mean and SEM.

Figure 5.

Cellular responses to type I and II IFNs. (A) T cell blast stimulation assay. T cells were expanded for ∼13 d and restimulated with the indicated reagents for 4 h. Gene expression levels were determined by qPCR. GUSB was used as an internal control. (B–D) Monocyte stimulation assay. CD14⁺ monocytes were sorted, rested overnight, and then stimulated with the indicated reagents for 4 h. (B) Secreted cytokines as measured by a LEGENDplex assay. Statistical significance of the difference between RELA DN patients and healthy controls was determined by two-tailed Wilcoxon’s rank sum test with FDR adjustment. (C and D) RNA sequencing. (C) Global transcriptional profiles were summarized via uniform manifold approximation and projection (UMAP). (D) Differential expression analysis. Genes with log₂ fold-changes below −1 or above 1, together with FDR-adjusted P values below 0.05, are colored. In A and B, bars represent the mean and SEM.

Figure 6.

Type I IFN signatures across lymphoid and myeloid leukocyte subsets. scRNASeq analysis was performed on cryopreserved PBMCs from P1/2/3/4/5, patients with STAT1 GOF (N = 2), or STAT3 DN (N = 1) mutations, and healthy adult and pediatric controls. (A) Unsupervised clustering. Graph-based clustering was performed after batch correction with Harmony, and clusters were manually identified with the aid of the SingleR pipeline informed by the MonacoImmuneDataset. (B) GSEA. Genes were ranked based on the fold-change estimated through pseudobulk differential expression analysis between patients and healthy pediatric controls. Gene ranking was projected against the Hallmark gene sets (http://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp?collection=H). Only gene sets with FDR-adjusted P values below 10⁻²⁰ in at least one cell type are shown. NES, normalized enrichment score. (C) Heatmap showing the log₂ fold-change values between patients and healthy pediatric controls for the GSEA leading-edge genes for the hallmark IFN-α signaling gene set in classical monocytes. (D) Intercellular communication analysis with CellChat. Statistical significance for the difference between RELA DN patients and healthy children was determined through two-tailed Wilcoxon’s rank sum test with FDR adjustment. Bars represent the mean and SEM.

Figure S4.

Single-cell transcriptomic analysis of leukocyte subsets. (A and B) Cluster annotations. (A) Fraction of cell subsets defined based on the combination of canonical surface markers in 10X public CITE-Seq datasets superimposed onto each manually annotated cluster in our in-house dataset. Treg, regulatory T cell; CM, central memory T cell; EM, effector memory T cell; TEMRA, effector memory-expressing CD45RA T cell; MAIT, mucosal-associated invariant T cell, B Nv, naïve B cell; B Mm, memory B cell; ClasMono, classical monocyte; NClasMono, non-classical monocyte; cDC, classical dendritic cell. (B) Expression levels of select marker genes in each manually annotated cluster. (C) Relative abundance of cell types defined through clustering. Bars represent the mean and SEM. (D) GSEA for hallmark gene sets compared to healthy adult controls, as described in Fig. 6 B. (E) GSEA for TLR-related gene sets taken from the MsigDB database. Statistically nonsignificant (FDR-adjusted P values of 0.05 or higher) pathway–cell type pairs are shown by a small dot. NES, normalized enrichment score.

Figure 7.

Enhanced expression of molecular components involved in TLR7 signaling. (A) Heatmap showing log₂ fold-change values between RELA DN patients and healthy pediatric controls for pseudobulk GSEA leading-edge genes for the Kyoto Encyclopedia of Genes and Genomes (KEGG) TLR signaling gene set in pDCs. (B) Fold-change induction over nonstimulated conditions of IRF7 mRNA in PBMCs from RELA DN patients (P2/3/5; biological duplicates for P3) and healthy controls stimulated with TLR agonists for 24 h. GUSB was used as an internal control. (C) Fraction of cells expressing mRNA for genes involved in TLR7 signaling. Statistical significance for the difference between RELA DN patients and healthy children was determined through a two-tailed Wilcoxon’s rank sum test with FDR adjustment. Bars represent the mean and SEM.

Figure S5.

Skin manifestation and pathological findings at skin biopsy of P4. (A–C) Subcutaneous nodule on the trunk (A) and rash on the left upper arm (B) and left dorsum of the foot (C). (D–G) H&E staining (D–F) and Alcian blue staining (G) of skin biopsies. The pathological findings showed slight infiltration of mononuclear cells around the small vessels of the upper dermis of the trunk (D) and in the samples from the upper dermis of the left upper arm (E; scale bar is 500 μm; original magnification, 2×); higher magnification (F) clearly showed some spongiosis in the epidermis (arrow) and slight infiltration of mononuclear cells around the small vessels (arrowheads). Sparse spaces between collagen fibers observed in F were not stained with Alcian blue (G; scale bar is 100 μm; original magnification, 40×), which may be an artifact of specimen preparation.