FOXN1 compound heterozygous mutations cause selective thymic hypoplasia in humans

Abstract

We report on 2 patients with compound heterozygous mutations in forkhead box N1 (FOXN1), a transcription factor essential for thymic epithelial cell (TEC) differentiation. TECs are critical for T cell development. Both patients had a presentation consistent with T-/loB+NK+ SCID, with normal hair and nails, distinct from the classic nude/SCID phenotype in individuals with autosomal-recessive FOXN1 mutations. To understand the basis of this phenotype and the effects of the mutations on FOXN1, we generated mice using CRISPR-Cas9 technology to genocopy mutations in 1 of the patients. The mice with the Foxn1 compound heterozygous mutations had thymic hypoplasia, causing a T-B+NK+ SCID phenotype, whereas the hair and nails of these mice were normal. Characterization of the functional changes due to the Foxn1 mutations revealed a 5-amino acid segment at the end of the DNA-binding domain essential for the development of TECs but not keratinocytes. The transcriptional activity of this Foxn1 mutant was partly retained, indicating a region that specifies TEC functions. Analysis of an additional 9 FOXN1 mutations identified in multiple unrelated patients revealed distinct functional consequences contingent on the impact of the mutation on the DNA-binding and transactivation domains of FOXN1.

Keywords: Genetic diseases; Genetics; Immunology; Monogenic diseases; T cell development.

Figure 1. Compound heterozygous mutations in FOXN1 identified in 2 patients.

(A and B) Pt. 1 and Pt. 2 were identified from 2 independent and unrelated families. (C) Domain structure of FOXN1, with the 2 domains characterized to date: a DNA-binding region in the middle of the protein and a transactivation domain near the COOH terminus. Three FOXN1 autosomal-recessive mutations previously reported in patients with nude/SCID phenotypes are listed in red. Two patients, Pt. 1 and Pt. 2, presented with compound heterozygous mutations in FOXN1 at distinct sites, which are indicated above the exon assembly. (D and E) The DNA sequence mutations in FOXN1 and the corresponding effects on the amino acid sequence are shown for Pt. 1 (D) and Pt. 2 (E). The amino acid changes resulting from the various FOXN1 mutations are illustrated.

Figure 2. Human FOXN1 compound heterozygous mutations genocopied in mice cause thymic aplasia with normal fur and nails.

(A) The human FOXN1 mutations for Pt. 1 were introduced into the murine genome by CRISPR-Cas9 technology. The DNA repair template used for each allele is shown. Silent mutations were introduced into the murine sequence to facilitate genotyping and to prevent premature stop codons. (B) Images of F2-generation mice: WT Foxn1 (Foxn1^WT/WT), homozygous mutant (Foxn1^933/933 and Foxn1^1089/1089), and compound heterozygous (Foxn1^933/1089) mice, the latter genocopying Pt. 1. The genocopied mice are indicated in red font. The images are representative of 5 independently characterized mice. (C) The overall sizes of the thymi from the various mouse lines are shown for comparative purposes. (D) Thymus weights and overall thymic cellularity were calculated. Data represent the mean ± SEM. n = 22 Foxn1^WT/WT, n = 6 Foxn1^933/933, n = 10 Foxn1^1089/1089, and n = 10 Foxn1^933/1089 mice. P values of less than 0.05 were considered significant. For the comparisons shown, a Brown-Forsythe and Welch’s 1-way ANOVA was applied.

Figure 3. Thymic hypoplasia results from compound heterozygous mutations in Foxn1.

(A–D) WT Foxn1 (Foxn1^WT/WT), homozygous mutant (Foxn1^933/933, Foxn1^1089/1089), and compound heterozygous (Foxn1^933/1089) mice, the latter genocopying Pt. 1, were obtained by intercrossing the Foxn1^933/WT and Foxn1^1089/WT lines. The thymus was isolated and single-cell suspensions prepared for flow cytometry. (A) Fluorochrome-labeled antibodies against CD4 and CD8 were used to detect DN, DP, and SP thymocyte subsets. (B) The progression of thymocytes from the DN1–DN4 stages of thymocytes was assessed by cell-surface staining for CD44 and CD25 following exclusion of cells expressing CD3, CD4, CD8, B220, NK1.1, TCRγδ, Ter199, CD11b, and CD11c. (C) Positive selection of thymocytes was assessed by staining thymocytes for TCRβ and CD69. (D) Graphs show the percentage of various cell subsets, determined from pooled experiments using a minimum of 5 mice per line. Data indicate the mean ± SEM. n = 22 Foxn1^WT/WT mice, n = 6 Foxn1^933/933) mice, n = 6 Foxn1^1089/1089 mice, and n = 5 Foxn1^933/1089 mice. For the comparisons shown, a Brown-Forsythe and Welch’s 1-way ANOVA was applied. P values of less than 0.05 were considered significant. ****P < 0.0001 for most comparisons, with the following exceptions: P = 0.002 for DN comparisons of Foxn1^933/933 versus Foxn1^1089/1089; P = NS for DP comparisons of Foxn1^WT/WT versus Foxn1^1089/1089 and Foxn1^933/933 versus Foxn1^933/1089; P = 0.002 for CD4⁺ SP Foxn1^933/933 versus Foxn1^1089/1089 and Foxn1^1089/1089 versus Foxn1^933/1089; P = NS for Foxn1^933/933 versus Foxn1^933/1089; P = 0.05 for CD8⁺ SP Foxn1^933/933 versus Foxn1^933/1089. Comparisons for DN1–DN4 were as follows: P = NS for DN1 differences between Foxn1^WT/WT and Foxn1^1089/1089; P = 0.008 for DN2 comparisons of Foxn1^WT/WT versus Foxn1^1089/1089, P = 0.002 for DN2 comparisons of Foxn1^933/933 versus Foxn1^1089/1089, and P = NS for DN2 comparisons of Foxn1^1089/1089 versus Foxn1^933/1089; P = 0.002 for DN3 comparisons of Foxn1^933/933 versus Foxn1^1089/1089 and P = NS for DN3 comparisons of Foxn1^WT/WT versus Foxn1^1089/1089; P = 0.002 for DN4 comparisons of Foxn1^933/933 versus Foxn1^933/1089 and P = NS for DN4 comparisons of Foxn1^WT/WT versus Foxn1^1089/1089. ND, not detected; PE, phycoerythrin.

Figure 4. Mice with compound heterozygous mutations in Foxn1 have severe peripheral T cell lymphopenia.

(A–C) Lymph nodes were collected from the indicated mice and stained with antibodies detecting cell-surface CD4, CD8, B220, TCRβ, and NK1.1 expression. The cells were analyzed by flow cytometry comparing the cell-surface expression of (A) CD4⁺ and CD8⁺ SP cells; (B) B220 (marker of B cells) and CD3ε cell-surface expression; and (C) NK1.1 cell-surface expression and TCRβ. n = 3 to 26 mice per genotype for A–C. APC, allophycocyanin. (D) The percentages of CD4⁺ SP cells, CD8⁺ SP cells, B200⁺ B cells, and CD3ε⁺ T cells were calculated; n = 24 Foxn1^WT/WT, n = 5 Foxn1^933/933, n = 11 Foxn1^1089/1089, and n = 21 Foxn1^933/1089 mice. The percentages of NK1.1⁺ TCRβ^– cells were calculated; n = 26 Foxn1^WT/WT, n = 3 Foxn1^933/933, n = 11 Foxn1^1089/1089, and n = 11 Foxn1^933/1089 mice. For the comparisons shown, a Brown-Forsythe and Welch’s 1-way ANOVA was applied. P values of less than 0.05 were considered significant.

Figure 5. The thymic architecture is severely disrupted in mice with homozygous and compound heterozygous Foxn1 mutations that genocopied Pt. 1.

(A) Thymi were isolated from mice of the indicated genotypes (32–45 days old) and processed for H&E staining. Sections are shown at different magnifications to illustrate the severe thymic aplasia in the Foxn1^933/933 and Foxn1^933/1089 genotypes. Scale bars: 1 mm. (B) IHC was performed on thymic tissue sections with antibodies detecting cTECs (cytokeratin 8) and mTECs (cytokeratin 5). Two independent tissue samples were used per genotype and were labeled as the first set and second set. Scale bars: 0.5 mm. (C) Dispersed thymic tissues were prepared from mice of the indicated genotypes, and TECs were compared by flow cytometry, first by gating on EpCAM⁺CD45^– cells with antibodies detecting cTECs (BP-1) and mTECs (UEA-1) along with the cell-surface levels of MHC CII. (D and E) mTEC populations (UEA-1) were costained with antibodies detecting MHC CII in mice of the indicated genotypes, and dot blot comparisons are shown. (E) The MHC CII expression was compared between control littermates (black line) and Foxn1^1089/1089 mice (gray line). (F) The percentage of the different cTEC and mTEC subsets in the thymus of the indicated mice was calculated (n = 4 mice/group). Statistical analysis was performed using a Student’s t test.

Figure 6. Functional impairment of Foxn1 is dependent on the location and type of mutation.

(A–C) Transient transfection assays in HEK293T cells were performed with expression vectors for WT Foxn1 or Foxn1 constructs harboring the indicated mutations that matched the FOXN1 mutations identified in patients. The mutations were divided into those identified in Pt. 1 (A) (Foxn1⁹³³, Foxn1¹⁰⁸⁹) and Pt. 2 (B) (Foxn1¹²⁸⁸, Foxn1¹⁴⁶⁵) as well as the indicated controls. Forty-eight hours after transfection, the cells were lysed, and the proteins were extracted and resolved by SDS-PAGE. Western blotting was performed with antibodies against Foxn1, followed by antibodies detecting GAPDH, which was used as a loading control. Blots are representative of 4 independent experiments. (C and D) HEK293T cells were transfected with the indicated constructs along with a Psmb11 luciferase reporter construct and a β-gal vector. The mutations (mFoxn1) were grouped for those identified in Pt. 1 (C) (Foxn1⁹³³, Foxn1¹⁰⁸⁹) and Pt. 2 (D) (Foxn1¹²⁸⁸, Foxn1¹⁴⁶⁵) along with controls (D). Forty-eight hours after transfection, the cells were harvested, and luciferase activity was measured. The luciferase activity was normalized to β-gal, which was used as an internal control. Data are representative of triplicate samples from each group using 3 independent transfections per group. P values were determined using a standard 1-way ANOVA.

Figure 7. Differential functions of Foxn1 in TECs versus keratinocytes.

(A) Gene expression comparisons were made with 3 independently isolated fetal thymi, obtained from E13.5 embryos of the indicated genotypes. The presence of a hypoplastic thymi was confirmed by subsequent genotyping to select for Foxn1⁹³³ and Foxn1¹⁰⁸⁹ alleles. A heatmap revealed a subset of up- and downregulated genes, with a selection criterion of a 1.5-fold difference as a cutoff. (B) Data on differentially expressed transcripts, particularly those with reported functions in embryonic thymic development. (C) Images of postembryonic day–4 mice of the indicated genotypes were processed with bright-field imaging on a dark background, revealing hair and fur extending out from the skin. Scale bars: 1 mm. (D and E) qRT-PCR was performed to compare the levels of genes previously reported to be involved in hair shaft extension. This was performed to compare (D) 3 littermate controls and 3 Foxn1^933/933 mice and, in a separate experiment, (E) 3 controls and 3 Foxn1^933/1089 mice. The data shown reflect results from 1 of 2 independent experiments with 2 to 3 mice per group. ^#P = 0.02, ^##P = 0.001, and ^####P = 0.00006, by Student’s t test. There were no significant differences in any of the transcripts compared between the 2 mouse lines in E.