Research ArticleTHYMUS

PAX1 is essential for development and function of the human thymus

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Science Immunology  28 Feb 2020:
Vol. 5, Issue 44, eaax1036
DOI: 10.1126/sciimmunol.aax1036
  • Fig. 1 Pedigrees and PAX1 genetic studies.

    (A) Pedigrees and results of Sanger sequencing in patients with PAX1 variants and in healthy controls. For both family A and family B, results of Sanger sequencing in the heterozygous parents are also shown. (B) Schematic representation of the PAX1 protein and location of the variants identified in affected individuals.

  • Fig. 2 Molecular and functional analysis of PAX1 mutant proteins.

    (A) Western blot showing expression of WT and mutant human PAX1 proteins upon transient transfection in 293T cells. (B) Left: Intracellular protein localization upon transfection of HA-tagged WT and mutant PAX1 constructs into 293T cells, followed by staining with TRITC anti-HA. Right: Counterstaining with DAPI, demonstrating that the mutant PAX1 protein retains nuclear translocation capacity. Scale bar, 10 μm. (C) Results of a luciferase reporter assay demonstrating reduced transcriptional activity of mutant PAX1 proteins, corresponding to the PAX1 variants detected in patients. The promoter region of Nkx3-2 was used to drive luciferase expression. Results of six independent experiments (each run in triplicate) are shown (means ± SEM). P value was calculated with one-way ANOVA and adjusted by Dunnett’s multiple comparisons test. **P < 0.01; ***P < 0.0001.

  • Fig. 3 In silico analysis of the PAX1 paired box domain in WT and mutant proteins.

    (A) Molecular modeling of the paired box domain of WT and mutant PAX1 proteins, showing the presence of two globular domain separated by a linker. Note that the asparagine residue at position 155 is adjacent to linker domain, and its deletion results in shortening of the last turn of the third α helix in the first globular domain of the paired box domain. (B) Molecular superimposition of WT (in light blue) and mutant PAX1 variants after MD simulation, showing that both the Val147Leu and Asn155del variants predominantly affect the conformation of the C-terminal globular domain, whereas both globular domains are affected by the Gly166Val variant. (C) RMSF values of WT PAX1 and of the Val147Leu, Asn155del, and Gly166Val variants during MD simulations. RMSF values are used here as a measure of the flexibility of different regions of the protein during the MD simulations. The Y axes indicate the magnitude of the fluctuation, whereas the X axes indicate the specific location of each amino acid within the paired box domain.

  • Fig. 4 Interface analysis of PAX1 paired box domain and DNA interaction.

    Nucleotide residues, in which the paired box domain of either WT or PAX1 mutant proteins establishes interaction, are shown in black. The amino acids contacting nucleotides of target DNA are indicated on the Y axis for each PAX1 protein. The red and green colors indicate loss and gain of DNA binding, respectively.

  • Fig. 5 Differentially expressed genes between control and patient TEPs.

    (A) Heatmap of differentially expressed genes between iPS and TEP stage as determined by RNA-seq. Each heatmap shows the top 3000 genes, which were differentially expressed between iPS and TEP cells, with a significance (q < 0.01) by the two-group comparison (t test). Genes whose expression was found to be up-regulated at the TEP stage included epithelial cell markers (EPCAM, KRT8, and KRT19) as well as several genes (PSEN1, HES1, ASXL1, HOXA3, HAND2, EPHB3, and GATA3), which appeared at the leading edge of GSEA of thymus development in (B). (B and C) GSEA on thymus development gene set by preranked genes according to signed −log10 adjusted P value. The adjusted P value was acquired by DEseq2 analysis using normalized read count of RNA seq data. FDR, false discovery rate. (D) qRT-PCR analysis of FOXN1 and DLL4 expression at TEP stage of differentiation. Results are from five independent experiments for control and P1, and four independent experiments for control and P4, with triplicates in each case (mean ± SEM). The P value was calculated with two-tailed paired t test. P < 0.05 was considered to be significant. (E) Thymus development genes with evidence of differential expression between patient and control cells (adjusted P < 0.1 and concordant pattern of expression in both RNA-seq experiments). For this comparison, we considered genes that were part of the Thymus development gene set in MSigDB v7.0, and in the top 30 FOXN1 target genes reported in (19). The values displayed are the signed −log10 adjusted P value for differential expression.

  • Table 1 Laboratory data at presentation.

    AEoC, absolute eosinophil count; ALC, absolute lymphocyte count; ANC, absolute neutrophil count; n.d.: not done; cpm, counts per minute.

    Patient (age)P1
    (9 months)
    P2
    (14 days)
    P3
    (17 days)
    P4
    (1 month)
    P5
    (6 months)
    P6
    (1 month)
    Control
    values
    ANC (cells × 10−3/μl)0.522.776.361.223.83.961.0–9.0
    ALC (cells × 10−3/μl)2.092.182.462.3446.71.553.4–7.6
    AEoC (cells × 10−3/μl)5.860.220.670.389.60.860.05–0.7
    Platelets (× 10−9/liter)8389667324780449150–400
    CD3+ cells/μl1777625843,985182,500–5,500
    CD4+ cells/μl1038705010,762181,600–4,000
    CD8+ cells/μl291410017,3130560–1,700
    CD19+ cells/μl6181,4821,107946936811300–2,000
    CD16/56+ cells/μl2083591,1074821,4041,205170–1,100
    In vitro proliferation
    to PHA (cpm × 10−3)
    19.8*5.5n.d.2.162.50.65(≥ 94.9)
    IgG (mg/dl)1,450745658690170650611–1,542
    IgA (mg/dl)27<6.6<6<25<25<257–37
    IgM (mg/dl)21251749117<2526–122
    IgE (kU/liter)1,241730<2<210,080<20–9

    *Measured as % of CD3+ cells incorporating Edu (normal value: ≥58.5).

    • Table 2 HSCT in patients with pathogenic PAX1 variants.

      ATG, anti-thymocyte globulin; PBSC, peripheral blood stem cells; URD, unrelated donor.

      PatientP1P2P3P4
      Age at transplantation12 months4.5 months3.5 months4 months
      Transplant characteristics
        Donor type, HLA-matchingURD, 9/10Mother, 4/8Mother, 4/8URD, 4/6
        Source of stem cellsBone marrowT cell–depleted bone
      marrow
      PBSCCord blood
        Conditioning regimenAlemtuzumab 1 mg/kg
      Fludarabine 150 mg/m2
      Melphalan 140 mg/m2
      Busulfan 8 mg/kg
      Cyclophosphamide 200 mg/kg
      First HSCT:
        Treosulfan 30 g/m2
        Fludarabine 120 mg/m2
        ATG 4 mg/kg
      Second HSCT (7 months):
        Thiotepa 10 mg/kg
        Cyclophosphamide 150 mg/kg
        ATG 25 mg/kg
      Busulfan 16 mg/kg
      Fludarabine 160 mg/m2
      ATG 20 mg/kg
      Engraftment and immune reconstitution
        Time post-transplant9 months2 months11.5 months after 2nd HSCT4 years
        Chimerism (source)100% Donor, whole bloodAutologous78% recipient; 22% donor100% donor
        ANC (×10−3/μl)1.964.53.272.99
        ALC (×10−3/μl)0.710.452.70.44
        CD3+ cells/μl24795424
        CD4+ cells/μl7641321
        CD8+ cells/μl1281304
        CD19+ cells/μl1893622650*
        CD16/56+ cells/μl91n.d.32386
      Outcome, age,
      and cause of death
      Alive, 6 years and 4 months,
      waiting for thymic
      transplantation
      Deceased, 9.5 months,
      respiratory distress
      Deceased, 4 years and 7 months,
      septic shock
      Alive, 4 years and 4 months

      *Post-rituximab for severe autoimmune hemolytic anemia.

      Supplementary Materials

      • immunology.sciencemag.org/cgi/content/full/5/44/eaax1036/DC1

        Materials and Methods

        Fig. S1. Somatic and skeletal anomalies of P1 and P3.

        Fig. S2. Immunological phenotype of P1.

        Fig. S3. Lymph node pathology in P2.

        Fig. S4. Scheme of filtering used for analysis of WES data in patients P1, P2, and P4.

        Fig. S5. Evolutionary conservation of PAX1 Val147, Asn155, Gly166, and Cys368 amino acid residues.

        Fig. S6. Protein stability of mouse PAX1 mutants corresponding to human PAX1 variants.

        Fig. S7. Ramachandran plot of PAX1 structural modeling.

        Fig. S8. MD simulation of PAX1 conformations.

        Fig. S9. DNA-PAX1 paired box domain variant complexes after MD simulations.

        Fig. S10. Stemness profile of iPSCs.

        Fig. S11. Differentiation of iPSCs into TEP cells in control donor.

        Fig. S12. Immunofluorescence analysis of KRT8 protein expression of control (C) and patient (P1) TEP cells.

        Fig. S13. Differentially expressed genes between control and patient cells at TEP stage of differentiation.

        Table S1. List of candidate genes after filtering WES data.

        Table S2. Other genes involved in thymic development, with concordant differential expression in P1 and P4 versus control TEP cells.

        Data file S1. Raw data (Excel).

      • Supplementary Materials

        The PDF file includes:

        • Materials and Methods
        • Fig. S1. Somatic and skeletal anomalies of P1 and P3.
        • Fig. S2. Immunological phenotype of P1.
        • Fig. S3. Lymph node pathology in P2.
        • Fig. S4. Scheme of filtering used for analysis of WES data in patients P1, P2, and P4.
        • Fig. S5. Evolutionary conservation of PAX1 Val147, Asn155, Gly166, and Cys368 amino acid residues.
        • Fig. S6. Protein stability of mouse PAX1 mutants corresponding to human PAX1 variants.
        • Fig. S7. Ramachandran plot of PAX1 structural modeling.
        • Fig. S8. MD simulation of PAX1 conformations.
        • Fig. S9. DNA-PAX1 paired box domain variant complexes after MD simulations.
        • Fig. S10. Stemness profile of iPSCs.
        • Fig. S11. Differentiation of iPSCs into TEP cells in control donor.
        • Fig. S12. Immunofluorescence analysis of KRT8 protein expression of control (C) and patient (P1) TEP cells.
        • Fig. S13. Differentially expressed genes between control and patient cells at TEP stage of differentiation.
        • Table S1. List of candidate genes after filtering WES data.
        • Table S2. Other genes involved in thymic development, with concordant differential expression in P1 and P4 versus control TEP cells.

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