IRF4 haploinsufficiency in a family with Whipple’s disease
- INSERM U1163, France
- Paris Descartes University, France
- Sidra Medicine, Qatar
- The Rockefeller University, United States
- University Aix-Marseille, URMITE, UM63, CNRS 7278, IRD 198, France
- Garvan Institute of Medical Research, Australia
- University of New South Wales, Australia
- Memorial Sloan Kettering Cancer Center, United States
- Institut Pasteur, France
- CNRS UMR2000, France
- Cochin Hospital, France
- Weill Cornell Graduate School of Medical Sciences, United States
- Assistance Publique-Hôpitaux de Paris, Necker Hospital for Sick Children, France
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
Autosomal dominant IRF4 deficiency.
(A) Pedigree of the kindred, with allele segregation. Generations are designated by Roman numerals (I, II, III, IV, V and VI), and each individual is designated by an Arabic numeral (from left to …
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Figure 1—source data 1
Kindred information summary.
For each subject, Tw carriage status, IRF4 genotype, clinical status and date of birth (DOB) are reported. NA: not available; Pos: positive; Neg: negative; Tw: Tropheryma whipplei; WD: Whipple’s disease; E?: genotype not assessed.
- https://doi.org/10.7554/eLife.32340.004
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Figure 1—source data 2
Non-synonymous variants within the linkage regions found in WES data from patients.
- https://doi.org/10.7554/eLife.32340.005
Figure 1—figure supplement 1
Genome-wide linkage and whole-exome sequencing analyses.
(A) Genome-wide linkage analysis was performed by combining genome-wide array and whole-exome sequencing (WES) data, assuming an autosomal dominant (AD) mode of inheritance. LOD (logarithm of odds) …
Figure 2 with 1 supplement
Analysis in silico of IRF4 variants.
Minor allele frequency (MAF) and combined annotation–dependent depletion (CADD) score of all coding variants previously reported in a public database (gnomAD) (http://gnomad.broadinstitute.org) and …
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Figure 2—source data 1
156 non-synonymous heterozygous coding or splice variants reported in the gnomAD or HGID databases.
†: non-canonical transcript predicted to undergo nonsense mediated decay.
- https://doi.org/10.7554/eLife.32340.008
Figure 2—figure supplement 1
List of variants and strength of purifying selection on IRF4.
Genome-wide distribution of the strength of purifying selection, estimated by the f parameter (Eilertson et al., 2012), acting on 14,993 human genes. IRF4 is at the 9.4th percentile of the …
Figure 3 with 5 supplements
Molecular characterization of the R98W IRF4 mutation (loss of DNA binding).
(A) HEK293T cells were transfected with the pcDNA3.1 empty vector (E) or plasmids encoding IRF4 WT, IRF4 R98W or IRF4 R98A-C99A. Total cell extracts were subjected to western blotting; the upper …
Figure 3—figure supplement 1
Functional activity of IRF4.
(A) Luciferase activity of HEK293T cells cotransfected with an (ISRE)3 reporter plasmid plus the pcDNA3.1 empty vector (E) and plasmids encoding IRF4 WT and/or IRF4 R98W or IRF4 R98A/C99A. The …
Figure 3—figure supplement 2
Protein levels of IRF4 variants previously reported in gnomAD database.
HEK293T cells were transfected with the pcDNA3.1 empty vector (E), or plasmids encoding IRF4 WT, IRF4 R98W or several IRF4 variants previously reported in the gnomAD database (see Figure 3). Total …
Figure 3—figure supplement 3
Protein levels of IRF4 variants from HGID database.
HEK293T cells were transfected with the pcDNA3.1 empty vector (E), or plasmids encoding IRF4 WT, IRF4 R98W or IRF4 variants from the HGID database (see Figure 3). Total cell extracts were subjected …
Figure 3—figure supplement 4
Functional impact of IRF4 variants previously reported in gnomAD database.
Luciferase activity of HEK293T cells cotransfected with an (ISRE)3 reporter plasmid plus the pcDNA3.1 empty vector (E) and plasmids encoding IRF4 WT, IRF4 R98W or several IRF4 variants previously …
Figure 3—figure supplement 5
Functional impact of IRF4 variants from HGID database.
Luciferase activity of HEK293T cells cotransfected with an (ISRE)3 reporter plasmid plus the pcDNA3.1 empty vector (E) and plasmids encoding IRF4 WT, IRF4 R98W or IRF4 variants from the HGID …
Figure 4 with 1 supplement
IRF4 mRNA levels in EBV-B cells.
(A) Total RNA extracted from healthy unrelated controls (n = 7; IRF4 WT/WT), patients diagnosed with Whipple’s disease (n = 25; WT/WT for all coding exons of IRF4) not related to this kindred, …
Figure 4—figure supplement 1
IRF4 protein levels in EBV-B cells.
(A–C) (Left) Total cell (A), cytoplasmic (B) and nuclear (C) extracts from five healthy unrelated controls (C1 to C5), three homozygous WT relatives (WT1, WT2, WT3), three patients (P1 to P3) and …
Figure 5 with 7 supplements
IRF4 protein levels in CD4+ T cells.
(A–C) (Left) Total-cell (A), cytoplasmic (B) and nuclear (C) extracts from CD4+ T cells from four healthy unrelated controls (C1 to C4) and two patients (P1 and P3) stimulated with activating …
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Figure 5—source data 1
Immunophenotyping of patients (P1, P2 and P3) and a WT homozygous relative.
All subjects had normal numbers and percentages of T, B, and NK cells for age.
- https://doi.org/10.7554/eLife.32340.029
Figure 5—figure supplement 1
IRF4 protein levels in PBMC subpopulations.
Total cell extracts from PBMC subpopulations (CD3+ T cells, CD56+ NK cells, CD19+ B cells, CD19+ CD27+ memory B cells, CD19+ CD27- naive B cells, CD14+ monocytes) from two healthy unrelated controls …
Figure 5—figure supplement 2
Percentage of dendritic cells and monocyte subtypes within total PBMCs.
(A) Percentage of CD11c+, myeloid dendritic cells (mDC1 and mDC2) and plasmacytoid dendritic cells (pDCs) (left), and monocyte subtypes (right) among total PBMCs from healthy unrelated controls, a …
Figure 5—figure supplement 3
IRF4 levels in controls and patient monocyte-derived macrophages.
(A) IRF4 protein levels, as determined by western blotting on total cell extracts from M2-like (left panel) or M1-like (right panel) monocyte-derived macrophages (MDMs) from two healthy unrelated …
Figure 5—figure supplement 4
Surface marker levels in controls and patient monocyte-derived macrophages.
CD11b, CD86, CD206, CD209 and HLA-DR mean fluorescence intensity (MFI) for M2-MDM (left) and M1-MDM (right) from P1 and two healthy unrelated controls (C1 and C2), either left non-stimulated (NS) or …
Figure 5—figure supplement 5
Percentage of memory B cells in PBMCs from controls and patients.
PBMCs from healthy unrelated controls and patients (P1, P2 and P3) were stained with antibodies against CD20, CD10 and CD27, IgM, IgD, IgG, or IgA. Percentages of memory B cells (CD20+ CD10- CD27+) …
Figure 5—figure supplement 6
In vitro differentiation of CD4+ T cells from patients and controls.
Naive and memory CD4+ T cells from healthy unrelated controls and patients (P2 and P3) were purified by sorting and cultured with TAE beads. The secretion of IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, …
Figure 5—figure supplement 7
Ex vivo cytokine production by CD4+ memory T cells from patients and controls.
Naive CD4+ T cells from healthy unrelated controls and patients (P2 and P3) were stimulated with TAE beads alone or under Th1, Th2, Th17 or Tfh polarizing conditions. The production of IL-10, IL-21, …
Figure 6
Overall transcriptional responsiveness of PBMCs following in vitro exposure to Tw and BCG and pathway activity analysis for genes responsive to BCG exposure.
(A) The overall responsiveness of individual subjects following stimulation with BCG and Tw, relative to non-stimulated conditions (along the horizontal axis) is shown as a heatmap. For each …
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Figure 6—source data 1
Differentially expressed (DE) genes found to be responsive to BCG in homozygous WT subjects using the criteria described in the Materials and methods section.
Genes are grouped by up- or down- regulation and ranked in alphabetical order.
- https://doi.org/10.7554/eLife.32340.027
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Figure 6—source data 2
Differentially expressed (DE) genes found to be responsive to Tw in homozygous WT subjects using the criteria described in the Materials and methods section.
Genes are grouped by up- or down- regulation and ranked in alphabetical order.
- https://doi.org/10.7554/eLife.32340.028
Author response image 1
Electrophoresis of the full-length IRF4 cDNA from EBV-B cell lines.
EBV-B cell lines from 25 patients diagnosed with Whipple’s disease (WD1 to WD25, WT/WT at all coding exons of IRF4) and from three patients (P1, P2 and P3), two asymptomatic heterozygous relatives …
Author response image 2
IRF4 protein levels in EBV-B cell lines.
Total lysates of EBV-B cells from five healthy controls (C1 to C5), four homozygous WT relatives (WT1 to WT4), two asymptomatic heterozygous relatives (HET1 and HET2), two patients (P1 and P3) and …
Author response image 3
IRF4 mRNA levels in transfected HEK293T cells.
Total RNA extracted from HEK293T cells transfected with E, WT, R98W, R98A-C99A or the five predicted LOF variant plasmids, was subjected to RT-qPCR for total IRF4 . Data are displayed as 2ΔΔCt after …
Author response image 4
Electrophoresis of full-length IRF4 cDNA from transfected HEK293T cells.
Total cDNA extracted from HEK293T cells transfected with E, WT, R98W, R98A-C99A or the five predicted LOF variant plasmids. A partial actin B cDNA was used as a loading control.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
| Gene (Human) | IRF4 (NM_002460.3) | This paper | Vector backbone: pcDNA 3.1D/V5-His-TOPO vector (Thermo Fisher Scientific) | |
| Gene (Human) | PU.1 (NM_001080547.1) | This paper | Vector backbone: pcDNA 3.1D/V5-His-TOPO vector (Thermo Fisher Scientific) | |
| Gene (Human) | BATF (NM_006399.3) | OriGene | RC207104 | |
| Gene (Human) | JUN (NM_002228.3) | OriGene | RC209804 | |
| Strain (Tropheryma whipplei), strain background (DIG APD 25) | Tw | This paper | NCBI taxon: 2039 | Obtained from ‘Research Unit of Infectious and Tropical Emerging Diseases, University Aix-Marseille, URMITE, UM63, CNRS 7278, IRD 198, 13005 Marseille, France, EU’. Strain isolated from mesenteric lymph node (29/01/09). |
| Strain (Mycobacterium bovis-Bacillus Calmette-Guerin) , strain background (pasteur) | BCG | doi: 10.1084/jem.20021769 | NCBI taxon: 33892 | |
| Cell line (Human) | HEK293T | ATCC | CRL-3216 | |
| Cell line (Human) | EBV-B cells | This paper | For each individual, purified B cells were immortalized with EBV in the laboratory | |
| Transfected construct (PGL4.10[luc2]) | (ISRE)3 reporter plasmid, | This paper, backbone: Promega | #E6651 | Obtained from ‘Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology’ |
| Transfected construct (PGL4.10[luc2]) | AICE reporter plasmid | This paper, backbone: Promega | #E6651 | Generated by metabion international ag |
| Transfected construct (pRL-SV40 vector) | pRL-SV40 vector | Promega | #E2231 | |
| Biological sample (Human) | Patients' blood samples | This paper | ||
| Biological sample (Human) | Controls' blood samples | This paper | ||
| Antibody | anti-IRF4 | Santa Cruz | M-17 | Dilution: 1/1000 |
| Antibody | anti-GAPDH | Santa Cruz | FL-335 | Dilution: 1/1000 |
| Antibody | anti-topoisomerase I | Santa Cruz | C-21 | Dilution: 1/1000 |
| Antibody | anti-lamin A/C | Santa Cruz | H-110 | Dilution: 1/1000 |
| Antibody | anti-CD11b | Miltenyi Biotec | # 130-110-611 | Fluorochrome: PE |
| Antibody | anti-CD86 | Miltenyi Biotec | #130-094-877 | Fluorochrome: PE |
| Antibody | anti-CD206 | Miltenyi Biotec | #130-099-732 | Fluorochrome: PE |
| Antibody | anti-CD209 | Miltenyi Biotec | #130-109-589 | Fluorochrome: PE |
| Antibody | anti-HLADR | Miltenyi Biotec | #130-111-789 | Fluorochrome: PE |
| Antibody | anti-CD20 | BD biosciences | Fluorochrome: PE; clone H1 | |
| Antibody | anti-CD10 | BD biosciences | Fluorochrome: APC, clone HI10a | |
| Antibody | anti-CD27 | BD biosciences | Fluorochrome: PerCP-Cy5.5; clone L128 | |
| Antibody | anti-IgM | Miltenyi | Clone PJ2-22H3 | |
| Antibody | anti-IgG | BD biosciences | Fluorochrome: BV605; clone G18-145 | |
| Antibody | anti-IgA | Miltenyi | Clone IS11-8E10 | |
| Antibody | anti-CD4 | eBioscience | Fluorochrome: Pacific blue; clone OKT4 | |
| Antibody | anti-CD45RA | BD biosciences | Fluorochrome: PerCP-Cy5.5, clone HI100 | |
| Antibody | anti-CCR7 | Sony | Fluorochrome: FITC; clone G043H7 | |
| Recombinant DNA reagent | pcDNA 3.1D/V5-His-TOPO vector | Thermo Fisher Scientific | #K4900-01 | |
| Sequence-based reagent | IRF4-specific primer | Thermo Fisher Scientific | #Hs01056533_m1 | |
| Sequence-based reagent | GUSB | Thermo Fisher Scientific | #4326320E | |
| Peptide, recombinant protein | rhGM-CSF | R and D System | #CAA26822 | |
| Peptide, recombinant protein | rhM-CSF | R and D System | #NP_757350 | |
| Peptide, recombinant protein | IFN-γ | Boehringer Ingelheim | Imukin | |
| Peptide, recombinant protein | rhIL4 | R and D System | #P05112 | |
| Commercial assay or kit | Lipofectamine LTX kit | Thermo Fisher Scientific | #15338100 | |
| Commercial assay or kit | Dual-Luciferase 1000 assay system kit | Promega | #E1980 | |
| Commercial assay or kit | ZR RNA Microprep kit | Zymo research | #R1061 | |
| Commercial assay or kit | High-Capacity RNA-to-cDNA kit | Thermo Fisher Scientific | #R4387406 | |
| Commercial assay or kit | TOPO TA cloning kit | Thermo Fisher Scientific | #K450001 | |
| Commercial assay or kit | directional TOPO expression kit | Thermo Fisher Scientific | #K4900-01 | |
| Commercial assay or kit | QuikChangeII XL Site-Directed Mutagenesis Kit | Agilent Technologies | #200522 | |
| Commercial assay or kit | LIVE/DEAD Fixable Aqua Dead Cell Stain Kit | Thermo Fisher Scientific | #L34957 | |
| Chemical compound, drug | Tris | MP biomedicals | #11TRIS01KG | |
| Chemical compound, drug | HCl | Sigma | #H1758 | |
| Chemical compound, drug | NaCl | Sigma | #S3014 | |
| Chemical compound, drug | Triton X-100 | Sigma | #T8532 | |
| Chemical compound, drug | EDTA | MP biomedicals | #11EDTA05M1 | |
| Chemical compound, drug | protease inhibitors Complete | Roche | #04693116001 | |
| Chemical compound, drug | Phosphatase inhibitor cocktail | Roche | #04906837001 | |
| Chemical compound, drug | DTT | Thermo Fisher Scientific | #20290 | |
| Chemical compound, drug | pepstatin A | Sigma | #P4265 | |
| Chemical compound, drug | leupeptin | Sigma | #L2884 | |
| Chemical compound, drug | antipain | Sigma | #A6191 | |
| Chemical compound, drug | Hepes | Sigma | #H3375 | |
| Chemical compound, drug | KCl | Sigma | #P9333 | |
| Chemical compound, drug | EGTA | Amresco | #0732 | |
| Chemical compound, drug | NP40 | Sigma | #N6507 | |
| Chemical compound, drug | NaF | Sigma | #S7920 | |
| Chemical compound, drug | PMSF | Sigma | #P7626 | |
| Chemical compound, drug | MgCl2 | Sigma | #M8266 | |
| Chemical compound, drug | Klenow fragment | NEB | #M0210S | |
| Chemical compound, drug | d-ATP-32P | PerkinElmer | #BLU012H250UC | |
| Chemical compound, drug | TBE migration buffer | Euromedex | #ET020-B | |
| Chemical compound, drug | acrylamide/bis-acrylamide 37.5:1 | Sigma | #A7168 | |
| Software, algorithm | affy R package | Gautier et al., 2004; Irizarry et al., 2003 | ||
| Software, algorithm | IPA software | Alsina et al., 2014 | ||
| Software, algorithm | Microsoft Excel | Microsoft | ||
| Software, algorithm | GraphPad Prism V7.0 | GraphPad | ||
| Software, algorithm | Image studio | Licor |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32340.030
