Ph.D Antoine Guérin

M.Sc Gaspard Kerner

Ph.D Nico Marr

Ph.D Janet G Markle

M.D Florence Fenollar

B. Sc. (Hons Natalie Wong

Ph.D Sabri Boughorbel

B. Sc Danielle T Avery

Ph.D Cindy S Ma

Ph.D Salim Bougarn

Ph.D Matthieu Bouaziz

Ph.D Vivien Béziat

Ph.D Erika Della Mina

M.Sc Carmen Oleaga-Quintas

B.Sc Tomi Lazarov

B.Sc. (Hons Lisa Worley

B.Sc. (Hons Tina Nguyen

Ph.D Etienne Patin

M.Sc Caroline Deswarte

Ph.D Rubén Martinez-Barricarte

M.D Soraya Boucherit

M.D Xavier Ayral

PharmD Sophie Edouard

Ph.D Stéphanie Boisson-Dupuis

M.Sc Vimel Rattina

M.Sc Benedetta Bigio

Ph.D Guillaume Vogt

M.D Frédéric Geissmann

Ph.D ° Lluis Quintana-Murci

Ph.D Damien Chaussabel

Ph.D Stuart G Tangye

M.D Didier Raoult

M.D Laurent Abel

M.D Jacinta Bustamante

M.D Jean-Laurent Casanova
email: casanova@rockefeller.edu

IRF4 haploinsufficiency in a family with Whipple's disease

Keywords: Whipple's disease, primary immunodeficiency, IRF4, haploinsufficiency

Most humans are exposed to Tropheryma whipplei (Tw). Whipple's disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance.

We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that IRF4 had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance.

Introduction

Whipple's disease (WD) was first described as an intestinal inflammatory disease by George H. [START_REF] Whipple | A hitherto undescribed disease characterized anatomically by deposits of fat and fattyacids in the intestinal and mesentericlymphatic tissues[END_REF][START_REF] Whipple | A hitherto undescribed disease characterized anatomically by deposits of fat and fattyacids in the intestinal and mesentericlymphatic tissues[END_REF]. Its infectious origin was suspected in 1961 [START_REF] Yardley | Combined electron and light microscopy in Whipple's disease. Demonstration of "bacillary bodies" in the intestine[END_REF], and the causal microbe, Tropheryma whipplei (Tw), a Grampositive actinomycete, was detected by PCR in 1992 [START_REF] Relman | Identification of the uncultured bacillus of Whipple's disease[END_REF], and cultured in 2000 [START_REF] Raoult | Cultivation of the bacillus of Whipple's disease[END_REF]. Tw is probably transmitted between humans via the oro-oral or feco-oral routes. WD is a chronic condition with a late onset (mean age at onset: 55 years) [START_REF] Braubach | Fluorescence In Situ Hybridization for Diagnosis of Whipple's Disease in Formalin-Fixed Paraffin-Embedded Tissue[END_REF] affecting multiple organs. The clinical manifestations of classical WD are arthralgia, diarrhea, abdominal pain, and weight loss [START_REF] Dobbins | Whipple's disease[END_REF][START_REF] Durand | Whipple disease. Clinical review of 52 cases. The SNFMI Research Group on Whipple Disease[END_REF][START_REF] Fleming | Whipple's disease: clinical, biochemical, and histopathologic features and assessment of treatment in 29 patients[END_REF][START_REF] Mahnel | Immunosuppressive therapy in Whipple's disease patients is associated with the appearance of gastrointestinal manifestations[END_REF][START_REF] Maizel | Whipple's disease: a review of 19 patients from one hospital and a review of the literature since 1950[END_REF]. However, about 25% of WD patients display no gastrointestinal or osteoarticular symptoms, instead presenting with cardiac and/or neurological manifestations [START_REF] Durand | Whipple disease. Clinical review of 52 cases. The SNFMI Research Group on Whipple Disease[END_REF][START_REF] Fenollar | Tropheryma whipplei and Whipple's disease[END_REF][START_REF] Fenollar | Whipple's endocarditis: review of the literature and comparisons with Q fever, Bartonella infection, and blood culture-positive endocarditis[END_REF][START_REF] Gubler | Whipple endocarditis without overt gastrointestinal disease: report of four cases[END_REF][START_REF] Schneider | Whipple's disease: new aspects of pathogenesis and treatment[END_REF]. WD is fatal if left untreated, and relapses occur in 2 to 33% of treated cases, even after prolonged appropriate antibiotic treatment [START_REF] Lagier | Evidence of lifetime susceptibility to Tropheryma whipplei in patients with Whipple's disease[END_REF][START_REF] Marumganti | Whipple's disease: neurological relapse presenting as headache for two years[END_REF]. WD is rare and has been estimated to affect about one in a million individuals [START_REF] Dobbins | Is there an immune deficit in Whipple's disease?[END_REF][START_REF] Dobbins | Whipple's disease[END_REF][START_REF] Fenollar | Whipple's disease[END_REF]. However, about two thousand cases have been reported in at least nine countries worldwide, mostly in North America and Western Europe [START_REF] Bakkali | Acquired resistance to trimethoprim-sulfamethoxazole during Whipple disease and expression of the causative target gene[END_REF][START_REF] Desnues | New insights into Whipple's disease and Tropheryma whipplei infections[END_REF]Fenollar et al., 2008a;[START_REF] Lagier | Failure and relapse after treatment with trimethoprim/sulfamethoxazole in classic Whipple's disease[END_REF][START_REF] Puechal | Whipple's arthritis[END_REF]Schneider et al., 2008). Chronic asymptomatic carriage of Tw is common in the general population, and this bacterium has been detected in feces, saliva, and intestinal mucosae. The prevalence of Tw carriage in the feces has been estimated at 2 to 11% for the general population, but can reach 26% in sewer workers and 37% in relatives of patients and carriers [START_REF] Amsler | Prevalence of Tropheryma whipplei DNA in patients with various gastrointestinal diseases and in healthy controls[END_REF][START_REF] Ehrbar | PCR-positive tests for Tropheryma whippelii in patients without Whipple's disease[END_REF][START_REF] Fenollar | Tropheryma whipplei and Whipple's disease[END_REF][START_REF] Fenollar | Whipple's disease[END_REF][START_REF] Maibach | Detection of Tropheryma whipplei DNA in feces by PCR using a target capture method[END_REF][START_REF] Rolain | False positive PCR detection of Tropheryma whipplei in the saliva of healthy people[END_REF][START_REF] Schneider | Whipple's disease: new aspects of pathogenesis and treatment[END_REF][START_REF] Street | Tropheryma whippelii DNA in saliva of healthy people[END_REF].

Seroprevalence for specific antibodies against Tw in the general population varies from 50% in France to 70% in Senegal [START_REF] Fenollar | A paradoxical Tropheryma whipplei western blot differentiates patients with whipple disease from asymptomatic carriers[END_REF][START_REF] Fenollar | Tropheryma whipplei and Whipple's disease[END_REF][START_REF] Raoult | Cultivation of the bacillus of Whipple's disease[END_REF][START_REF] Schneider | Whipple's disease: new aspects of pathogenesis and treatment[END_REF]. At least 75% of infected individuals clear Tw primary infections, but a minority (<25%) become asymptomatic carriers, a very small proportion of whom develop WD (<0.01%) (Fenollar et al., 2008b). Tw infection is, therefore, necessary, but not sufficient, for WD development, and it is unclear whether prolonged asymptomatic carriage necessarily precedes WD. The hypothesis that WD results from the emergence of a more pathogenic clonal strain of Tw was not supported by bacterial genotyping [START_REF] Li | Genotyping reveals a wide heterogeneity of Tropheryma whipplei[END_REF]. WD mostly affects individuals of European origin, but does not seem to be favored by specific environments. WD is typically sporadic, but six multiplex kindreds have been reported, with cases often diagnosed years apart, suggesting a possible genetic component [START_REF] Durand | Whipple disease. Clinical review of 52 cases. The SNFMI Research Group on Whipple Disease[END_REF][START_REF] Fenollar | Whipple's disease[END_REF][START_REF] Ponz De Leon | Whipple's disease in a father-son pair[END_REF]. WD patients are not prone to other severe infections [START_REF] Marth | Tropheryma whipplei infection and Whipple's disease[END_REF]. Moreover, WD has never been reported in patients with conventional primary immunodeficiencies (PIDs) [START_REF] Picard | International Union of Immunological Societies: 2017 Primary Immunodeficiency Diseases Committee Report on Inborn Errors of Immunity[END_REF]. This situation is reminiscent of other sporadic severe infections, such as herpes simplex virus-1 encephalitis, severe influenza, recurrent rhinovirus infection, severe varicella zoster disease, trypanosomiasis, invasive staphylococcal disease, and viral infections of the brainstem, which are caused by single-gene inborn errors of immunity in some patients [START_REF] Andersen | Functional IRF3 deficiency in a patient with herpes simplex encephalitis[END_REF]Casanova, 2015a;Casanova, 2015b;[START_REF] Ciancanelli | Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency[END_REF][START_REF] Israel | Human Adaptive Immunity Rescues an Inborn Error of Innate Immunity[END_REF][START_REF] Lamborn | Recurrent rhinovirus infections in a child with inherited MDA5 deficiency[END_REF][START_REF] Lenardo | Genomics of Immune Diseases and New Therapies[END_REF][START_REF] Ogunjimi | Inborn errors in RNA polymerase III underlie severe varicella zoster virus infections[END_REF][START_REF] Vanhollebeke | Human Trypanosoma evansi infection linked to a lack of apolipoprotein L-I[END_REF][START_REF] Zhang | Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection[END_REF]. We therefore hypothesized that WD might be due to monogenic inborn errors of immunity to Tw, with age-dependent incomplete penetrance.

Results

A multiplex kindred with WD

We investigated four related patients diagnosed with WD (P1, P2, P3, and P4) with a mean age at diagnosis of 58 years. They belong to a large non-consanguineous French kindred (Figure 1A). The proband (P1), a 69-year-old woman, presented with right knee arthritis in 2011, after recurrent episodes of arthritis of the right knee since 1980. Tw was detected in the synovial fluid by PCR and culture, but not in saliva, feces, or small intestine tissue by PCR.

Treatment with doxycycline and hydroxychloroquine was effective. At last follow-up, in 2016, P1 was well and Tw PCR on saliva and feces was negative. P2, a second cousin of P1, is a 76-year-old woman with classical WD diagnosed at 37 years of age in 1978 by periodic acid-Schiff (PAS) staining of a small intestine biopsy specimen. She was treated with sulfamethoxazole/trimethoprim. At last follow-up, in 2016, Tw PCR on saliva and feces was positive. P3, the father of P1, is a 92-year-old man with classical WD diagnosed at 62 years of age, in 1987, based on positive PAS staining of a small intestine biopsy specimen. Long-term sulfamethoxazole/trimethoprim treatment led to complete clinical and bacteriological remission. P4, the brother of P2, is a 70-year-old man who consulted in 2015 for arthralgia of the knees and right ulna-carpal joints. PCR and culture did not detect Tw in saliva and feces, but serological tests for Tw were positive. Treatment with methotrexate and steroids was initiated before antibiotics, the effect of which is currently being evaluated. All four patients are otherwise healthy. Saliva and/or feces samples from 18 other members of the family were tested for Tw (Figure 1A; Figure 1-source data 1). Five individuals are chronic carriers (mean age: 55 years) and 13 tested negative (mean age: 38 years). Nine additional relatives could not be tested. The distribution of WD in this kindred was suggestive of an AD trait with incomplete clinical penetrance.

A private heterozygous missense IRF4 variant segregates with WD

We analyzed the familial segregation of WD by genome-wide linkage (GWL), using information from both genome-wide single-nucleotide polymorphism (SNP) microarrays and whole-exome sequencing (WES) [START_REF] Belkadi | Whole-exome sequencing to analyze population structure, parental inbreeding, and familial linkage[END_REF]. Multipoint linkage analysis was performed under an AD model, with a very rare disease-causing allele (<10 -5 ) and agedependent incomplete penetrance. Twelve chromosomal regions linked to WD were identified on chromosomes 1 (x3), 2, 3, 6, 7, 8, 10, 11, 12 and 17, with a LOD score close (>1.90) to the maximum expected value (1.95) (Figure 1-figure supplement 1A). These regions covered 27.18 Mb and included 263 protein-coding genes. WES data analysis for these 263 genes identified 54 heterozygous non-synonymous coding variants common to all four WD patients (Figure 1-source data 2). Only one, a variant of the Interferon regulatory factor (IRF) 4 gene encoding a transcription factor from the IRF family [START_REF] Ikushima | The IRF family transcription factors at the interface of innate and adaptive immune responses[END_REF], located in a 200 kb linked region on chromosome 6 (Figure 1-figure supplement 1A, 1B), was very rare, and was even found to be private [not found in the gnomAD database, http://gnomad.broadinstitute.org, or in our in-house WES database (HGID)], whereas all other variants had a frequency >0.001, which is inconsistent with the frequency of WD and our hypothesis of a very rare (<10 -5 ) deleterious heterozygous allele. The variant is a c.292 C>T substitution in exon 3 of IRF4, replacing the arginine residue in position 98 with a tryptophan residue (R98W) (Figure 1A, 1B, 1C). IRF4 is a transcription factor with an important pleiotropic role in innate and adaptive immunity, at least in a few strains of inbred mice [START_REF] Shaffer | IRF4: Immunity. Malignancy! Therapy? Clinical cancer research : an official journal of the American Association for[END_REF]. Mice heterozygous for a null Irf4 mutation have not been studied, but homozygous null mice have various T-and B-cell abnormalities and are susceptible to both Leishmania and lymphocytic choriomeningitis virus [START_REF] Klein | Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination[END_REF][START_REF] Lohoff | Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4[END_REF][START_REF] Mittrucker | Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function[END_REF][START_REF] Suzuki | Critical roles of interferon regulatory factor 4 in CD11bhighCD8alpha-dendritic cell development[END_REF][START_REF] Tamura | IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity[END_REF][START_REF] Tominaga | Development of Th1 and not Th2 immune responses in mice lacking IFN-regulatory factor-4[END_REF]. We confirmed the IRF4 R98W mutation by Sanger sequencing genomic DNA from the blood of the four WD patients (Figure 1C). Thirteen relatives of the WD patients were WT/WT at the IRF4 locus, and 10 of these relatives (77%) tested negative for Tw carriage. Eight other relatives were heterozygous for the IRF4 R98W mutation, five of whom (62.5%) were Tw carriers (mean age: 55 years) (Figure 1A; Figure 1-source data 1). Overall, 12 individuals from the kindred, including the four patients, the five chronic carriers of Tw, two non-carriers of Tw and one relative not tested for Tw, were heterozygous for IRF4 R98W (Figure 1A;

Figure 1-source data 1). The familial segregation of the IRF4 R98W allele was therefore consistent with an AD pattern of WD inheritance with incomplete clinical penetrance. Chronic Tw carriage also followed an AD mode of inheritance.

R98W is predicted to be loss-of-function, unlike most other IRF4 variants

The R98 residue in the DNA-binding domain (DBD) of IRF4 is highly conserved in the 12 species for which IRF4 has been sequenced (Figure 1B, 1D). It has been suggested that this residue is essential for IRF4 DNA-binding activity, because the R98A-C99A double mutant is loss-of-function (Brass et al., 1999;[START_REF] Escalante | Crystal structure of PU.1/IRF-4/DNA ternary complex[END_REF]. The R98W mutation is predicted to be damaging by multiple programs [START_REF] Kircher | A general framework for estimating the relative pathogenicity of human genetic variants[END_REF]; it has a CADD score of R98W (26.5), well above the mutation significance cutoff (MSC) of IRF4 (11.125) (Figure 2) [START_REF] Itan | The mutation significance cutoff: gene-level thresholds for variant predictions[END_REF][START_REF] Kircher | A general framework for estimating the relative pathogenicity of human genetic variants[END_REF]. The R98W variant was not present in the gnomAD database or our in-house HGID database of more than 4,000 WES from patients with various infectious diseases. The mutant allele was not found in the sequences for the CEPH-HGDP panel of 1,052 controls from 52 ethnic groups, or in 100 French controls, confirming that this variant was very rare, probably private to this kindred. Therefore, the minor allele frequency (MAF) of this private allele is <4x10 -6 . Moreover, the IRF4 gene has a gene damage index (GDI) of 2.85, a neutrality index score of 0.15 [START_REF] Itan | The human gene damage index as a gene-level approach to prioritizing exome variants[END_REF], and a purifying selection f parameter of 0.32 (among the <10% of genes in the genome subject to the greatest constraint; Figure 2-figure supplement 1), strongly suggesting that IRF4 has evolved under purifying selection (i.e., strong evolutionary constraints) [START_REF] Eilertson | SnIPRE: selection inference using a Poisson random effects model[END_REF]. Biologically disruptive heterozygous mutations of IRF4 are therefore likely to have clinical effects. We identified 156 other high-confidence heterozygous non-synonymous coding or splice variants of IRF4 (Figure 2-source data 1) in public (gnomAD: 153 variants, all with MAF<0.009) and HGID (3 variants) databases: 147 were missense variants (two of which were also found in the homozygous state: p.S149N and p.A370V), four were frameshift indels leading to premature stop codons (p.W27YfsTer50, p.W74GfsTer28, p.Y152LeufsTer60, and S160RfsTer11), three were in-frame indels (p.E46del, p.G279_H280del, and S435del), one was a nonsense variant (p.R82*), one was an essential splice variant (c.403+2T>C), and two were missense variants found only in a non-canonical transcript predicted to undergo nonsense-mediated decay (p.L406P and p.R407W). Up to 150 of the 156 variants are predicted to be benign, whereas only six were predicted to be potentially loss-of-function (LOF) according to the gnomAD database classification (the four frameshift indels, the nonsense variant, and the essential splice variant). Comparison of the CADD score and MAF of these IRF4 variants showed R98W to have the second highest CADD score of the four variants with a MAF < 4 x10 -6 (Figure 2). These findings suggest that the private heterozygous IRF4 variant of this kindred is biochemically deleterious, unlike most other rare (MAF<0.009) non-synonymous variants in the general population, 150 of 156 of which were predicted to be benign [START_REF] Lek | Analysis of proteincoding genetic variation in 60,706 humans[END_REF].

R98W is loss-of-function, unlike most other previously observed IRF4 variants

We first characterized IRF4 R98W production and function in vitro, in an overexpression system. We assessed the effect of the IRF4 R98W mutation on IRF4 levels by transiently expressing WT or mutant R98W in HEK293T cells. IRF4 R98A-C99A, which is LOF for DNA binding (Brass et al., 1999), was included as a negative control. In total cell extracts, mutant IRF4 proteins were more abundant than the WT protein, and had the expected molecular weight (MW) of 51 kDa, as shown by western blotting (Figure 3A). The R98 residue has been shown to be located in a nuclear localization signal, the complete disruption of which results in a loss of IRF4 retention in the nucleus [START_REF] Lau | Interferon regulatory factor subcellular localization is determined by a bipartite nuclear localization signal in the DNA-binding domain and interaction with cytoplasmic retention factors[END_REF]. We therefore analyzed the subcellular distribution of IRF4 WT and R98W proteins, in total, cytoplasmic, and nuclear extracts from transiently transfected HEK293T cells. The R98W mutant was more abundant than the WT protein in total cell and cytoplasmic extracts, but these proteins were similarly abundant in nuclear extracts (Figure 3B). We performed luciferase reporter assays to assess the ability of the mutant IRF4 protein to induce transcription from interferon-stimulated response element (ISRE) motif-containing promoters.

Unlike the WT protein, both R98W and R98A-C99A failed to activate the (ISRE) 3 promoter (Figure 3C). We also assessed the ability of IRF4 to induce transcription from an (AP-1)-IRF composite element (AICE) motif-containing promoter [START_REF] Li | BATF-JUN is critical for IRF4-mediated transcription in T cells[END_REF]. Both R98W and R98A-C99A failed to activate the AICE promoter, WT protein (Figure 3D). Moreover, we observed no dominant-negative effect of the IRF4 R98W protein, with either the ISRE or AICE motif-containing promoter (Figure 3-figure supplement 1A, 1B). We assessed the ability of R98W to bind DNA, in an electrophoretic mobility shift assay (EMSA) (Figure 3E, 3F). Signal specificity was assessed by analyzing both supershift with an IRF4-specific antibody and by competition with an unlabeled competitor probe. The R98W mutation abolished IRF4 binding to the ISRE cis element (Figure 3E), and binding of the IRF4-PU.1 complex to interferon composite elements (EICEs) containing both IRF4 and PU.1 recognition motifs (Brass et al., 1999) (Figure 3F). The R98W allele of IRF4 is therefore LOF for both DNA binding and the induction of transcription. We then tested 153 of the other 156 IRF4 variants: 150 variants previously described in the gnomAD database and three variants found in the HGID database. The essential splice variant and the two variants present only in a non-canonical transcript were not tested. All the variants tested were normally expressed (two with a higher MW), except for the five predicted LOF variants tested (the epitope of the antibody being at the C-terminus of IRF4) (Figure 3-figure supplement 2, 3). Transfection with the five predicted LOF plasmids was efficient, as assessed by cDNA amplification and sequencing (data not shown). When tested for (ISRE) 3 promoter activation, the five stop-gain or frameshift variants predicted to be LOF in the gnomAD database (very rare variants, MAF< 6x10 -6 ) were found to be LOF. We also showed that amongall the non-synonymous coding variants tested, only one rare very rare (MAF 9x10 -6 ) inframe deletion of one amino acid (E46del) reported in the gnomAD database, was hypomorphic, and another variant from our in-house WES database (G279_H280 del, private to one family) was LOF (Figure 3figure supplement 4, 5). The cumulative frequency of these seven LOF (n=6) or hypomorphic (n=1) variants was <4x10 -5 , fully consistent with the frequency of WD (occurring only in adults chronically infected with Tw). Overall, our data show that the R98W IRF4 allele is LOF, like only six other very rare non-synonymous IRF4 coding variants of the 153 variants tested. Moreover, R98W is not dominant negative.

AD IRF4 deficiency phenotypes in heterozygous EBV-B cells

We investigated the cellular phenotype of heterozygosity for the R98W allele in EBVtransformed B-cell lines (EBV-B cells) from patients. We performed reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) on EBV-B cells from P1, P3, two healthy heterozygous relatives (IRF4 WT/R98W), four healthy IRF4-WT homozygous relatives, and seven healthy unrelated controls. We also investigated 25 unrelated WD patients with Tw carriage. We sequenced all IRF4 coding exons for these patients, who were found to be WT. They were also found to have an intact IRF4 cDNA structure and normal IRF4 protein levels in EBV-B cells (data not shown). Cells from individuals heterozygous for the R98W mutation (patients and healthy carriers) had higher IRF4 mRNA levels than those from WT homozygous relatives, unrelated WD cohort patients and EBV-B cells from healthy unrelated controls (Figure 4A). We compared the relative abundances of WT and R98W IRF4 mRNA in EBV-B cells from heterozygous carriers of the mutation, by performing TA-cloning experiments on P1, P3, one healthy heterozygous relative, one relative homozygous for WT IRF4, and two previously tested healthy unrelated controls. In heterozygous carriers of the mutation (patients and healthy relatives) the R98W mutation was present in 48.1%-60% of the total IRF4 mRNA, whereas the rest was WT (Figure 4B). We evaluated the levels and distribution of IRF4 protein by western blotting on EBV-B cells from P1, P2, P3, one healthy heterozygous relative, three healthy homozygous WT relatives and five unrelated healthy individuals. As in transfected HEK293T cells, IRF4 protein levels were high both in total cell extract and even more so in cytoplasmic extracts of EBV-B cells from heterozygous carriers (Figure 4-figure supplement 1A, 1B). By contrast, IRF4 protein levels in EBV-B cell nuclei were similar in heterozygous carriers and controls (Figure 4-figure supplement 1C). As IRF4 is a transcription factor, we then analyzed the steady-state transcriptome of EBV-B cells from three healthy homozygous WT relatives and three WT/R98W heterozygotes (P1, P3, VI.6).

We identified 37 protein-coding genes as differentially expressed between subjects heterozygous for IRF4 and those homozygous WT for IRF4 (18 upregulated and 19 downregulated; data not shown). We identified no marked pathway enrichment based on these genes. EBV-B cells from individuals heterozygous for IRF4 had a detectable phenotype, in terms of IRF4 production and function, consistent with AD IRF4 deficiency underlying WD.

AD IRF4 deficiency phenotypes in heterozygous leukocytes

We assessed IRF4 levels in peripheral mononuclear blood cells (PBMCs) from healthy controls (Figure 5-figure supplement 1). IRF4 were also expressed in CD4 + T cells, particularly after stimulation with activating anti-CD2/CD3/CD28 monoclonal antibodycoated (mAb-coated) beads (data not shown) We therefore assessed the IRF4 protein expression profile in CD4 + T cells from four healthy unrelated controls, P1 and P3, with and without (non-stimulated, NS) stimulation with activating anti-CD2/CD3/CD28 mAb-coated beads. The results were consistent with those for transfected HEK293T and EBV-B cells, as IRF4 levels were higher in activated CD4 + T cells from P1 and P3 than in controls, both for total cell extracts, and even more so for the cytoplasmic compartment (Figure 5A, 5B). By contrast, IRF4 levels in the nucleus were similar and, possibly, even slightly lower in patients than in controls (Figure 5C). We also investigated peripheral myeloid (Figure 5-figure supplement 1-4) and lymphoid blood cell subsets in patients (Figure 5-figure supplement 5-7;

Figure 5-data source 1) which display an apparently normal development compared to healthy controls' cells. Then,we checked for transcriptomic differences associated with genotype and/or infection, by investigating the transcriptomes of PBMCsfrom six IRF4-heterozygous individuals (three patients, P1-P3; and three healthy relatives, HET1-HET3) and six IRF4 WT-homozygous individuals (four healthy relatives, WT1-WT4; and two healthy unrelated controls, C1-C2) with and without in vitro infection with Tw, or Mycobacterium bovis-Bacillus Calmette-Guerin (BCG), which, like Tw, belongs to phylum Actinobacteria, for 24 hours. We performed unsupervised hierarchical clustering of the differentially expressed (DE) transcripts (infected versus uninfected) to analyze the overall responsiveness of PBMCs from individual subjects to BCG and Tw infections in vitro. Heterozygous individuals clearly clustered separately from homozygous WT individuals (Figure 6A), revealing a correlation between genotype and response to infection. Overall, we found that 402 transcripts from 193 unique genes were responsive to BCG infection (Figure 6-source data 1), and 119 transcripts from 29 unique genes were responsive to Tw infection (Figure 6-source data 2) in homozygous WT subjects, according to the criteria described in the materials and methods.

Due to the small number of Tw-responsive transcripts linked to unique genes, we were unable to detect any pathway enrichment for this specific condition. However, we identified 24 canonical pathways as enriched after the exposure of PBMCs to BCG. We ranked these pathways according to the difference in mean z-score between homozygous WT and heterozygous subjects (Figure 6B). The top 10 pathways included the interferon signaling network, the Th1 pathway network, the HMGB1 signaling network, the p38 MAPK signaling network, the NF-B signaling network, the dendritic cell maturation network and the network responsible for producing nitric oxide and reactive oxygen species. These pathways were highly ranked mostly due to IFNG and STAT1, which were strongly downregulated in IRF4 heterozygotes, particularly in P1, P2 and P3, relative to WT homozygotes. IRF4 is predicted to bind the promoter regions of 47% of the genes identified in the BCG study (91 of 193 genes), including those of IFNG and STAT1. Subjects heterozygous for IRF4 also had lower levels of LTA expression, and lower levels of IL2RA expression were observed specifically in patients (GSE102862). These data suggest a general impairment of the T-cell response in subjects heterozygous for IRF4 upon BCG infection in vitro. Moreover, the lower levels of CD80 expression suggest a possible impairment of myeloid and/or antigen-presenting cell function upon BCG infection in patients, but not in healthy heterozygous or homozygous WT subjects (GSE102862). Peripheral leukocytes from IRF4-heterozygous individuals therefore had a phenotype in terms of IRF4 production and function.

Discussion

WD was initially described as an inflammatory disease [START_REF] Whipple | A hitherto undescribed disease characterized anatomically by deposits of fat and fattyacids in the intestinal and mesentericlymphatic tissues[END_REF], but was subsequently shown to be infectious [START_REF] Raoult | Cultivation of the bacillus of Whipple's disease[END_REF][START_REF] Relman | Identification of the uncultured bacillus of Whipple's disease[END_REF][START_REF] Yardley | Combined electron and light microscopy in Whipple's disease. Demonstration of "bacillary bodies" in the intestine[END_REF]. We provide evidence that WD is also a genetic disorder. We show here that, in a large multiplex kindred, heterozygosity for the private, loss-of-function R98W mutation of IRF4 underlies an AD form of WD with incomplete penetrance. The causal relationship between IRF4 genotype and WD was demonstrated as follows. First, the IRF4 R98W mutation is the only non-synonymous rare variant segregating with WD in this kindred.

Second, the mutation was demonstrated experimentally to be LOF, unlike 146 of 153 other non-synonymous coding IRF4 variants in the general population. Only seven of the 153 nonsynonymous coding variants identified (including the five predicted to be LOF in the gnomAD database) were found to be LOF (n=6) or hypomorphic (n=1) and they were all extremely rare (cumulative MAF <4x10 -5 ). Moreover, IRF4 has evolved under purifying selection, suggesting that deleterious heterozygous variants of this gene entail fitness costs [START_REF] Barreiro | From evolutionary genetics to human immunology: how selection shapes host defence genes[END_REF][START_REF] Quintana-Murci | Population genetic tools for dissecting innate immunity in humans[END_REF][START_REF] Rieux-Laucat | Immunology. Autoimmunity by haploinsufficiency[END_REF]. Third, EBV-B cells and activated CD4 + T cells heterozygous for IRF4 R98W have a distinctive phenotype, particularly for IRF4 expression in the cytoplasm. This mutation also has a strong functional impact on gene expression in IRF4 R98W-heterozygous PBMCs stimulated with BCG or Tw. These findings unequivocally show that heterozygosity for the R98W allele of IRF4 is the genetic etiology of WD in this kindred. Although we did not find any IRF4 mutations in a pilot cohort of 25 patients with sporadic WD, these and other patients may also develop WD due to other inborn errors of immunity, possibly related to IRF4, as suggested by the apparent genetic heterogeneity and physiological homogeneity underlying severe infectious diseases (Casanova, 2015a;Casanova, 2015b). This observation therefore extends our model, in which life-threatening infectious diseases striking otherwise healthy individuals during primary infection can result from single-gene inborn errors of immunity.

In this kindred with AD IRF4 deficiency, haploinsufficiency was identified as the key mechanism, although IRF4 protein levels in the cytoplasmic compartment were higher in patients with the mutation than in wild-type homozygotes. The protein was not more abundant in the nucleus, where IRF4 exerts its effects on transcription. Moreover, half of the IRF4 mRNA in EBV-B cells from heterozygous subjects is WT. The total amount of IRF4 mRNA was higher in the EBV-B cells of heterozygous subjects, but the total amount of IRF4 protein in the nuclear compartment of heterozygous EBV-B and activated CD4 + T cells was similar to that in WT homozygous cells. These data suggest that no more than half the IRF4 protein in the nucleus is WT in heterozygous cells. In addition, not only is IRF4 subject to purifying selection, but the R98W mutation is itself LOF, with no detectable dominant-negative effect at cell level. Haploinsufficiency is an increasingly recognized mechanism underlying AD inborn errors of immunity [START_REF] Afzali | BACH2 immunodeficiency illustrates an association between super-enhancers and haploinsufficiency[END_REF][START_REF] Rieux-Laucat | Immunology. Autoimmunity by haploinsufficiency[END_REF]. It is commonly due to loss-of-expression alleles, contrasting with the negative dominance typically exerted by expressed proteins, but many mutations are known to cause haploinsufficiency without actually preventing protein production [START_REF] Afzali | BACH2 immunodeficiency illustrates an association between super-enhancers and haploinsufficiency[END_REF][START_REF] Perez De Diego | Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis[END_REF][START_REF] Rieux-Laucat | Immunology. Autoimmunity by haploinsufficiency[END_REF]. Haploinsufficiency in this kindred is not due to loss of expression of IRF4. Instead, it results from a lack of activity of the R98W IRF4 proteins present in the nucleus.

Incomplete penetrance is common in conditions resulting from haploinsufficiency. In this kindred, incomplete penetrance may result from a lack of Tw infection (in heterozygous individuals IV.5 and VI.7), or a lack of WD development in infected individuals (in heterozygous individuals III.6, IV.4, V.3, V.4, VI.6). All five chronic carriers of Tw were heterozygous for the IRF4 R98W mutation, suggesting that AD IRF4 deficiency also favors the development of chronic Tw carriage. The five asymptomatic carriers were 24 to 82 years old, whereas the four patients were 69 to 92 years old. The impact of IRF4 R98W may therefore increase with age, initially facilitating chronic carriage in Tw-infected individuals, and subsequently predisposing chronic carriers to the development of WD. We cannot exclude the possibility that a modifier allele at another locus contributes to the development of WD in infected heterozygous individuals with IRF4 mutations. Future studies will attempt to define the cellular basis of WD in individuals with IRF4 mutations. The apparently normal development of all peripheral myeloid and lymphoid blood cell subsets studied in patients, and the selective predisposition of these individuals to WD suggest that the disease mechanism is subtle and specifically affects protective immunity to Tw, and that it may act in the gastrointestinal (GI) tract. Interestingly, the data of several public databases indicate that IRF4 RNA is expressed in the stomach, colon, and small intestine (https://www.gtexportal.org, http://biogps), and that the IRF4 protein is expressed in glandular cells from the stomach, duodenum, small intestine, and rectum (https://www.proteinatlas.org).

In addition, a recent analysis of human intestinal macrophage subsets [START_REF] Bujko | Whole-exome sequencing-based discovery of STIM1 deficiency in a child with fatal classic Kaposi sarcoma[END_REF] showed IRF4 RNA to be expressed in human intestinal myeloid resident cells. Further studies of GI tract-resident cells, including myeloid and lymphoid cells in particular, should make it possible to decipher the molecular and cellular mechanisms by which human IRF4 haploinsufficiency underlies WD upon infection by Tw.

Materials and Methods

Patients and family

All members of the multiplex kindred studied, the pedigree of which is shown in P4 had no rheumatoid factor, anti-cyclic citrullinated peptide antibodies (anti-CCP), or antinuclear antibodies. The fluid aspirated from the right knee contained 4,800 erythrocytes/mm 3 and 10,900 leukocytes/mm 3 (91% neutrophils and 9% lymphocytes) without crystals. Blood tests revealed an ESR of 30 mm/h and a CRP concentration of 50 mg/l, with no rheumatoid factor, anti-cyclic citrullinated peptide antibodies (anti-CCP) or anti-nuclear antibodies. An X ray revealed a narrowing of the joint space in the knees and vertebral hyperostosis was visible. The joints of the hands were unaffected. The patient was treated with antiinflammatory drugs, without success. Treatment with methotrexate and steroids was introduced, followed by antibiotics, the effect of which is currently being evaluated.

Saliva and/or feces samples from 18 other members of the family were checked for the presence of Tw, by a PCR specifically targeting T. whipplei, as previously described (Figure 1A, table 1) [START_REF] Edouard | The rise of Tropheryma whipplei: a 12-year retrospective study of PCR diagnoses in our reference center[END_REF]. Five individuals were found to be chronic carriers (mean age: 55 years) and 13 were not (mean age: 38 years). Testing was not possible for nine other relatives. The overall distribution of WD in this kindred was suggestive of an AD trait with incomplete penetrance.Genome-wide analysis Genome-wide linkage analysis was performed by combining genome-wide array and whole-exome sequencing (WES) data [START_REF] Belkadi | Whole-exome sequencing to analyze population structure, parental inbreeding, and familial linkage[END_REF]. In total, nine family members were genotyped with the Genome-Wide Human SNP Array 6.0. Genotype calling was achieved with the Affymetrix Power Tools Software Package (http://www.affymetrix.com/estore/partners_programs/programs/developer/tools/powertools.a ffx). SNPs were selected with population-based filters [START_REF] Purcell | PLINK: a tool set for whole-genome association and population-based linkage analyses[END_REF], resulting in the use of 905,420 SNPs for linkage analysis. WES was performed as described in the corresponding section, in four family members, P1, P2, P3 and P4. In total, 64,348 WES variants were retained after application of the following filtering criteria: genotype quality (GQ) > 40, minor read ratio (MRR) > 0.3, individual depth (DP) > 20x, retaining only diallelic variants with an existing RS number and a call rate of 100%. Parametric multipoint linkage analysis was performed with the Merlin program [START_REF] Abecasis | Merlin--rapid analysis of dense genetic maps using sparse gene flow trees[END_REF], using the combined set of 960,267 variants. We assumed an AD mode of inheritance, with a frequency of the deleterious allele of 10 -5 and a penetrance varying with age (0.8 above the age of 65 years, and 0.02 below this threshold). Data for the family and for Europeans from the 1000G project were used to estimate allele frequencies and to define linkage clusters, with an r 2 threshold of 0.4.

The method used for WES has been described elsewhere [START_REF] Bogunovic | Mycobacterial Disease and Impaired IFN-gamma Immunity in Humans with Inherited ISG15 Deficiency. Science Brass[END_REF][START_REF] Bujko | Whole-exome sequencing-based discovery of STIM1 deficiency in a child with fatal classic Kaposi sarcoma[END_REF]. Briefly, genomic DNA extracted from the patients' blood cells was sheared with a Covaris S2 Ultrasonicator (Covaris). An adapter-ligated library was prepared with the Paired-End Sample Prep kit V1 (Illumina). Exome capture was performed with the SureSelect Human All Exon kit (71 Mb version -Agilent Technologies). Paired-end sequencing was performed on an Illumina Genome Analyzer IIx (Illumina), generating 72-or 100-base reads. We used a BWA-MEM aligner (Li and Durbin, 2009)

Tw detection

PCR and serological tests for Tw were performed as previously described [START_REF] Fenollar | A paradoxical Tropheryma whipplei western blot differentiates patients with whipple disease from asymptomatic carriers[END_REF].

Cell culture and subpopulation separation

PBMCs were isolated by Ficoll-Hypaque density centrifugation (GE Healthcare) from cytopheresis or whole-blood samples obtained from healthy volunteers and patients, respectively. PBMCs and EBV-B cells (B-cell lines homemade transformed with EBV) were cultured in RPMI medium supplemented with 10% FBS, whereas HEK293T cells (ATCC;

CRL-3216) were cultured in DMEM medium supplemented with 10% FBS. Subsets were separated by MACS, using magnetic beads conjugated with the appropriate antibody (Miltenyi Biotec) according to the manufacturer's protocol. All cells used in this study were tested for mycoplasma contamination and found to be negative.

Site-directed mutagenesis and transient transfection

The full-length cDNA of IRF4 and PU.1 was inserted into the pcDNA ™ 3.1D/V5-His-TOPO ® vector with the directional TOPO expression kit (Thermo Fisher Scientific). BATF anf JUN were obtained from Origene companie (#RC207104 and #RC209804 respectively).

Constructs carrying mutant alleles were generated from this plasmid by mutagenesis with a site-directed mutagenesis kit (QuikChangeII XL; Agilent Technologies), according to the manufacturer's instructions. HEKβλγT cells were transiently transfected with the various constructs, using the Lipofectamine LTX kit (Thermo Fisher Scientific) in accordance with the manufacturer's instructions.

Cell lysis and western blotting

Total protein extracts were prepared by mixing cells with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, and 2 mM EDTA) supplemented with protease inhibitors (Complete, Roche) and phosphatase inhibitor cocktail (PhoStop, Roche), 0.1 mM dithiothreitol DTT (Life Technologies), 10 µg/ml pepstatin A (Sigma, #P4265), 10 µg/ml leupeptin (Sigma, #L2884), 10 µg/ml antipain dihydrochloride (Sigma, #A6191) and incubating for 40 minutes on ice. A two-step extraction was performed to separate the cytoplasmic and nuclear content of the cells; cells were first mixed with a membrane lysis buffer (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.05 % NP40, 25 mM NaF supplemented with 1 mM PMSF, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin) and incubated for 30 minutes on ice. The lysate was centrifuged at 10,000 x g. The supernatant, corresponding to the cytoplasm-enriched fraction, was collected and the nuclear pellet was mixed with nuclear lysis buffer (20 mM Hepes pH 7.9, 0.4 M NaCl, 1,mM EDTA, 1, mM EGTA, 25% glycerol supplemented with 1 mM PMSF, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Equal amounts of protein, according to a Bradford protein assay (BioRad, Hercules, California, USA), were resolved by SDS-PAGE in a Criterion ™ TGX ™ 10% precast gel (Biorad) and transferred to a low-fluorescence PVDF membrane.

Membranes were probed with unconjugated antibody: anti-IRF4 (Santa Cruz, M-17) antibody was used at a dilution of 1:1000 and antibodies against GAPDH (Santa Cruz, FL-335), topoisomerase I (Santa Cruz, C-21), and/or lamin A/C (Santa Cruz, H-110) were used as loading controls. The appropriate HRP-conjugated or infrared dye (IRDye)-conjugated secondary antibodies were incubated with the membrane for the detection of antibody binding by the ChemiDoc MP (Biorad) or Licor Odyssey CLx system (Li-Cor, Lincoln, Nebraska, USA) respectively.

EMSA

Double-stranded unlabeled oligonucleotides (cold probes) were generated by annealing in TE buffer (pH 7.9) supplemented with 33.3 mM NaCl and 0.67 mM MgCl 2 . The annealing conditions were100°C for 5 minutes, followed by cooling overnight at room temperature. After centrifugation at 3,000 x g at 4°C for 30 minutes, the pellet was suspended in water. We labeled 0.1 µg of cold probe in Klenow buffer supplemented with 9.99 mM dNTP without ATP, 10 U Klenow fragment (NEB) and 50 µCi d-ATP-32 P, at 37°C for 60 minutes. Labeled probes were purified on Illustra MicroSpin G-25 Columns (GE Healthcare Life Sciences), according to the manufacturer's protocol. We incubated 10 µg of nuclear protein lysate on ice for 30 minutes with a 32 P-labeled (a-dATP) ISRE probe (5'gat cGG GAA AGG GAA ACC GAA ACT GAA-γ') designed on the basis of the ISG15 promoter or the B probe (5'gat cGC TCT TTA TTT TCC TTC ACT TTG GTT AC-γ') described by Brass et al. in 1999(Brass et al., 1999). For supershift assays, nuclear protein lysates were incubated for 30 minutes on ice with 2 µg of anti-IRF4 (Santa Cruz, M-17) antibody or antigoat Ig (Santa Cruz) antibody. Protein/oligonucleotide mixtures were then subjected to electrophoresis in 12.5% acrylamide/bis-acrylamide 37.5:1 gels in 0.5% TBE migration buffer for 80 minutes at 200 mA. Gels were dried on Whatman paper at 80°C for 30 minutes and placed in a phosphor-screen cassette for five days. Radioactivity levels were analyzed with the Fluorescent Image Analyzer FLA-3000 system (Fujifilm).

Luciferase reporter assays

The according to the manufacturer's instructions. For the AICE assay, we used the same protocol, but with the addition of BATF and JUN expression plasmids (25 ng/well each). Cells were used 24 h after transfection for the ISRE assay and 48 h after transfection for the AICE assay, with the Dual-Luciferase ® 1000 assay system kit (Promega #E1980), according to the manufacturer's protocol. Signal intensity was determined with a Victor ™ X4 plate reader (Perkin Elmer). Experiments were performed in triplicate and reporter activity is expressed as fold-induction relative to cells transfected with the empty vector. Negative dominance was assessed by performing the same protocol with the following modifications: (ISRE) 3 reporter plasmid (100 ng/well for a 96-well plate), pRL-SV40 vector (40 ng/well) WT and mutant plasmids were used to cotransfect cells, with a constant amount of WT plasmid (25 ng/well) but various amounts of mutant plasmid (25 ng/well alone made up to 100 ng with empty plasmid or with the indicated amount, made up to 100 ng with empty plasmid) amounting to a total of 240 ng/well. For the AICE assay, the same protocol was used, but with the addition of BATF and JUN expression plasmids (25 ng/well each).

Microarrays

For the microarray analysis of PBMCs, cells from six IRF4-heterozygous individuals (three patients, P1-P3; and three asymptomatic relatives, HET1-HET3) and six IRF4 WThomozygous individuals (four healthy relatives, WT1-WT4; and two healthy unrelated controls, C1-C2) were dispensed into a 96-well plate at a density of 200,000 cells/well and were infected in vitro with live Tw at a multiplicity of infection (MOI) of 1, or with live BCG (M. bovis-BCG, Pasteur substrain) at a MOI of 20, or were left uninfected (mock). Two wells per condition were combined 24 h post-infection for total RNA isolation with the ZR RNA Microprep ™ kit (Zymo Research). For the microarray on EBV-B cells, we used 400,000 cells from three IRF4-heterozygous mutation carriers and three WT individuals from the kindred for total RNA isolation with the ZR RNA Microprep ™ kit (Zymo Research). Microarray experiments on both PBMCs and EBV-B cells were performed with the Affymetrix Human Gene 2.0 ST Array. Raw expression data were normalized by the robust multi-array average expression (RMA) method implemented in the affy R package [START_REF] Gautier | affy--analysis of Affymetrix GeneChip data at the probe level[END_REF][START_REF] Irizarry | Exploration, normalization, and summaries of high density oligonucleotide array probe level data[END_REF]. Normalized expression data were processed as previously described [START_REF] Alsina | A narrow repertoire of transcriptional modules responsive to pyogenic bacteria is impaired in patients carrying loss-of-function mutations in MYD88 or IRAK4[END_REF] and briefly summarized here. First, fold-changes (FC) in expression between mockinfected and BCG-infected or Tw-infected conditions were calculated for each individual separately. For each set of conditions, transcripts were further filtered based on a minimal 1.5

FC in expression (up-or downregulation). In a final stage, transcripts satisfying the previous filters in at least four of the six homozygous WT individuals for each in vitro infection condition were retained for downstream analysis. We counted the differentially expressed

IRF4 qPCR

Total RNA was prepared from the EBV-B cells of individuals heterozygous for IRF4 mutations (two patients and two asymptomatic relatives), and healthy homozygous WT relatives (n=4). We also included samples from unrelated individuals (seven healthy controls and 25 patients with Tw carriage; all IRF4 coding exons for each individual were sequenced and shown to be WT). Moreover, the 25 WD patients included in this experiment were found to have an intact IRF4 cDNA structure and normal levels of IRF4 protein production in EBV-B cells. RNA was prepared from 500,000 cells with the ZR RNA Microprep ™ kit (Zymo Research), according to the manufacturer's instructions. A mixture of random octamers and oligo dT-16 was used, with the MuLV reverse transcriptase (High-Capacity RNA-to-cDNA ™ kit, Thermo Fisher Scientific), to generate cDNA. Quantitative real-time PCR was performed with the TaqMan ® Universal PCR Master Mix (Thermo Fisher Scientific), the IRF4-specific primer (Hs01056533_m1, Thermo Fisher Scientific) and the endogenous humanglucuronidase (GUSB) as a control (4326320E, Thermo Fisher Scientific). Data were analyzed by the ΔΔCt method, with normalization against GUSB.

IRF4 TA-cloning

The full-length cDNA generated from the EBV-B cells of IRF4-heterozygous and WT homozygous individuals was used for the PCR amplification of exon 3 of IRF4. The products obtained were cloned with the TOPO TA cloning kit (pCR2.1®-TOPO® TA vector, Thermo

Fisher Scientific), according to the manufacturer's instructions. They were then used to transform chemically competent bacteria, and 100 clones per individual were Sangersequenced with M13 primers (forward and reverse).

CD4 + T-cell stimulation by activating anti-CD2/CD3/CD28 mAb-coated beads

Isolated CD4 + T cells from patients (P1 and P3) and from healthy unrelated controls (C1-C4) were either left unstimulated (NS) or stimulated with activating anti-CD2/CD3/CD28 mAb-coated beads (Miltenyi Biotec) for 24 h in RPMI medium supplemented with 10% FBS (24-well plate).

Differentiation and activation of in vitro monocyte-derived macrophages (MDMs)

After isolation, monocytes from P1 or two healthy unrelated controls were plated (24well plate; 600,000 cells/well) in RPMI medium supplemented with 40% human serum (M1like) or 10% FBS (M2-like). Differentiation cytokines (R&D Systems) were immediately added: 0.5 ng/ml rhGM-CSF (M1-like) or 20 ng/ml rhM-CSF (M2-like). Every three days, we replaced 30% of the medium with fresh complete medium supplemented with the appropriate cytokines. After 14 days of differentiation, cells were left unstimulated (NS) or were activated by incubation with 2.5 ng/ml IFN-(M1-like) or 50 ng/ml rh-IL-4 (M2-like) for 48 h.

MDM surface marker expression

Differentiated/activated MDMs were detached by treatment with trypsin (1.6 µg/ml) in PBS. Cells were treated with Fc receptor blocking agent (Miltenyi Biotec) and Aqua Dead Cell Stain kit (Thermo Fisher Scientific) for 1 h. They were then washed and stained for 1 h at room temperature with appropriate antibodies (see Figure 5-figure supplement 4) and appropriate isotype controls (BD biosciences). Samples were analyzed on a Beckman Coulter Gallios flow cytometer.

Percentage of memory B cells

PBMCs from healthy unrelated controls and patients (P1, P2 and P3) were stained with antibodies against CD20, CD10 and CD27, and IgM, IgD, IgG or IgA. The percentages of transitional (CD20 + CD10 + CD27 -), naïve (CD20 + CD10 -CD27 -) and memory (CD20 + CD10 -CD27 + ) B cells were determined by flow cytometry. We then assessed the IgM/IgD or IgG or IgA expression of the memory B cells, to determine the extent of Ig isotype switching in the memory compartment.

Ex vivo naïve and effector/memory CD4 + T-cell stimulation

CD4 + T cells were isolated as previously described [START_REF] Ma | Functional STAT3 deficiency compromises the generation of human T follicular helper cells[END_REF]. Briefly, cells were labeled with anti-CD4, anti-CD45RA, and anti-CCR7 antibodies, and naïve (defined as CD45RA + CCR7 + CD4 + ) T cells or effector/memory T cells (defined as CD45RA -CCR7 ± CD4 + ) were isolated (> 98% purity) with a FACS Aria cell sorter (BD Biosciences). Purified naïve or effector/memory CD4 + T cells were cultured with T-cell activation and expansion beads (anti-CD2/CD3/CD28; Miltenyi Biotec) for five days. Culture supernatants were then used to assess the secretion of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IFN and TNF in a cytometric bead array (BD biosciences), and the secretion of IL-22, by ELISA (Peprotech).

In vitro differentiation of naïve CD4 + T cells

Naïve CD4 + T cells (CD45RA + CCR7 + ) were isolated (> 98% purity) from healthy unrelated controls or patients, with a FACS Aria sorter (BD Biosciences). They were cultured under polarizing conditions, as previously described [START_REF] Ma | Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets[END_REF]. Briefly, cells were [START_REF] Eilertson | SnIPRE: selection inference using a Poisson random effects model[END_REF], acting on 14,993 human genes. IRF4 is at the 9.4 th percentile of the distribution, indicating that it is more constrained than most human genes. 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 were subjected to western blotting. The upper panel shows IRF4 levels and the lower panel shows the levels of GAPDH, used as a loading control. The results shown are representative of two independent experiments. We showed that IRF4 was produced in large amounts in total B lymphocytes (CD19 + ), but also in naïve and memory B lymphocytes (CD19 + CD27 -and CD19 + CD27 + , respectively). IRF4 was less strongly expressed in CD3 + T lymphocytes and was not detectable in CD14 + monocytes or CD56 + natural killer (NK) cells. PBMCs from healthy unrelated controls, a patient (P1) and a homozygous WT relative (WT1). We showed that the frequencies of these subsets in P1 were similar to those in healthy controls B. Gating strategy to define the dendritic cell and monocyte subtypes.

Key resources table

Figure

  Figure 1A, live in France and are of French descent. Informed consent was obtained from all

  (ISRE) 3 reporter plasmid (pGL4.10[luc2] backbone, Promega #E6651), which contains three repeats of the ISRE sequence separated by spacers, was kindly provided by Prof. Aviva Azriel (Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology). The AICE reporter plasmid (pGL4.10[luc2] backbone, Promega #E6651) contains part of the IL23R promoter (-254 to -216). HEK293T cells were transiently transfected with the (ISRE) 3 reporter plasmid (100 ng/well on a 96-well plate), the pRL-SV40 vector (Promega # E2231, 40 ng/well) and a IRF4 WT or mutant pcDNA ™ 3.1D/V5-His-TOPO ® plasmid (Invitrogen #K4900-01, 25 ng/well or the amount indicated, made up to 100 ng with empty plasmid), with the Lipofectamine LTX kit (Thermo Fisher Scientific),

(

  DE) transcripts affected by stimulation in samples from all subjects for each stimulus, and determined the mean counts for these DE transcripts in all homozygotes. The mean values obtained were then used to normalize the counts of DE transcripts, yielding an overall transcriptional responsiveness for each individual separately, and for each stimulus. This overall responsiveness of subjects to either Tw or BCG is shown as a heatmap, and individual subjects were grouped by unsupervised hierarchical clustering. Responsive transcripts were further analyzed with Ingenuity Pathway Analysis (IPA) Software, Version 28820210 (QIAGEN)[START_REF] Alsina | A narrow repertoire of transcriptional modules responsive to pyogenic bacteria is impaired in patients carrying loss-of-function mutations in MYD88 or IRAK4[END_REF] for functional interpretation. In brief, the FC values for each individual and treatment were used as input data for the identification of canonical pathway enrichment (z-score cut-off set at 0.1). The activation z-score values calculated for the identified pathways were exported from IPA and used to calculate mean values and differences between WT homozygotes and heterozygotes, and for graphical representation, with Microsoft Excel and GraphPad Prism Version 7.0, respectively. The direction of the difference was not considered further. Negative mean difference values were converted into positive values before the ranking of the canonical pathways according to the difference between the genotypes. The microarray data used in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) database, under accession number GSE102862.

  cultured with T-cell activation and expansion beads (anti-CD2/CD3/CD28; Miltenyi Biotec) alone or under Th1 (IL-1β [β0 ng/ml; R&D Systems]) or Th17 (TGF , IL-1 [β0 ng/ml; Peprotech], IL-6 [50 ng/ml; PeproTech], IL-21 [50 ng/ml; PeproTech], IL-23 [20 ng/ml; eBioscience], anti-IL-4 [5 g/ml], and anti-IFN-[5 g/ml; eBioscience]) polarizing conditions. After five days, culture supernatants were used to assess the secretion of the cytokines indicated, by ELISA (IL-22), or with a cytometric bead array (all other cytokines).

Figure legends Figure 1 .

 legends1 Figure legends

Figure 1 -

 1 Figure 1-figure supplement 1. Genome-wide linkage and whole-exome sequencing

Figure 1 -

 1 Figure 1-source data 1. Kindred information summary. For each subject, Tw carriage

Figure 1 -

 1 Figure 1-source data 2. Non-synonymous variants within the linkage regions found in

Figure 2 .

 2 Figure 2. Analysis in silico of IRF4 variants. A. Minor allele frequency and combined

Figure 2 -

 2 Figure 2-figure supplement 1. List of variants and strength of purifying selection on

Figure 2 -

 2 Figure 2-source data 1. 156 non-synonymous heterozygous coding or splice variants reported in the gnomAD or HGID databases. †μ non-canonical transcript predicted to

Figure 3 .

 3 Figure 3. Molecular characterization of the R98W IRF4 mutation (loss of DNA binding).

Figure 3 -

 3 Figure 3-figure supplement 1. Functional activity of IRF4. A. Luciferase activity of

Figure 3 -

 3 Figure 3-figure supplement 2. Protein levels of IRF4 variants previously reported in

Figure 3 -

 3 Figure 3-figure supplement 3. Protein levels of IRF4 variants from HGID database.

Figure 3 -

 3 Figure 3-figure supplement 4. Functional impact of IRF4 variants previously reported

Figure 3 -

 3 Figure 3-figure supplement 5. Functional impact of IRF4 variants from HGID database.

Figure 4 .

 4 Figure 4. 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;

Figure 4 -

 4 Figure 4-figure supplement 1. IRF4 protein levels in EBV-B cells. A.-C. (Left) Total cell

Figure 5 .

 5 Figure 5. IRF4 protein levels in CD4 + T cells. A.-C. (Left) Total-cell (A), cytoplasmic (B) and nuclear (C) extracts from CD4 + T cells from

Figure 5 -

 5 Figure 5-figure supplement 1. IRF4 protein levels in PBMC subpopulations.

Figure 5 -

 5 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)

Figure 5 -

 5 Figure 5-figure supplement 3. IRF4 levels in controls and patient monocyte-derived

Figure 5 -

 5 Figure 5-figure supplement 4. Surface marker levels in controls and patient monocyte-

Figure 5 -

 5 Figure 5-figure supplement 5. Percentage of memory B cells in PBMCs from controls

Figure 5 -

 5 Figure 5-figure supplement 6. In vitro differentiation of CD4 + T cells from patients and controls. Naïve and memory CD4 + T cells from healthy unrelated controls and patients (P2

Figure 5 -

 5 Figure 5-figure supplement 7. Ex vivo cytokine production by CD4 + memory T cells

Figure 5 -

 5 Figure 5-source data 1. Immunophenotyping of patients (P1, P2 and P3) and a WT

Figure 6 .

 6 Figure 6. Overall transcriptional responsiveness of PBMCs following in vitro exposure to

Figure 6 -

 6 Figure 6-data source 1. Differentially expressed (DE) genes found to be responsive to

Figure 6 -

 6 Figure 6-data source 2. Differentially expressed (DE) genes found to be responsive to Tw

  to align the sequences with the human genome reference sequence (hg19 build). Downstream processing was carried out with the Genome analysis toolkit (GATK)[START_REF] Mckenna | The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data[END_REF] SAMtools(Li et al., 2009), and Picard Tools (http://picard.sourceforge.net). Substitution calls were made with a GATK UnifiedGenotyper, whereas indel calls were made with a SomaticIndelDetectorV2. All calls with a read coverage <2x and a Phredscaled SNP quality <20 were filtered out. Single-

	nucleotide	variants	(SNV)	were	filtered	on	the	basis	of	dbSNP135

(http://www.ncbi.nlm.nih.gov/SNP/) and 1000 Genomes (http:browser.1000genomes.org/index.html) data. All variants were annotated with ANNOVAR

[START_REF] Wang | ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data[END_REF]

. All IRF4 mutations identified by WES were confirmed by Sanger sequencing.

Acknowledgments

We thank the patients and their families for participating in the study. We thank Yelena Nemirovskaya, Tatiana Kochetkov, Lahouari Amar, Cécile Patissier, Céline Desvallées, Dominick Papandrea, Mark Woollett, and Amy Gall for technical and secretarial assistance and all members of the Laboratory of Human Genetics of Infectious Diseases for helpful discussions. We acknowledge the use of the biological resources of the Imagine Institute DNA biobank (BB-33-00065). A.G. was supported by ANR-IFNPHOX (ANR-13-ISV3-0001-01), ANR-GENMSMD (ANR-16-CE17-0005-01) and the Imagine Institute. The Laboratory of Human Genetics of Infectious Diseases is supported in part by grants from the St. Giles Foundation, The Rockefeller University, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris Descartes University, and the European Research Council (ERC), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID) and the French National Research Agency (ANR) under the "Investments for the future" program (grant number ANR-10-IAHU-01), ANR-IFNPHOX (ANR-13-ISV3-0001-01, to J.B.), ANR-GENMSMD (ANR-16-CE17-0005-01, to J.B.). Research in the Quintana-Murci laboratory was supported by the Pasteur Institute, the Centre National de la Recherche Scientifique (CNRS), the French Government's Investissement d'Avenir program, (ANR-10-LABX-62-IBEID), IEIHSEER (ANR-14-CE14-0008-02) and TBPATHGEN (ANR-14-CE14-0007-0β), and the European Union's Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement No. 281297. S.G.T. and C.S.M. are supported by grants and fellowship awarded by the National Health and Medical Research Council of Australia (1113904, 1042925) and the Office of Health and Medical Research of the New South Wales State Government. T.N. and L.W. are supported by Australian Postgraduate Research Awards from the University of NSW.


Julien Bertolino

Yasmine Beaussault

Nolwenn Sautereau

Audrey Benyamine

Philippe Berbis

Brigitte Granel
email: bgranel@ap-hm.fr

Une érythrodermie typique

Keywords: Ofuji's papuloerythroderma, erythroderma, deck chair sign

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

L'histoire

Un homme de 70 ans, sans antécédents, consultait pour une dermatose érythémato-papuleuse prurigineuse généralisée évoluant depuis trois mois. Les premières lésions des avant-bras étaient survenues après avoir coupé des figuiers. Dans l'hypothèse d'une urticaire, un traitement par ébastine était proposé. L'atteinte cutanée s'aggravait et prédominait sur le tronc avec un aspect infiltré de la peau qui respectait les grands plis, notamment les plis abdominaux (Figure 1). On notait des adénopathies inguinales infra centimétriques.

La biologie retrouvait une hyperéosinophilie modérée (1,3 giga/L), une discrète lymphopénie, une augmentation modérée des IgE totales, sans syndrome inflammatoire. La recherche de cellules de Sézary circulantes, le typage lymphocytaire et l'électrophorèse des protéines sériques ne montraient pas d'anomalie. L'exploration immunologique retrouvait la présence isolée d'anticorps anti-nucléaires de fluorescence mouchetée (titre à 1/320) sans spécificité antigénique. L'histologie cutanée montrait une capillarite dermite à prédominance de polynucléaires éosinophiles non spécifique.

Le diagnostic

La papulo-érythrodermie d'Ofuji était diagnostiquée sur l'érythrodermie épargnant les plis cutanés, et notamment les grands plis de flexion et les plis cutanés abdominaux avec le signe de la « chaise longue », bien visible sur la Figure 1.

Les commentaires

La papulo-érythrodermie d'Ofuji se caractérise par des papules érythémateuses (voir brunâtres), prurigineuses dont la coalescence évolue rapidement vers une éruption étendue, touchant surtout le tronc, voire un tableau d'érythrodermie, respectant les grands plis de flexion, et notamment les plis cutanés abdominaux [START_REF] Torchia | Papuloerythroderma 2009: two new cases and systematic review of the worldwide literature 25 years after its identification by Ofuji[END_REF]. C'est une érythrodermie d'évolution chronique, avec présence d'adénopathies périphériques dans la moitié des cas, touchant le plus souvent des hommes d'âge mûr. Il existe une hyperéosinophilie dans plus de 80% des cas et une élévation des IgE totales dans des cas. Une lymphopénie est moins fréquente. L'histologie est non spécifique, mais permet d'exclure un lymphome ou une autre pathologie cutanée.

Dans la littérature, la papulo-érythrodermie d'Ofuji pourrait être associée à des cas d'hémopathies malignes, ainsi qu'à des néoplasies viscérales et notamment des cancers gastriques [START_REF] Teraki | High incidence of internal malignancy in papuloerythroderma of Ofuji: a case series from Japan[END_REF]. Chez le patient, un bilan exhaustif comprenant un scanner thoraco-abdominopelvien et une endoscopie oeso-gastro-duodénale, revenait négatif. La corticothérapie locale, générale et la PUVA thérapie [START_REF] Mutluer | Treatment of papuloerythroderma of Ofuji with Re-PUVA: a case report and review of the therapy[END_REF] sont les principaux traitements.

Pour le patient, une corticothérapie locale a permis d'améliorer les lésions cutanées, mais un effet rebond a été observé à la décroissance de celle-ci. Dans la littérature, des traitements par ciclosporine, interféron alfa et azathioprine ont été proposés en cas de corticorésistance [START_REF] Jullien | Ofuji's papuloerythroderma: treatment with cyclosporin A[END_REF][START_REF] Plantin | Ofuji's papuloerythroderma: efficacy of interferon-alpha[END_REF].

Les auteurs ne déclarent aucun conflit d'intérêt.

Légende de la figure