Camille Louvrier

Eman Assrawi

Elma El Khouri

PhD Isabelle Melki

PhD Bruno Copin

Emmanuelle Bourrat

MD Noémie Lachaume

MD Bérengère Cador-Rousseau

MD Philippe Duquesnoy

William Piterboth

Eman Pharmd

Assrawi

PhD Elma El Khouri

Fawaz Awad

PhD Claire Jumeau

PhD Marie Legendre

PhD Gilles Grateau

MD Sophie Georgin- Lavialle

MD PhD Sonia A Karabina

PhD Serge Amselem
email: serge.amselem@inserm.fr

PhD Irina Giurgea
email: irina.giurgea@inserm.fr

NLRP3-associated autoinflammatory diseases: phenotypic and molecular characteristics of germline versus somatic mutations

KEY MESSAGES

 This study, which reviews and reinterprets all previously reported NLRP3 variations, shows on the human NLRP3 cryo-electron microscopy structure that the mosaic mutations are mainly situated in the core of the NLRP3-inflammasome activating domain, while germline mutations are scattered throughout this domain.

 Only a minority of NLRP3 mutations is found both in germinal and in somatic mosaic state. Noteworthy, in the germinal state, those mutations were all found in sporadic cases presenting with the most severe AID phenotype. The mutation localization in the NLRP3 protein but also the aminoacid change, could account for the occurrence of mutations in mosaic or in germinal state.

CAPSULE SUMMARY

The mosaic/germinal status and localization of the underlying mutation participate in the phenotypic spectrum of NLRP3-AID patients. The identification of novel germline variations should not hamper the search for mosaic mutations. Functional studies are mandatory.

KEY WORDS

NLRP3-associated autoinflammatory diseases, NLRP3, somatic mosaic mutations, NLRP3inflammasome activation, mutational hotspot. NACHT is an adenosine triphosphatase (ATPase) domain including a nucleotide binding domain (NBD) that contains a Walker A (ATP binding) and a Walker B (Mg2+ binding) catalytic motifs, the helical domain 1 (HD1), a winged helix domain (WHD), and the helical domain 2 (HD2). Through the NBD, HD2 and LRRs regions, NLRP3 interacts with the Ser/Thr kinase NEK7 that mediates NLRP3 inflammasome activation 3 .

NLRP3-AIDs, previously known as cryopyrin-associated periodic syndromes (CAPSs), include three overlapping entities with increasing severity: (i) the familial cold autoinflammatory syndrome (FCAS, OMIM#120100) characterized by cold-induced inflammatory episodes of fever, arthralgia and urticaria; (ii) the Muckle-Wells syndrome (MWS, OMIM#191900) associating recurrent attacks of fever, urticaria and arthritis with subsequent sensorineural hearing loss; and (iii) the chronic infantile neurological, cutaneous and articular syndrome (CINCA, OMIM#607115) characterized by central nervous system involvement, continuous urticarial rash and deformative joint arthritis.

Primarily related to germline heterozygous NLRP3 mutations, NLRP3-AIDs were described in familial forms of AIDs transmitted as an autosomal dominant trait [START_REF] Hoffman | Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome[END_REF] , as well as in sporadic cases due to de novo NLRP3 mutations [START_REF] Aksentijevich | De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases[END_REF][START_REF] Feldmann | Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes[END_REF] . The identification of somatic mosaic NLRP3 mutations has further broadened the clinical spectrum of NLRP3-AIDs [START_REF] Labrousse | Mosaicism in autoinflammatory diseases: Cryopyrin-associated periodic syndromes (CAPS) and beyond. A systematic review[END_REF][START_REF] Nishikomori | Low-frequency mosaicism in cryopyrin-associated periodic fever syndrome: mosaicism in systemic autoinflammatory diseases[END_REF] with the reports of late onset diseases [START_REF] Zhou | Brief Report: Cryopyrin-Associated Periodic Syndrome Caused by a Myeloid-Restricted Somatic NLRP3 Mutation[END_REF][START_REF] Mensa-Vilaro | Brief Report: Late-Onset Cryopyrin-Associated Periodic Syndrome Due to Myeloid-Restricted Somatic NLRP3 Mosaicism[END_REF][START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF] and paucisymptomatic forms [START_REF] Assrawi | Somatic mosaic NLRP3 mutations and inflammasome activation in late-onset chronic urticaria[END_REF] . The advances in next generation sequencing (NGS) technologies have led to the identification of an increasing number of NLRP3 sequence variations in AID patients, raising the question of the pathogenicity of the identified sequence variations.

In this study, in an attempt to identify possible germline-specific and mosaic-specific features of NLRP3 mutations, we re-assessed all previously reported NLRP3 variations identified in NLRP3-AID patients. To evaluate the functional impact of the mutations, we studied their molecular characteristics such as their location on the 3D structure of the NLRP3 protein and the aminoacid change, as well as their associated phenotypic severity. In this study, we also took into account three unrelated NLRP3-AID patients with mosaic variations involving the same aminoacid. One of those patients also carried a novel germline variation.

We assessed the pathogenicity of the identified variants through intra-familial segregation for the germline one, and by performing inflammasome-activation studies.

PATIENTS AND METHODS

Patients

Clinical features were collected through a standardized form. The study conforms to the Helsinki declaration regarding ethical principles for medical research. Written informed consents were obtained from each patient or their parents.

Study of the NLRP3 gene

Genomic DNA was extracted from peripheral blood leukocytes of patients and parents from Proband III using standard procedures. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Pancoll density gradient (PAN-Biotech). PBMCs were separated by positive selection using immunomagnetic microbeads (MACS, Miltenyi Biotec), anti-CD14 (monocytes) followed by anti-CD15 (neutrophils), anti-CD19 (B cells) and anti-CD3 (T cells). DNA was then extracted from monocytes, neutrophils, B cells, T cells, urine and buccal cells using proteinase K digestion followed by a phenol-chloroform extraction protocol.

Sanger sequencing: NLRP3 exon 3 and its intronic junctions were amplified by polymerase chain reaction (PCR) and sequenced using the Big Dye Terminator sequencing kit (Applied Biosystems) on an ABI 3130XL automated capillary DNA sequencer (Applied Biosystems). Sequences were analyzed against the reference sequence (NM_004895).

Next generation sequencing (NGS): NGS was performed using a custom sequence capture (Nimblegen SeqCap EZ Choice system, Roche) of the exons and the flanking intronic sequences of the main AID-causing genes: ADA2, CARD14, IL1RN, IL36RN, LACC2, LPIN2, MEFV, MVK, NLRC4, NLRP12, NLRP3, NOD2, PLCG2, PSMB8, PSTPIP1, RBCK1, TMEM173, TNFAIP3, TNFRSF11A and TNFRSF1A. Sequencing was performed on MiSeq or Nextseq500 (Illumina) platforms according to the manufacturer's instructions. The conventional bioinformatics pipeline, previously described [START_REF] Jeanson | Mutations in GAS8, a Gene Encoding a Nexin-Dynein Regulatory Complex Subunit, Cause Primary Ciliary Dyskinesia with Axonemal Disorganization[END_REF] , was used to study germline mutations. For the mosaic-dedicated bioinformatics pipeline sequence reads in fastq format were aligned to the reference human genome (hg19) with Bowtie2. Variant calling was performed with VarScan with a threshold for the minimum mutated allele percentage (MAP) of 1%. Variant calls in VCF format were then annotated through ANNOVAR. All results obtained by NGS were confirmed by Sanger sequencing.

Mutagenesis, cell culture and functional tests

Site-directed mutagenesis of the NLRP3 wild-type expression vector (pNLRP3-WT) was performed to generate the plasmids carrying the missense variations identified in the patients: pNLRP3-Glu569Gly, pNLRP3-Glu569Lys and pNLRP3-Gly769Ser.

HEK293T cells stably expressing green fluorescent protein (GFP)-tagged-ASC and FLAG-tagged pro-caspase1 (designated ASC-GFP_C1-FLAG) were transfected with 375 ng of mentioned plasmids using FUGENE HD (Promega). After 24h, the transfected cells were observed using a Nikon Eclipse TS100 inverted fluorescent microscope. Manual counting of cells containing ASC-GFP specks was then performed in 5 randomly selected fields at 20 X magnification. The percentage of speck-positive cells was calculated as the number of specks divided by the total number of counted cells. THP1 cells were initially primed with 100 ng/ml PMA (Phorbol 12-Myristate 13-Acetate) for 3h, then transfected with 500 ng of the plasmids using the FF-100 program in the 4D-Nucleofector, Amaxa (Lonza). Transfected THP1 cells were treated directly with 100 ng/ml LPS at 37C°. After 24h, cytokine measurements by ELISA assays were performed following manufacturer's instructions (R&D).

Methodology for literature review of NLRP3 mutations

Literature review of NLRP3 mutations was performed in Pubmed using the terms "NLRP3" and "mutation". Based on the classification of the American College of Medical Genetics (ACMG) [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF] , only pathogenic and likely pathogenic mutations were taken into account.

NLRP3 crystal structure analysis NLRP3 crystal structure analysis was performed using the human NLRP3 crystal (Protein Data Bank accession number 6NPY) by Sharif et al [START_REF] Sharif | Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome[END_REF] . The 3D structure was visualized with the Pymol software.

Statistical analyses

Differences were analyzed using the unpaired student t-test and were plotted with the Prism 5, GraphPad software, (San Diego, CA). A p value <0.05 was considered as statistically significant.

RESULTS

Clinical phenotype of NLRP3-AID patients

We studied three unrelated patients presenting with early onset of fever, urticaria, arthralgia and inflammatory biological profile. Probands I and II, both 50 years old, also developed sensorineural hearing loss around the age of 30 and were, therefore, suggestive of MWS. Proband III, a 4-year-old boy, displayed a clinical phenotype evocative of CINCA shortly after birth (table 1 and supplemental data 1). None of the patients had a family history of AID.

To identify the molecular defect responsible for the patients' phenotype, we followed a NGS approach based on the targeted sequencing of the main genes so far involved in AIDs.

For Probands I and II, no mutation was detected by a previous NLRP3 analysis by Sanger sequencing.

The c.1705G>A, p.(Glu569Lys) missense variation with a mutated allele percentage (MAP) of 7% and 13% was identified in peripheral blood DNA of Probands I and II, respectively (table 2, supplemental figure 1). This variation, which is absent from the genome aggregation database (gnomAD), has already been reported in several NLRP3-AID patients [START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF][START_REF] Assrawi | Somatic mosaic NLRP3 mutations and inflammasome activation in late-onset chronic urticaria[END_REF][START_REF] Saito | Disease-associated CIAS1 mutations induce monocyte death, revealing low-level mosaicism in mutation-negative cryopyrin-associated periodic syndrome patients[END_REF][START_REF] Tanaka | High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study[END_REF][START_REF] Izawa | Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing[END_REF][START_REF] Nakagawa | Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes[END_REF] .

In Proband III, the NGS analysis revealed a novel heterozygous NLRP3 variation, the c.2305G>A, p.(Gly769Ser) which is not described in gnomAD and, according to the MaxEntScan software, is not predicted to affect splicing. In the 3D structure of NLRP3, Gly769 is located on an external part of a LRR motif (supplemental figure 2), outside the region that interacts with NEK7. Secondarily, the access to parents' DNA samples revealed that p.(Gly769Ser) was inherited from the asymptomatic father, which argues against its involvement in the AID phenotype. The NGS data were therefore subsequently reanalyzed using a mosaic-specific pipeline and the c.1706A>G, p.(Glu569Gly) variation with a MAP of 9% in peripheral blood DNA was detected. This variation, absent from gnomAD, has never been reported in NLRP3-AID patients.

The cellular distribution of the identified mosaic variations was then determined by NGS and Sanger sequencing of DNA isolated from cells of different embryonic origins. In Proband I, who carries the p.(Glu569Lys) variation, a wide cellular distribution of the mutated allele was found with MAP ranging from 5% to 19%. It ranged from 11% to 15% in Proband II who also carries the p.(Glu569Lys) variation. Noteworthy, Proband III, who had the most severe phenotype (CINCA) and carries the p.(Glu569Gly) missense variation, displayed the lowest rate of the mutated allele (ranging from 7% to 10%, depending on the analyzed cell types) (table 2, supplemental figure 1).

Functional consequences of identified NLRP3 variations

To assess the pathogenicity of the identified NLRP3 variations, we studied ASC-speck formation, a common readout of inflammasome activation [START_REF] Man | Regulation of inflammasome activation[END_REF] . In agreement with the known role of NLRP3 in inflammasome assembly and ASC-speck formation, cells transfected with pNLRP3-WT displayed a significantly higher percentage of ASC-specks as compared to cells transfected with the empty vector (EV) alone. However, no significant difference was observed between the cells transfected with the constructs carrying either the germline variant (pNLRP3-Gly769Ser) or the wild-type. In contrast, cells transfected with pNLRP3-Glu569Gly or with pNLRP3-Glu569Lys (used as a positive control of inflammasome activation) showed a significantly higher percentage of ASC-specks as compared to cells transfected with pNLRP3-WT. Therefore, a gain-of-function effect on inflammasome activation was observed for p.(Glu569Gly), leading us to classify this variation as pathogenic (figure 1).

To further assess the functional consequences of the p.(Gly769Ser) variation, we tested its impact on cytokine secretion. To this end, THP1 cells were transfected with the EV, pNLRP3-WT, pNLRP3-Glu569Lys (used as positive control) or pNLRP3-Gly769Ser and treated with lipopolysaccharide (LPS), a well-known activator of the NLRP3 inflammasome.

IL1β secretion was measured in the cell culture supernatants and significantly higher levels were found in the supernatant of cells transfected with pNLRP3-Glu569Lys as compared to cells transfected either with pNLRP3-WT or with the EV. When compared to WT-NLRP3, similar amounts of secreted IL1β were measured for the p.(Gly769Ser) variant (supplemental figure 3). Taken together, these results led us to classify the p.(Gly769Ser) variant as probably benign.

Different NLRP3 mutations occur in somatic mosaic and germinal state

In an attempt to identify possible germline-specific and mosaic-specific characteristics of NLRP3 mutations, we re-interpreted all previously reported NLRP3 variations according to both the associated disease severity and their localization in the protein domains. The three unrelated NLRP3-AID patients with mutations involving the same residue but associated with different phenotypic severity were incorporated in this study. Based on the ACMG classification of sequence variations [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF] , we classified as likely pathogenic or pathogenic 90 distinct NLRP3 mutations identified in 277 patients: 33 somatic mosaic mutations in 54 unrelated patients and 65 germline mutations reported in 223 unrelated patients (figure 2, supplemental table 1). Noteworthy, half (27/54 patients) of the reported cases with mosaic mutations carry mutations located in the HD2 domain of NLRP3, including the p.(Gly569Lys) mutation, whereas only 9% (20/223 patients) of the patients with germline mutations carry mutations located in this region (figure 2). A second mosaic mutational hotspot (10/54 -18.5%) involves Phe304 to Gly309 aminoacids that overlaps the Walker B motif of the NBD. Among all reported mutations, only 8 of them have been found in both a mosaic and a germinal state (figure 3). These 8 mutations were identified in a germinal state in 16 patients (16/223 -7.2%) and, noteworthy, all these patients presented with the severe CINCA phenotype. The same mutations were identified in mosaic state in 22 patients (22/54 -40.7%) who displayed moderate (MWS) to severe (CINCA) phenotypes (figure 3). On the other hand, the most frequent germline mutations, such as p.(Thr350Met), p.(Arg262Trp), p.(Asp305Asn), p.(Leu355Pro) and p.(Ala441Val), have never been reported in a mosaic state (figure 4a), whereas some of these mutations involve aminoacids that are changed in other substitutions in mosaic state [p.(Arg262Pro), p.(Asp305His), p.(Asp305Ala) and p.(Ala441Pro)] (figure 2).

With the aim to further assess the functional impact of the mutations, we located on the crystal structure of NLRP3 3 the five aminoacids involved in the most frequently reported mutations in mosaic and germinal state. Noteworthy, we found that the mosaic mutations are located in the core of the NACHT domain close to the ATPase domain, while the germline mutations are scattered throughout the NACHT domain (figure 4b).

DISCUSSION

For an optimal medical care of patients with NLRP3-AID, an early molecular NLRP3 diagnosis is essential. Indeed, only one of the three patients identified in the current study received an effective anti-IL1 treatment prior to the identification of a NLRP3 mutation.

Although the use of NGS-based technologies increases the sensitivity of detection of somatic mutations usually undetectable by Sanger sequencing, two critical points are to be followed for an accurate molecular diagnosis of NLRP3-AIDs: i) high-depth sequencing and specific bioinformatics pipelines are necessary to detect low level mosaicisms; ii) intra-familial segregation studies of germline variations and functional assays of all newly identified variations are mandatory, because, as shown here, a benign sequence variation can hide the pathogenic one.

By studying the level of mosaicism of the identified mosaic mutations in different cell types, we observed a wide distribution, indicating that the mutational event arose early during embryogenesis. However in Proband I, a higher percentage of the mutated allele was observed in myeloid cells and B lymphocytes. A similar pattern was already observed in adult patients with a late onset of the disease [START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF] . We did not observe any correlation between the level of mosaicism and the severity of the disease phenotype, neither among the three patients, nor in the previously reported ones (figure 3), thereby suggesting that additional genetic and/or environmental factors might modulate the expressivity of NLRP3-AIDs.

The aminoacid Glu569 of NLRP3 is the most frequently affected residue involved in mosaic mutations [START_REF] Labrousse | Mosaicism in autoinflammatory diseases: Cryopyrin-associated periodic syndromes (CAPS) and beyond. A systematic review[END_REF] . The c.1705G>A p.(Glu569Lys) mutation has been reported (including the current study) in ten patients [START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF][START_REF] Assrawi | Somatic mosaic NLRP3 mutations and inflammasome activation in late-onset chronic urticaria[END_REF][START_REF] Saito | Disease-associated CIAS1 mutations induce monocyte death, revealing low-level mosaicism in mutation-negative cryopyrin-associated periodic syndrome patients[END_REF][START_REF] Tanaka | High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study[END_REF][START_REF] Izawa | Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing[END_REF][START_REF] Nakagawa | Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes[END_REF] . Of note, this G>A transition is located on a CpG dimer that favors this type of mutational event [START_REF] Cooper | On the sequence-directed nature of human gene mutation: the role of genomic architecture and the local DNA sequence environment in mediating gene mutations underlying human inherited disease[END_REF] . Two other mosaic mutations implicate the same aminoacid, c.1705G>C p.(Glu569Gln) [START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF] and c.1706A>G p.(Glu569Gly) (Proband III) without involving a CpG dimer. Overall, Glu569 is affected in 22% (12/54) of patients carrying a mosaic mutation and in only 0.4% (1/223) of the patients with a germline mutation. The patients carrying a mosaic mutation involving Glu569 had either a moderate (n=7) or a severe phenotype (n=5) [START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF][START_REF] Assrawi | Somatic mosaic NLRP3 mutations and inflammasome activation in late-onset chronic urticaria[END_REF][START_REF] Saito | Disease-associated CIAS1 mutations induce monocyte death, revealing low-level mosaicism in mutation-negative cryopyrin-associated periodic syndrome patients[END_REF][START_REF] Tanaka | High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study[END_REF][START_REF] Izawa | Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing[END_REF][START_REF] Nakagawa | Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes[END_REF] . Noteworthy, the single patient with a germline mutation involving this residue had a very severe neonatal CINCA syndrome [START_REF] Paccaud | Neonatal treatment of CINCA syndrome[END_REF] , thereby suggesting that mutations found only in mosaic state could be incompatible with life if present in germinal state.

While taking into account all reported NLRP3 mutations (n=90) identified in 277 patients, further observations can be made. Firstly, we noticed that in half of the patients with a mosaic mutation (27/54), the affected aminoacid lies on a short segment (Tyr565 to Tyr572) of the HD2 subdomain of the NACHT domain, whereas only 9% (20/223) of the patients with a germline mutation carry a mutation in that region. In an attempt to explain this striking observation, we highlighted the mutated aminoacids on the crystal structure of NLRP3. The region spanning Tyr565 to Tyr572 is located in the core of the NACHT domain facing the Walker B motif, a sequence involved in ATP hydrolysis and essential for NLRP3inflammasome activation [START_REF] Neven | Molecular basis of the spectral expression of CIAS1 mutations associated with phagocytic cellmediated autoinflammatory disorders CINCA/NOMID, MWS, and FCU[END_REF] . Interestingly, the region of the Walker B motif includes the second hotspot (Phe304 to Gly309) for mosaic mutations (18.5%, 10/54 patients). The two mutational hotspots account for 68.5% (37/54) of the patients with mosaic mutations, but only 26.9% (60/223) of those with germline mutations. Secondly, only a minority of the reported NLRP3 mutations (8/90) were identified in both somatic mosaic and germinal state. In germinal state, these 8 mutations concern 16 unrelated patients (16/223 -7.1%) who are all sporadic cases presenting with the severe CINCA phenotype [START_REF] Aksentijevich | De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases[END_REF][START_REF] Paccaud | Neonatal treatment of CINCA syndrome[END_REF][START_REF] Neven | Molecular basis of the spectral expression of CIAS1 mutations associated with phagocytic cellmediated autoinflammatory disorders CINCA/NOMID, MWS, and FCU[END_REF][START_REF] Jesus | Phenotype-genotype analysis of cryopyrin-associated periodic syndromes (CAPS): description of a rare non-exon 3 and a novel CIAS1 missense mutation[END_REF][START_REF] Jesus | Hereditary autoinflammatory syndromes: a Brazilian multicenter study[END_REF][START_REF] Aksentijevich | The clinical continuum of cryopyrinopathies: novel CIAS1 mutations in North American patients and a new cryopyrin model[END_REF][START_REF] Rosen-Wolff | Two German CINCA (NOMID) patients with different clinical severity and response to anti-inflammatory treatment[END_REF][START_REF] Matsubara | A severe case of chronic infantile neurologic, cutaneous, articular syndrome treated with biologic agents[END_REF][START_REF] Haverkamp | Impaired cytokine responses in patients with cryopyrin-associated periodic syndrome (CAPS)[END_REF] . Thirdly, the most frequent germline mutations have never been reported in NLRP3-AID patients in a mosaic state despite their location on CpG dimers for four of them (figure 2 and4a). It is tempting to speculate that those mutations reported in a germinal state only, may display a very mild phenotype (or even be asymptomatic) if present in a mosaic state and could, therefore, escape diagnosis. In keeping with this hypothesis, the single report of the p.(Thr350Met) mutation in a mosaic state concerns an asymptomatic mother who transmitted the mutation to her child who presented with a MWS phenotype [START_REF] Jimenez-Trevino | First report of vertical transmission of a somatic NLRP3 mutation in cryopyrinassociated periodic syndromes[END_REF] . Conversely, somatic mosaic mutations could be responsible for severe phenotypes or be incompatible with life if present in a germinal state [START_REF] Nakagawa | Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes[END_REF] .

A negative selection of gametes carrying the most activating mutations is another attractive hypothesis. Indeed, recent studies have shown a prominent NLRP3 staining of peritubular cells of testis from patients with impaired spermatogenesis [START_REF] Walenta | NLRP3 in somatic non-immune cells of rodent and primate testes[END_REF][START_REF] Fan | Obesity or Overweight, a Chronic Inflammatory Status in Male Reproductive System, Leads to Mice and Human Subfertility[END_REF] . Moreover, two different studies suggested hypofertility in NLRP3-AID patients [START_REF] Leslie | Phenotype, genotype, and sustained response to anakinra in 22 patients with autoinflammatory disease associated with CIAS-1/NALP3 mutations[END_REF][START_REF] Tran | Muckle-Wells syndrome and male hypofertility: a case series[END_REF] .

From a general viewpoint, it is therefore important to underline that the phenotypes observed in NLRP3-AID patients (from FACS to CINCA) represent only a part of the phenotypic spectrum associated with a mutation in NLRP3. Indeed, on the basis of the epidemiological data presented in this study, it appears that the NLRP3 mutations cover a phenotypic spectrum larger than that revealed by the patients' phenotype: some mutations could be lethal or asymptomatic (and therefore not reported) depending on their molecular characteristics (i.e. germinal/mosaic status, localization in the protein and aminoacid change).

In conclusion, as illustrated here, the early detection of an NLRP3 mosaicism is of key importance to establish the diagnosis of NLRP3-AID and to initiate anti-IL1 treatment. Our study also sheds light on one diagnostic pitfall due to the identification of several NLRP3 sequence variations in the same patient, thereby underlining the importance of functional and segregation studies to assess the pathogenicity of the identified sequence variations. The two hotspots for mosaic mutations are located around the Walker B motif, essential for NLRP3inflammasome activation. The phenotypic spectrum of NLRP3-AIDs appears to be related to the germinal/mosaic status and localization of the underlying mutations.

ABBREVIATIONSACMG:

  ABBREVIATIONS

Figure 1 .

 1 Figure 1. Impact of NLRP3 mutations on ASC speck formation. HEK (ASC-GFP_C1-FLAG) cells were transfected with 375ng of the expression plasmids encoding the wild-type NLRP3 or the NLRP3 protein carrying the missense variations p.(Glu569Gly), p.(Glu569Lys) and p.(Gly769Ser) or the empty vector (EV). The percentage of ASC specks was calculated as described in "Patients and Methods". Results represent the mean + SD from 3 independent experiments. P values were calculated using unpaired Student's t test as compared to the wildtype. * indicates significant P value <0.05; ** P value <0.01; ns indicates non-significant P value.

Figure 2 .

 2 Figure 2. Somatic mosaic and germline NLRP3 mutations on a domain organization model of the protein. Top: all mutations reported in a mosaic state. Bottom: all mutations reported in a germinal state. The five most frequently reported mutated aminoacids are in red. The asterisks denote mutations identified in this study. PYD: Pyrin domain; NBD: Nucleotide binding domain; HD1: Helical domain 1; WHD: Winged helix domain; HD2: Helical domain 2; LRR: Leucine-rich repeat domain. The Walker A and Walker B motifs are in yellow and green respectively.

Figure 3 .

 3 Figure 3. Characteristics of the NLRP3 mutations reported in both mosaic and germline state. Venn diagram of mosaic and somatic NLRP3 mutations is represented on left part of the figure. The 8 common mutations carried by 38 patients in either a mosaic or a germinal state and the phenotypes of the corresponding patients are detailed on the right part of the figure.

For

  the p.(Glu569Lys) mutation (E569K) the levels of mosaicism are also given. MAP: mutated allele percentage

Figure 4 .

 4 Figure 4. Patients' phenotypes and localization in the 3D structure of NLRP3 of the most frequently reported mutations. a. Characteristics associated with the five most frequent mosaic and germline mutations b. Highlight of the five most frequently mutated aminoacids in mosaic state (in red), in germline state (in blue) and in both mosaic and germinal state (in green). Cyan: adenosine di-Phosphate (ADP); yellow: Walker A motif; green: Walker B motif.

ACKNOWLEDGMENTS

We are grateful to the patients and the parents whose cooperation made this study possible.

This study was funded by the Agence Nationale de la Recherche, France, ANR-17-CE17-0021-01 and H2020, European Union, Grant Agreement N°779295. CL was supported by INSERM, France (Poste d'accueil, 00552404).

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.