In-vitro NLRP3 functional test assists the diagnosis and therapeutic choice of cryopyrin-associated periodic syndrome (CAPS) patients: a Brazilian cooperation

doi:10.21203/rs.3.rs-1731906/v1

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In-vitro NLRP3 functional test assists the diagnosis and therapeutic choice of cryopyrin-associated periodic syndrome (CAPS) patients: a Brazilian cooperation

https://doi.org/10.21203/rs.3.rs-1731906/v1

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Objective To report our five-years experience on the use of NLRP3 inflammasome functional assays in the differential diagnosis of Brazilian patients with a clinical suspicion of CAPS.

Patients and methods: The study included 9 patients belonging to 2 families (I, II) and 7 unrelated patients with a clinical suspicion of AID according to Eurofever/PRINTO classification, recruited between 2017 and 2022. The control group for the NLRP3 functional assay consisted of 10 healthy donors and for the CBA cytokines measurement of 19 healthy controls. Patients underwent clinical evaluation, genetic and functional analysis.

Results: All members of the family I received the diagnosis of Muckle-Wells Syndrome (MWS), carried the NLRP3 Thr348Met variant and resulted positive for the functional assay. The 2 patients of the family II resulted negative for the mutational screening but positive for the functional assay compatible with a MWS clinical phenotype. In 2 unrelated patients with NLRP3 mutations, including a novel mutation (Gly309Val, Asp303His), a positive functional test confirmed the clinical diagnosis of NOMID. 3 unrelated MWS and 1 FCAS patients resulted negative to the genetic screening and positive for the functional test. One patient with a FCAS-like phenotype harbored the NLRP12 His304Tyr variant confirming the diagnosis of FCAS2.

Conclusion: The NLRP3 inflammasome functional assay can assist the clinical diagnosis of CAPS even in patients with unknown genetic defects.

autoinflammatory diseases

CAPS

NLRP3

inflammossome

functional assay

cryopyrin-associated periodic syndrome

Autoinflammatory diseases (AIDs) or Systemic Autoinflammatory Diseases (SAIDs) are a group of disorders that belong to the inborn errors of immunity (IEI) and around 25% of them may present with any autoinflammatory phenomena.¹ The inflammasomopathies comprises a group of disorders that arise from aberrant inflammasome activation in the cytosol and are driven by molecules such as IL-1ß or IL-18, or TNF, in a monogenic or multifactorial mode depending of the involved pathway. Clinical diagnosis of AIDs is challenging, due to overlap of symptoms among themselves and with other diseases. ²

CAPS (Cryopyrin-associated Periodic Syndrome) refers to three specific clinical conditions with a continuous severity spectrum: familial cold autoinflammatory syndrome (FCAS - MIM #120100), Muckle-Wells syndrome (MWS - MIM #191900) and neonatal-onset-multisystem inflammatory disease (NOMID; also known as chronic infantile neurological cutaneous articular syndrome, CINCA - MIM #607115). Common features include recurrent episodes of urticarial rashes, fever and arthralgia whereas progressive sensorineural hearing loss and AA amyloidosis are feared complications in MWS and NOMID patients. Gain-of-function mutations in NLRP3 gene are found in about 50% of CAPS patients inherited in an autosomal dominant fashion, even if in NOMID patients the mutations are always de-novo variants or somatic mosaicism. 3,4 Moreover, in the FCAS phenotype, mutations in NLRP12 and NLRC4 gene have been reported, leading to the classification of FCAS2 (MIM #611762) and FCAS4 (MIM #616115), respectively. While the gain-of-function mutations reported in NLRP3 and NLRC4 resulted in the hyper-activation of inflammasome complexes and share similarities in CAPS, the exact pathogenic mechanism of the mutated NLRP12 is still uncertain ^5,6.

CAPS diagnosis relies on clinical and genetic evaluation and due to heterogeneity of clinical presentation, overlapping of signs and symptoms with other AIDs, CAPS diagnosis is still challenging. Several groups from high-income countries have developed functional assays to discriminate AIDs patients, sustain diagnosis and a personalized therapy, but none is still worldwide nor commercially available nor in routine practices ⁷.

In this work we report our five-years´ experience on the use of a functional assay for the differential diagnosis of Brazilian patients with a clinical suspicion of CAPS.

Ethics approval and consent to participate.

The research was in compliance with the Declaration of Helsinki and was approved by the Institutional Review Board of the “Hospital das Clínicas” Faculty of Medicine, University of Sao Paulo (HC/FMUSP, São Paulo, Brazil). (Number : CAAE 74305817.0.0000.0068) and the Ethical Committee of the Institute of Biomedical Sciences of the University of Sao Paulo (ICB-USP, São Paulo, Brazil) (No. 5.241.841). Written informed consents were collected from all the participated subjects and/or their legal guardian.

Patients and controls.

A total of 16 patients (F/M: 7/9; 29,5: 2-74 years) who fulfilled the diagnostic criteria for CAPS were recruited from Brazilian reference centers in Inborn Errors of Immunity at the Ambulatory of Autoinflammatory and Immunedysregulatory Diseases at the Division of Clinical Immunology and Allergy of the HC/FMUSP (São Paulo, Brazil). CAPS patients were defined accordingly to the diagnosis criteria proposed by (Gattorno et al 2019)⁸ characterized by raised acute phase reactants and at least 2 of the following clinical manifestations: recurrent fever, urticarial-like skin rashes, cold or stress triggered attacks, sensorineural hearing deficit, chronic aseptic meningitis, skeletomuscular symptoms or abnormalities, regardless of their family history. Blood samples and skin biopsies were collected from patients with active disease before treatment initiation. Infections and/or hypersensitivity were excluded from all individuals based on clinical history and sample's collection. A group of healthy volunteers (n = 10; F/M: 6/4; age: 31.6 ± 7.1 years) were included as controls.

Genetic analysis.

All coding exons (2-10) of the NLRP3 gene (Refseq: NM_001243133.2) were sequenced by Sanger Sequencing following standard procedures (see Supplementary File 1 for primers sequenced). NLRP3 negative patients were additionally screened using a commercially available Next Generation Sequencing (NGS) panel covering 11 autoinflammatory genes L1RN (NM_173841), LACC1 (NM_153218), LPIN2 (NM_014646), MEFV (NM_000243), MVK (NM_000431), NLRC4 (NM_021209), NLRP12 (NM_144687), NLRP3 (NM_001243133), NOD2 (NM_022162), PSTPIP1 (NM_003978) e TNFRSF1A (NM_001065), at the Genomika-Diagnosticos (Rusmini et al 2016) ⁹.

In vitro cellular assay.

After isolating human peripheral blood mononuclear cells (PBMC) via Ficoll-Paque gradient centrifugation, cells were counted and seeded onto 96-well plates (Corning-Costar) at a concentration of 4x 10⁶ cells/ml in RPMI-1640 (Gibco, Thermo Fisher Scientific). PBMC were incubated for 90 minutes at 37°C 5% CO_2,then non-adherent cells were removed, and adherent cells (mainly monocytes) were cultured in culture medium (RPMI-1640 medium with 10% fetal bovine serum/FBS) (Gibco, Thermo Fisher Scientific). Monocytes were treated with 1 μg/ml bacterial lipopolysaccharide (LPS from Escherichia coli O111:B4; Sigma-Aldrich/Merck) for 3 hours and then with 1 mM ATP (Sigma-Aldrich/Merck) for another 15 minutes as described in (Gattorno et al, 2007) ¹⁰. Supernatants collected without stimulation or just with 1 mM ATP were used as untreated controls (UN) and ATP controls (ATP), respectively. The culture supernatants were collected for the analysis of IL-1β and IL-18 release. All tests were performed in duplicates.

Cytokines measurements.

IL-1β and IL-18 were measured by sandwich enzyme-linked immunosorbent assay (ELISA) commercial kits (Biolegend and R&D System, respectively), according to the manufacturer’s instructions. For a subgroup of patients (n=9) plasma levels of IFN-γ, TNF-α, IL-17A, IL-10, IL-6, IL-4 and IL-2 were measured using a BD™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 (BD Biosciences) according to the manufacturer’s instructions

Statistical analysis.

X² test was applied to compare categorical variables. A 1-Way ANOVA test followed by multi comparisons analysis was executed to compare cytokines levels among different conditions. The level of significance was 0.05. Statistical analyses and graphic presentations were carried out using Prism 9.3.1 software (GraphPad).

Demographic, clinical and laboratorial characteristics of CAPS patients

Demographic and clinical data of the 16 CAPS patients recruited for the current study were retrospectively summarized in Table 1. 7 out of 16 patients were sporadic cases with a broad clinical spectrum of CAPS. Familial clusters were observed for 9 patients with Muckle-Wells Syndrome in Family I (P03, P05, P06, P07, P08, P09, P11) and Family II (P12, P13), respectively (Figure 1A-B). In most of the patients (13/16, 81%) the first symptoms were observed before 16 years of life, while all NOMID patients (P01-02) had neonatal onset. Of note, P14 presented an adult onset (20 years old) of symptoms. (Table 1)

Recurrent fever was present in all but 2 patients (87.5%), whose were genetically negative. Urticaria-like rash could be observed in all but one patient (P14), who presented with an uncommon phenotype characterized by a sudden sensorineural hearing loss (SNHL) with neutrophilic meningitis. Arthritis and bone overgrowth could be observed in 10 (62.5%) and 1 (6.3%) patients, respectively. Deafness was observed in 10 out of 16 (62.5%) patients, supporting the MWS diagnosis (Table 1). All patients had positive acute reactant markers (Table 1), but now, no patients suffer with amyloidosis. 14 patients (87,5%) reported cold exposure as a common flares’ trigger (all with FCAS and MWS profile) and vaccine induced flare could be observed frequently just in one patient (P10). 14 patients were vaccinated to COVID-19 (10 Pfizer/Biontech; 4 Aztrazeneca) and none of them experienced flare nor fever triggered after immunization.

Skin biopsy was performed in 6 patients (3 from Family I and 3 unrelated patients) and for 5 out of 6 (91%) the results were compatible with a neutrophilic dermatosis (Table 2; Figure 1 D-L), as already described.¹¹ Intriguingly, a lymphocytic skin infiltration was observed in P12 (Table 2)

Mutational screening identified yet known pathogenic variants of NLRP3 in 9 out of 16 patients (53%) (Table 2). The Thr348Met mutation was detected in all the members of Family I, and the Gly307Val and Asp303His, were reported as de novo variants in NOMID patients P01 and P02, respectively. All mutations found in the NLRP3 gene are localized within the central NACHT domain (exon 4 of reference sequence NM_001243133.2; from http:// infevers.umai-montpellier.fr) (Figure 1C). P15 harbors the His304Tyr pathogenic variant in the NLRP12 gene, demonstrated a neutrophilic infiltration of the skin and received the diagnosis of FCAS2 (Table 1-2).

For 9 patients, namely family I members (P03, P05, P06, P07, P08, P09, P11), the NOMID patient P01, and the MWS patient P14, a deep investigation of plasma Th1/Th17/Th2 cytokines profile was realized and compared to healthy donors (Table 2, Figure 4).The proinflammatory cytokine IL-6 resulted high in all the patients (61.4 ± 83.9 pg/mL) and significantly higher than HD (8.2 ± 7.4; p = 0.007) (Figure 2). 4 out of 7 members of Family I and P21 showed medium-to-high levels of IL-17A (20.7 - 131.0 pg/mL, average level: 68.3 ± 50.3 pg/mL), which also resulted increased compared to HD (values below the detection limit; p = 0.003). P23 showed an increased level of TNF-α (17.9 pg/mL) but not of IL-17A (Figure 2A-B). IFN-γ, IL-10, IL-4 and IL-2 were undetectable in all our samples.Statistical differences of plasma levels of IL-6 in children (p < 0.0007) and adults (p < 0.001) could be observed when compared to healthy controls. For IL-17A statistical difference could be observed only in adults (p<0.0213), also compared to healthy controls. (Figure 2C-D)

Regarding the treatment, all patients from Family I as well as P16, P10, P01, P02 were on anti-IL1 therapy (Canakinumab). The other CAPS patients were using steroids on flare. FCAS2 patient (P15) was currently under anti-IL1 receptor antagonist therapy (Anakinra) (Table 1).

In vitro functional assay

NLRP3 acts as a sensor of cytosolic perturbances, such as K+ efflux, mitochondrial and/or lysosomal damage, and, through the interaction of the adaptor protein ASC, recruits and activates the pro-inflammatory caspase-1, which in turn is responsible for the cleavage of the immature proforms of IL-1ß and IL-18 and of the release of the biologic active cytokines. Several models of NLRP3 inflammasome activation have been proposed, however the most commonly used in vitro relies on the “two-step“ challenge performed by treating peripheral blood monocytes with bacterial lipopolysaccharide (LPS) followed by adenosintriphosphate (ATP). While LPS induces the expression of inflammasome components through the Toll-Like Receptor (TLR) 4-mediated pathway, the ATP activates the purinergic receptor P2X7, which mediates the K+ efflux responsible for NLRP3 activation.¹²

In healthy individuals, monocytes produce a large amount of IL-1ß in response to LPS, which is dramatically augmented when ATP is added to the culture. Monocytes from CAPS patients, however, tend to present a normal-to-higher release of IL-1ß in response to LPS compared to healthy donors, but fail in responding to ATP stimulation. ¹³This characteristic behavior may be used in vitro to identify defects in the NLRP3 inflammasome. Several protocols have been described in literature with few differences, and we decided to realize the assay as reported by and briefly described in the Material and Method section. ¹⁰

As shown in Figure 5A, all patients' monocytes after 3 hours of LPS-treatment released equal or higher levels of IL-1ß compared to healthy donors’ cells. However, differently to HD, the addition of ATP was not able to further augment the cytokine production in CAPS monocytes, with the exception of P15, who harbors the NLRP12 His304Tyr variant and behaves like a HD in this test. Low or undetectable levels of IL-18 were produced by monocytes regardless of LPS and/or ATP stimuli (data not shown). The difference between the IL-1ß production in LPS and LPS+ATP treated monocytes is statistically significant just in healthy donors’ cells but not in CAPS ones (Figure 5B). Interestingly, but not surprisingly, we did not observe significant differences between CAPS patients carrying or not pathogenic mutation in NLRP3 (Figure 5C).

As the response to LPS is known to be heterogeneous in the general population, we therefore consider for our analysis to calculate the ratio between IL-1ß level in LPS+ATP and LPS in patients and controls. In monocytes isolated from HD the IL-1ß release in response to LPS+ATP and LPS gave a ratio > 2 (Figure 3D). The ratio was < 2 for all CAPS samples, independently from the presence of NLRP3 mutation (Figure 3D). Of note, in P15 the LPS+ATP/LPS ratio is 2.51.

In agreement with our results, other authors have emphasized that the dysregulation of the NLRP3 inflammasome may be detected in cellular assays also in mutation-negative individuals.¹⁴

Diagnosis of AIDs is still a big challenge in clinical management of chronic inflamed patients. Specially for CAPS patients many difficulties arise because of the overlapping findings with other clinical conditions such as chronic spontaneous urticaria, neonatal sepsis and spontaneous neurosensorial deafness without urticaria as presented here. The delay in diagnosis for CAPS patients in the world varies from 1,4 years in the more severe phenotypes to 23 years in the mild phenotypes, similarly to our findings.¹⁵ Direct consequences of not properly diagnosed and treated CAPS patients are irreversible disabilities such as deafness, found in around 60% of our patients. If anti-IL1 can prevent the installation of deafness is still a question to be studied, but here we present two patients with NOMID (P01 and P02) phenotype that received canakinumab, the only anti-IL1 available in Brazil, before the 1 year of life and signs of deafness could not be observed over a median period of 3 years of follow-up. The pathogenic mechanism of deafness in CAPS patients is presumed to be related to local IL-1 production by the inner ear macrophages in the central nervous system, but whether it it's direct activation of the NLRP3 inflammasome or if it is derived by the infiltration of the circulating immune cells is yet not clear. ¹⁶ In our case series we observed high levels of IL-17 just in adult MWS patients all with deafness but not in children with a severe phenotype and we surrogate if the presence of IL-17 in adult CAPS patients may have an implication in deafness. If IL-17 production is a direct consequence of NLRP3 activation, premature imuno inflammaging or a combination of both, it is still to be elucidated.

In Brazil, from 2009, AIDs have started to be diagnosed mainly by the identification of genetic defects.¹⁷ In more than a decade the increasing knowledge of AIDs and of diagnostic criteria within Brazilian physicians increased the identification of patients with suspected AIDs. Due to the quite recent awareness of these diseases among physicians, it is interesting to notice that many adults suffering with recurrent fever and systemic inflammation, often with unusual/atypical presentation, are also being taken in account for AIDs diagnosis. ¹⁸ This observation is reinforced by a patient presented here in P09 of Family I diagnosed at the age of 74 years. Therefore, disease awareness is still necessary for Autoinflammatory Diseases in Brazil.

The genomic sequencing allowed the dissection of the genes involved in the AIDs and became an extremely important diagnostic tool, but in 50% or more of the patients it does not fulfill genotype and phenotype correlation, indicating that, even if genetic analysis is performed, it may not be resolutive for the diagnosis in all the patients.¹⁹ So together with the effort in expanding the clinical and genetic basis of AIDs, some research groups have been developing in vitro functional assays to support diagnosis.^{10, 20, 21, 22} Another clinical problem raised after the general use of genetic sequencing is the report of VUS (variants of uncertain significance) including in the NLRP3 gene. Therefore, functional assays must be strongly recommended at least as an initial screening to avoid misdiagnosis and the economic burden of unnecessary therapies.

As recently discussed by Gattorno et al 2019, several clinical diagnostic criteria have been proposed for monogenic “classical” AIDs, such as CAPS, and they have been re-evaluated taking in account the mutational screening. However, even if patients with the syndromic phenotype but lacking the genetic finding can still fulfill the diagnosis criteria proposed they will miss a proof of NLRP3 dysfunction, important to treatment access. Another consideration is the neutrophilic infiltration of the skin, that is one of the diagnostic criteria for CAPS and many technical issues must be considered for a non-neutrophilic finding in suspected CAPS lacking the genetic proof, as we observed in P19. Subsequent biopsies could be considered in such a specific situation which is invasive, risky and it's difficult to obtain from children. Therefore, we here demonstrate that the NLRP3 functional assays can be of great help in establishing a definitive diagnosis for these situations.

All these considered, in the last five years we have applied the functional assay for the support of the diagnosis of CAPS patients in a single reference center in Brazil. In the present study we reported the evaluation of IL-1ß release in monocytes isolated from fresh blood stimulated in vitro with LPS and ATP. As expected, CAPS patients, either harboring or not NLRP3 mutations, presented a defective response to ATP in LPS-treated monocytes resulting in positive clinical evidence to diagnosis for 50% of the CAPS patients studied. While in the case of NLRP3 mutated patients, the functional test just confirms the diagnosis of CAPS, in genetic negative patients it represents a fundamental tool to support clinical evaluation and the diagnosis.

As we demonstrate an assay to functionally access disrupts in the NLRP3 inflammasome have enormous clinical impacts in CAPS diagnosis but some limitations for its realization must be considered. The main difficulty is to simplify the procedure to be able to introduce it into routine laboratory tests as it takes at least four days straight, including the realization of ELISA for IL-1ß detection and needs qualified personnel. Now the functional assay for NLRP3 inflammasome is just realized in academic laboratories and funded by research grants.

Funding

This work was supported by the “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP) research grant n.2019/06363-4 and n.2015/23395-6. V.N.C.L. is a recipient of a Post-doc Fellowship from FAPESP. M.E.G.V.R. and R.C.C. are recipient of a fellowship from the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES) A.P. is recipient of a Fellowship from Conselho Nacional de Pesquisa e Tecnologia (CNPq).

Competing Interests

L.O.M. received fees as speaker and advisory board of Novartis Brazil. The other authors declare no competing interests.

Availability of data and material

The authors declares that the original data are available under request to the corresponding author.

Ethics approval

This work was approved by Hospital das Clinicas of the Faculdade de Medicina da USP CAAAE number: (Number : CAAE 74305817.0.0000.0068) and the Ethical Committee of the Institute of Biomedical Sciences of the University of Sao Paulo (ICB-USP, São Paulo, Brazil) (No. 5.241.841).

Consent to participate

All patients consented to participate to this project.

Consent for publication

All patients consented to the publication of this article.

Acknowledgment

We thank the Ambulatory of Immunology and Allergy of the “Hospital das Clinicas” for the support and contribution in sample collection and to all families that participated in this work.

Author contributions

L.O.M. and A.P. have designed, drafted the work and have finalized the manuscript for submission.

L.O.M.,M.A.M.T.B., E.T., I.C.M. F., A.S.G., N.O.P, F.F.M.C. have assist patients recruitment and for patients care

L.O.M., S.F.B., A.M. have realized the Sanger sequencing of genomic DNA and have performed the dosage of plasma cytokines by CBA.

V.N.C.L., M.E.G.V.R. and R.C.C have performed the in vitro functional assay.

M.C.R.M. have executed the skin biopsies and histochemical analysis.

J.K. have contributed as a senior and critical revision of the manuscript

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Table 1 - Demographic and clinical characteristics of the study cases. Patients are identified as numbers (Identification, ID) and age and age of onset are expressed in years.

Patient ID	Sex	Age	Age at onset	DIagnosis	Fever	Rash	Osteo articular	Bone Overgrowth	Deafness	Conjuntivitis	Meningitis	Treatment
1	F	4	0	NOMID	x	x	x	-	-	x	x	Canakinumab
2	M	4	0	NOMID	x	x	x	x	-	x	x	Canakinumab
3	M	17	0	MWS	x	x	x	-	x	x	-	Canakinumab
4	F	46	2	MWS	x	x	-	-	x	-	-	Steroids on flare
5	M	48	4	MWS	x	x	x	-	x	x	-	Canakinumab
6	M	4	5	MWS	x	x	x	-	x	x	-	Canakinumab
7	M	45	0	MWS	x	x	-	-	-	-	-	Canakinumab
8	M	44	14	MWS	x	x	-	-	x	x	-	Canakinumab
9	F	74	1	MWS	x	x	x	-	x	x	-	Canakinumab
10	M	29	4	MWS	x	x	-	-	x	x	-	Canakinumab
11	M	50	4	MWS	x	x	x	-	x	x	-	Canakinumab
12	F	8	1	MWS	-	x	x	-	x	x	x	Steroids on flare
13	M	10	1	MWS	-	x	x	-	-	x	-	Steroids on flare
14	F	2	20	MWS	x		-	-	x	x	x	Steroids on flare
15	F	53	10	FCAS	x	x	x	-	-	x	-	Anakinra
16	F	30	1	FCAS	x	x	-	-	-	-	-	Canakinumab

List of abbreviations: NOMID (Neonatal, multisystemic, inflammatory disorder); MWS (Muckle-Wells Syndrome); FCAS (Familial Cold Autoinflammatory Syndrome)

Table 2 - Laboratory results for the CAPS Patients. Patient ID, clinical diagnosis are reported as well as results from mutational screening , plasma CBA analysis and in vitro functional assay (reported as IL-ß ratio between LPS+ATP and LPS stimulated cells)

Patient ID	Diagnosis	Gene / Mutation	Plasma cytokine levels	IL-1ß ratio
01	NOMID	NLRP3 - c.920 G>T; p.Gly307Val	↑↑ IL-6, IL-17A	1,2
02	NOMID	NLRP3 - c.907 G>C p.Asp303His	n.r.	1,3
03	MWS	NLRP3 - c.1043 C>T ; p.Thr348Met	↑↑ IL-6, IL-17A	1,4
04	MWS	Negative	n.r.	1,3
05	MWS	NLRP3 - c.1043 C>T ; p.Thr348Met	↑↑ IL-6, IL-17A	1,1
06	MWS	NLRP3 - c.1043 C>T ; p.Thr348Met	↑↑ IL-6, IL-17A	1,2
07	MWS	NLRP3 - c.1043 C>T ; p.Thr348Met	↑↑ IL-6, IL-17A	1,1
08	MWS	NLRP3 - c.1043 C>T ; p.Thr348Met	↑↑ IL-6	1,9
09	MWS	NLRP3 - c.1043 C>T ; p.Thr348Met	↑↑ IL-6, IL-17A	1,3
10	MWS	Negative	n.r.	1,5
11	MWS	NLRP3 - c.1043 C>T ; p.Thr348Met	↑↑ IL-6	1,1
12	MWS	Negative	n.r.	1,0
13	MWS	Negative	n.r.	1,2
14	MWS	Negative	↑↑ IL-6, IL-17A, TNF	1,6
15	FCAS2	NLRP12 - c.910 C>T; p. His304Tyr	n.r.	2,5
16	FCAS	Negative	n.r.	1,1

.List of abbreviations: NOMID (Neonatal, multisystemic, inflammatory disorder); MWS (Muckle-Wells Syndrome); FCAS (Familial Cold-Associated Syndrome). n.r. (not realized)

SupplementaryFiles.docx
Supplementary File 1. NLRP3 coding exons primers used to genetic sequencingSequence of Forward (Fw) and Reverse (Rev) primers and respective melting temperatures (Tm) are reported for the coding sequence of NLRP3 gene (Refseq: NM_001243133.2)Supplementary File 2 . CBA raw data Th1/Th17/Th2 CBA assay result per patient. List of abreviations MWS: Muckle Wells Syndrome; NOMID: Neonatal onset multi organ inflammatory disease; HD: healthy child; HDa: healthy adult; ctrl: control group.

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In-vitro NLRP3 functional test assists the diagnosis and therapeutic choice of cryopyrin-associated periodic syndrome (CAPS) patients: a Brazilian cooperation

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Ethics approval and consent to participate.

Patients and controls.

Genetic analysis.

In vitro cellular assay.

Cytokines measurements.

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