Periodic Fever with Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome Is Associated with a CARD8 Variant Unable To Bind the NLRP3 This information is current as of September 24, 2021. Ming Sin Cheung, Katerina Theodoropoulou, Jérôme Lugrin, Fabio Martinon, Nathalie Busso and Michaël Hofer J Immunol 2017; 198:2063-2069; Prepublished online 30 January 2017; doi: 10.4049/jimmunol.1600760 Downloaded from http://www.jimmunol.org/content/198/5/2063

Supplementary http://www.jimmunol.org/content/suppl/2017/01/27/jimmunol.160076 http://www.jimmunol.org/ Material 0.DCSupplemental References This article cites 29 articles, 5 of which you can access for free at: http://www.jimmunol.org/content/198/5/2063.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Periodic Fever with Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome Is Associated with a CARD8 Variant Unable To Bind the NLRP3 Inflammasome

Ming Sin Cheung,*,1 Katerina Theodoropoulou,†,‡,1 Je´roˆme Lugrin,x Fabio Martinon,x Nathalie Busso,{ and Michae¨l Hofer†,‡

Periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is a relatively common autoinflammatory condition that primarily affects children. Although tendencies were reported for this syndrome, genetic variations influencing risk and disease progression are poorly understood. In this study, we performed next-generation sequencing for 82 unrelated PFAPA patients and identified a frameshift variant in the CARD8 (CARD8-FS). Subsequently, we compared the frequency of

CARD8-FS carriers in our PFAPA cohort (13.9%) with a healthy local population group (3.2%) and found a significant associ- Downloaded from ation between the CARD8-FS polymorphism and risk for PFAPA syndrome (p = 0.012; odds ratio: 4.96 [95% confidence interval, 1.33–18.47]). Moreover, CARD8-FS carriers display a distinct PFAPA phenotype that is characterized by a higher prevalence of symptoms out of flares and oral aphthosis (both p = 0.02 compared with PFAPA patients without the frameshift variant). CARD8 encodes a component of the NLRP3 inflammasome, which plays an important role in inflammation and contributes to the pathology of various autoinflammatory diseases. We found that the CARD8-FS variant led to a truncated CARD8 protein lacking the FIIND and CARD domains. As a result, the mutant CARD8 protein lost the ability to interact with the NOD domain of http://www.jimmunol.org/ NLRP3. In summary, these results identify a new CARD8 variant associated with PFAPA and further suggest that disruption of the interaction between CARD8 and NLRP3 can regulate autoinflammation in patients. The Journal of Immunology, 2017, 198: 2063–2069.

eriodic fever with aphthous stomatitis, pharyngitis, and PFAPA syndrome represents the most common cause of re- cervical adenitis (PFAPA) syndrome is characterized by current fever in children in European populations, and an annual P recurrent fever flares accompanied by pharyngitis, adenitis, incidence of 2.3 cases per 10,000 children per year was recently and/or aphthous stomatitis without evidence of infection, relatively reported (4). The diagnosis is based on clinical criteria and the asymptomatic intervals between the flares, and normal growth and exclusion of other causes of recurrent fever. Most of the time, by guest on September 24, 2021 development. Onset before the age of 5 y used to be considered a these febrile episodes resolve spontaneously before adulthood. criterion based on the first patients described; however, observa- The rapid response of the fever attacks to a single dose of corti- tions of classical PFAPA phenotype with later disease onset suggest costeroid points to an inflammatory origin, and the absence of an that relying on age at onset to confirm the diagnosis might not be infectious or autoimmune cause supports the hypothesis that appropriate (1–3). PFAPA syndrome is an autoinflammatory disease (5). Familial susceptibility suggests a potential genetic basis for some cases (5), although no causative gene defect has been identified. We *Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland; †Pediatric hypothesized that, as in other autoinflammatory diseases, IL- Rheumatology Unit of Western Switzerland, Pediatric Department, University Hospital 1b might play a central role in the pathogenesis of PFAPA Center Vaudois, University of Lausanne, 1011 Lausanne, Switzerland; ‡Pediatric De- partment, Geneva University Hospitals, 1211 Geneva, Switzerland; xDepartment of secondary to a genetic defect that leads to a dysregulation of IL- { Biochemistry, University of Lausanne, 1011 Lausanne, Switzerland; and Service of 1b secretion. This hypothesis was supported by the fact that, Rheumatology, Department of Musculoskeletal Health, Lausanne University b Hospital, University Hospital Center Vaudois, University of Lausanne, 1011 during PFAPA febrile attacks, an increase in IL-1 was shown at Lausanne, Switzerland the transcript and protein levels (6, 7). 1M.S.C. and K.T. contributed equally to this work. The NLRP3 inflammasome is a crucial multiprotein complex b ORCIDs: 0000-0003-3423-4609 (M.S.C.); 0000-0002-1178-2752 (J.L.); 0000-0002- involved in IL-1 secretion. Upon activation, the sensor 6969-822X (F.M.). protein NLRP3 assembles together with the adaptor protein ASC Received for publication April 29, 2016. Accepted for publication January 3, 2017. and procaspase-1 (reviewed in Ref. 8). Formation of the inflam- This work was supported by Swiss National Science Foundation Grant FN 310030- masome leads to the autoproteolytic maturation of caspase-1, 130085/1. which subsequently results in maturation and extracellular Address correspondence and reprint requests to Prof. Michae¨l Hofer, Pediatric Rheu- release of the proinflammatory cytokines IL-1b and IL-18. Gain- matology Unit of Western Switzerland, Pediatric Department, University Hospital of-function mutations in exon 3 of the NLRP3 gene Center Vaudois, 1011 Lausanne, Switzerland. E-mail address: [email protected] were implicated in hereditary inflammatory diseases, often The online version of this article contains supplemental material. classified as cryopyrin-associated periodic syndromes (CAPS), Abbreviations used in this article: CAPS, cryopyrin-associated periodic syndrome; CARD8-FS, frameshift variant in the CARD8 gene; FIIND, function to find; MAF, suggesting the presence of functionally important sites in this minor allele frequency; NGS, next-generation sequencing; PFAPA, periodic fever region (9). Interestingly, we previously found NLRP3 variants, with aphthous stomatitis, pharyngitis, and cervical adenitis; VSV, vesicular stomatitis mainly the polymorphism Q705K (rs35829419) in exon 3, virus. (6) which is a gain-of-function variant leading to moderately Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 increased IL-1b (10) that does not drive a typical CAPS www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600760 2064 CARD8 VARIANT WITH IMPAIRED NLRP3 INTERACTION IN PFAPA phenotype per se but was associated with PFAPA syndrome, Next-generation sequencing analysis suggesting that even moderate dysregulation of inflammasome Targeted gene capture and next-generation sequencing (NGS) were carried activation can contribute to the development of autoinflammatory out at the Novartis Institutes for Biomedical Research facility to sequence diseases. Moreover, it was suggested that CAPS-like disease 32 of interest (Supplemental Table I). Multiplexed sequencing li- manifestations in patients with low-penetrance NLRP3 variants braries were prepared from genomic DNA using the NuGEN Ovation are caused by a physiopathology other than solely classical Ultralow DR Multiplex protocol. Genomic regions of interest were selectively captured from the libraries using the Agilent Technologies caspase-1 hyperactivation (11), highlighting the multifaceted na- SureSelectXT Target Enrichment System protocol (SureSelect Design ID# ture of inflammatory diseases like CAPS and PFAPA. CARD8 0500861; 1.561 Mbp). The multiplexed sequencing libraries were se- (also called TUCAN or Cardinal) is a possible regulator of quenced on an Illumina HiSEquation 2500 instrument with a 2 3 76-bp inflammasome activation (12). CARD8 is composed of an read length. All sequence analyses performed for this study and genomic locations N-terminal function to find (FIIND) domain and a C-terminal reported were based on reference assembly HG19. Se- CARD domain. CARD8, through its interaction with caspase-1 quence reads were aligned to the reference human genome with the [via CARD–CARD interaction (13)] or with NLRP3 [via Burrows-Wheeler Aligner (16). Downstream processing was per- FIIND–NOD interaction (12)] was proposed to negatively reg- formed according to the Genome Analysis Toolkit (17) Best Prac- ulate the NLRP3 inflammasome. tices recommendations (18, 19) (https://www.broadinstitute.org/ gatk/guide/best-practices), including base quality score recalibration, We previously showed that a familial aggregation was observed indel realignment, and duplicate removal, using standard hard filtering in almost 50% of patients with PFAPA syndrome, indicating a parameters. Variant calls were made with the Genome Analysis Toolkit potential genetic origin. It was demonstrated recently that PFAPA UnifiedGenotyper and annotated with the Ensembl Variant Effect Predictor , 3 results from oligogenic or complex inheritance of variants in (20). All variants with base coverage 5 or variant quality score log- Downloaded from odds score , 2 were considered low quality and eliminated from subse- multiple disease genes (14). These observations, together with quent analyses. the association of dysregulated levels of IL-1b,raisedthe Our analysis strategy focused on identifying uncommon, protein-altering question about which genetic variants predispose an individual variants that are observed more frequently in our PFAPA cohort compared to PFAPA. Therefore, we screened for variants of genes involved with healthy normal controls. We only considered variants that are novel or # in pathways associated with autoinflammatory conditions, such with a minor allele frequency (MAF) 5% in an in-house database as- sembled using data from various sources, such as the 1000 Genome Project as familial Mediterranean fever (gene MEFV), TNFR-associated (17), the Single Nucleotide Polymorphism database (dbSNP) (21), and the http://www.jimmunol.org/ periodic syndrome (gene TNFRSF1A), and hyper-IgD syndrome National Heart, Lung, and Blood Institute GO Exome Sequencing Project (gene MVK).Wealsoextendedourscreeningtoinflammasome- (http://evs.gs.washington.edu/EVS/). We also kept variants for which no related genes, such as NLRPs (1–14), and some regulator genes MAF information is available and treated them as if MAF = 0. Further- more, we sought variants that are predicted to alter the amino acid se- to gain fundamental insights into genetic alterations that are quence of a resulting protein product and, thus, are likely to have a potential predisposing factors for PFAPA. functional impact on that protein. The frameshift variant in the CARD8 gene (CARD8-FS), identified by Materials and Methods NGS analysis, was validated by Sanger sequencing on a 3730XL Genetic Analyzer instrument (Thermo Fisher Scientific) at the Novartis Insti- Study population tutes for Biomedical Research facility. Mutation Surveyor (version 4.0.0; by guest on September 24, 2021 All children with PFAPA syndrome attending the pediatric rheumatology SoftGenetics) was used to perform variant detection on Sanger sequence consultation of Western Switzerland at the Lausanne University Hospital traces. and the Geneva University Hospital, from November 2009 to November Plasmids 2013, were asked to participate in this study. Previously published diag- nostic criteria (15) were applied to our patients. However, because PFAPA NLRP3, CARD8, ASC, and CASP1 were subcloned into mammalian ex- is not a well-defined disease, and there are no confirmatory tests, the power pression vector pCR3 in frame with N-terminal vesicular stomatitis virus of these criteria remains limited. A new classification based on the opinion (VSV) or FLAG tags, as previously described (22). CARD8-FS was intro- of a large panel of experts and confirmed in a large cohort of PFAPA duced into a CARD8-expressing pCR3 plasmid, and PCR was performed patients is underway and should provide improvements in the diagnosis of with 59-CCTCTTATGCTTCTAAAAAGTCTGTTTTGAGATCG-39 and PFAPA. Consequently, patients with minor symptoms between flares, 59-CGATCTCAAAACAGACTTTTTAGAAGCATAAGAGG-39 muta- mainly aphthous stomatitis and abdominal pain, and patients with disease genesis primer pair and Pfu Ultra AD enzyme (600385; Agilent). Template onset after 5 y of age were included according to the published interna- plasmid was digested with DpnI enzyme (R0176S; New England Biolabs). tional multicenter cohort of 301 patients (3). Thomas et al. (15), who Sequencing certified correct insertion of the frameshift mutation into proposed the criteria based on a series of 94 patients, also described CARD8. moderate symptoms outside of the flares in a few of them. Patients with all three constitutional symptoms were described as having complete cluster, Transient transfection and immunoprecipitation whereas incomplete cluster was used to describe patients with one or two HEK293T cells were transfected with the above-described plasmids using constitutional symptoms. All of these children had normal growth and polyethylenimine transfectant. Twenty-four hours after transfection, cells development and did not present any other symptoms suggesting an al- were washed with PBS and lysed by three freeze/thaw cycles in 1% Nonidet ternative diagnosis. Exclusion of monogenic autoinflammatory diseases P-40, 20 mM Tris-HCl (pH 7.4), and 150 mM NaCl (lysis buffer). A fraction was performed based on clinical evaluation. Genomic DNA was extracted of cell extracts was kept as input controls, and the rest was precleared by a from EDTA blood. Mutation hotspots in the four major genes associated 1-h incubation at 4˚C with Sepharose-6B beads (6B100; Sigma) before with monogenic periodic fevers (MEFV, TNFRSF1A, MVK, and NLRP3) subsequent pull-down by 1 h incubation at 4˚C with mouse anti-Flag M2 were analyzed using PCR and DNA sequencing. The regions analyzed beads (A2220; Sigma). M2 beads were washed three times with 0.1% Nonidet were as follows: MEFV exons 2 and 10 (detects .95% of known patho- P-40, 20 mM Tris-HCl (pH 7.4), and 150 mM NaCl (wash buffer). Beads and genic familial Mediterranean fever mutations), TNFRSF1A exons 2, 3, 4, inputs were loaded on SDS-PAGE. Rabbit anti-Flag (PA1-984B; Pierce) and 6 (detects close to 100% of pathogenic TNFR-associated periodic and rabbit anti-VSV (V4888; Sigma) Abs were used for immunoblotting. syndrome mutations), MVK exons 9 and 11 (detects ∼70% of pathogenic hyper-IgD syndrome), and NLRP3 exon 3 (detects close to 100% of Inflammasome assay in HEK293T cells pathogenic CAPS mutations). All subjects received code numbers to ensure anonymity. Approval for HEK293T cells were seeded in a 24-well plate at a density of 1 3 105 cells the study was obtained from the Cantons’ Ethical Committees in Lausanne per well 1 d before transfection in DMEM supplemented with 10% FCS and in Geneva, and the parents/caregivers gave written informed consent, and penicillin-streptomycin. Cells were transfected with 100 ng of plasmid according to local ethical regulations. encoding for NLRP3 or empty vector, 20 ng of CASP1, 50 ng of ASC, and DNA samples from 100 anonymous healthy Swiss volunteers used in the 200 ng of CARD8/CARD8-FS/pCR3 (empty vector). Mock transfections Sanger validation were obtained through Interregionale Blutspende (Bern, were done with 370 ng of pCR3. Transfections were done in Opti-MEM Switzerland). with a polyethylenimine/DNA ratio of 1:1 (w/w). Six hours after trans- The Journal of Immunology 2065 fection, medium was replaced with fresh DMEM with 10% FCS and However, no experimental validation or functional investigation penicillin-streptomycin. Cells were harvested 48 h after transfection, and on this mutation had been done. whole-cell lysates were loaded on 15% SDS-PAGE for Western blotting. We analyzed the samples for further validation by Sanger se- Immunoblots were done using anti-human caspase-1 (Adipogen) and anti- tubulin (Sigma). quencing. The Sanger-determined genotype calls for the CARD8- FS variant confirmed the NGS-determined genotype status for all Statistical methods subjects (i.e., 11 [13.9%] heterozygous carriers and 68 noncarriers), The Fisher exact test (two-tailed) was performed to investigate the asso- with the exception of three subjects for which the Sanger genotype ciation between the CARD8-FS polymorphism and risk for PFAPA syn- calls were inconclusive (Supplemental Table II). No individual had , drome. A p value 0.05 was considered statistically significant. a homozygous CARD8-FS variant. Subsequent statistical analyses Differences in clinical characteristics between CARD8-FS carriers and noncarriers were analyzed using a two-sample test of proportion with 95% comparing CARD8-FS carriers and noncarriers were performed confidence interval and the Wilcoxon signed-rank test. Caspase-1 cleavage using only Sanger-confirmed results. assays were analyzed using one-way ANOVA, followed by the Newman– To compare the frequencies of CARD8-FS carriers in our Keuls multiple-comparison test. PFAPA cohort with a general healthy population group, we also analyzed DNA samples of 100 healthy controls by Sanger se- Results quencing. Because ethnicity can be an important confounding CARD8-FS mutation is associated with PFAPA factor in genetic studies, control DNA samples were collected Eighty-two unrelated PFAPA patients were included in our cohort. from Swiss volunteers to match as closely as possible the genetic We performed targeted sequencing on 32 putative candidate genes backgrounds of the PFAPA cohort. Of the 100 healthy control that were previously found to be involved in autoinflammatory samples, 3 (3.2%) were heterozygous carriers of CARD8-FS, and Downloaded from diseases or encoding components of the inflammasome, such as 92 were noncarriers (inconclusive for the remaining 5). NLRPs and regulators of inflammasomes (Supplemental Table I). Comparing the frequency distribution of the two groups We only considered variants that are novel or with an MAF , 5%. (Supplemental Table III), our data show a statistically significant Furthermore, we sought variants that are predicted to alter the association between CARD8-FS polymorphism and risk for amino acid sequence of a resulting protein product and, thus, are PFAPA syndrome (p = 0.012; odds ratio, 4.96 [95% confi- likely to have a functional impact on that protein. The average dence interval 1.33–18.47]). http://www.jimmunol.org/ coverage on the targeted regions is between 40 and 1253 per CARD8-FS variant is associated with more severe PFAPA sample (with the exception of idv_27709, for which the coverage (higher incidence of symptoms out of flares and aphthosis) is 133 because of suboptimal DNA quality). In total, we identified 2613 small variant sites (,50 bp) in the targeted genes where at The clinical characteristics of the CARD8-FS carriers are shown in least one sample has a heterozygous genotype call after filtering Table I. The median age at onset of the disease was 1 y and 11 mo, out low-quality calls; 994 variants that are novel or with MAF , the male/female ratio was 0.8, the median interval between fever 5% were retained for further analyses. attacks was 30 d, and the median duration of flares was 4 d. The Of the 82 unrelated PFAPA patients in our cohort, 12 were found onset of flares occurred after the age of 5 y in two patients. All to carry a heterozygous variant in the CARD8 gene. The 2-bp TT patients presented with at least one constitutional symptom, and by guest on September 24, 2021 insertion at position chr19:48735017 was predicted to cause a six had a complete cluster. Seven patients had a family history frameshift in the reading frame, and the resulting transcript was of recurrent fever, recurrent pharyngitis, or tonsillectomy. All predicted to be a target of nonsense-mediated decay (Fig. 1). patients (7/7) who had been treated with steroids responded CARD8-FS was previously observed in the 1000 Genome Project within 12 h. One CARD8-FS patient also had the Q705K (17), estimated to have MAF = 4%, and annotated as rs140826611 NLRP3 genetic variant, another had Q705K NLRP3 and E148Q in the Single Nucleotide Polymorphism database (dbSNP) (21). MEFV genetic variants, and one had the c.769-7T . 6MVK variant. All three of these patients exhibited symptoms out of flares. We also studied the clinical characteristics of the CARD8-FS2 PFAPA patients in our cohort. The median age at onset of the disease was 1 y and 5 mo, the median interval between fever at- tacks was 30 d, and the median duration of flares was 3.5 d. The onset of flare occurred after the age of 5 y in 11 patients. All patients presented with at least one constitutional symptom, and 21 had a complete cluster. Thirty patients had a positive family history. Of 58 patients treated with steroids, 57 had a good response. A comparison of the clinical characteristics between CARD8- FS carriers and noncarriers is shown in Table II. There is no significant difference in the median age at onset or in the prev- FIGURE 1. Schematic exon organization of the CARD8 and CARD8-FS alence of onset after the age of 5 y. Furthermore, the median transcripts. Open reading frames are indicated by gray boxes. In CARD8, duration of fever attacks and the median interval between the the N-termini undergoing alternative splicing are referred to as the V re- flares are similar in both groups. However, we observed a sta- gion. The open reading frames following exon 7 are more conserved and tistically significantly higher incidence of symptoms out of code for the FIIND and CARD domains. The CARD8-FS variant harbors flares in CARD8-FS carriers in comparison with noncarriers the rs140826611 single-nucleotide polymorphism in exon 7. The resulting frameshift generates a unique CARD8-FS–specific C terminus (black box). (p = 0.02). The prevalence of oral aphthosis was also elevated in The prototypical CARD8-FS amino acid sequence is shown; the mutation the CARD8-FS PFAPA patients (p = 0.02). The frequencies of the is outlined by a square box, and the unique CARD8-FS C terminus is other constitutional symptoms and complete clusters are similar in underlined. The asterisk indicates a stop codon (in this instance created by both groups. Almost all patients in both groups had a good re- the frameshift mutation). sponse to steroids. 2066 CARD8 VARIANT WITH IMPAIRED NLRP3 INTERACTION IN PFAPA

No evidence for CARD8 C10X being a risk allele The most extensively studied polymorphism in the CARD8 gene is the C10X nonsense mutation (rs2043211; A . T) located in exon 5

6 het of the gene, which introduces a stop codon (Cys . Stop) that . results in a truncated CARD8 protein. The C10X polymorphism is present in the V region of CARD8. Alternative splicing can bypass the C10X stop codon by changing the reading frame of exon 5 E148Q het introducing a missense mutation (Phe . Ile) that does not affect expression of a full-length protein (23). The C10X polymorphism is a very common variant, with an MAF = 31.7% reported by the NLRP3, MEFV, MVK, TNFRSF1A Presence of Other Variants in Genes: 1000 Genome Project, and 9% of a control population was found to be homozygous for the Stop/Phe . Ile allele (23). However, the functional consequences of the C10X polymorphism remain un- clear (23–25). No No No No No No Because the C10X variant has a MAF well above 5%, it was not AR, APH

between Flares included in our initial discovery analysis, which specifically tar- Minor Symptoms geted uncommon variants. Nevertheless, we had good coverage on the variant position in our NGS data (average 993); the genotype Downloaded from a a a a a a a calls are listed in Supplemental Table II. In our cohort of 82 un- NA No NA AbdP NLRP3: Q705K het MEFV: Yes Yes Yes Yes related PFAPA subjects, 36 are homozygous for the reference Response to Steroids allele, 34 are heterozygous, and 12 are homozygous for the Stop/ Phe . Ile allele. The MAF derived from our cohort is 35.4%, which is close to what was reported by the 1000 Genome Project. There is no evidence to suggest that CARD8 C10X is a risk allele http://www.jimmunol.org/ + for PFAPA. Functional studies of CARD8-FS None HLAB27 tonsillectomy tonsillectomy tonsillectomy tonsillectomy To study the function of CARD8-FS, we generated a construct expressing a tagged version of the protein. This variant was very

during childhood, uveitis, unstable compared with CARD8, but its expression could be de- tected by Western blot (Fig. 2). The CARD8-FS gene variant does not code for the CARD8 functional domains CARD and FIIND, which were shown to regulate the inflammasome (12, 13, 22). Therefore, this variant is predicted to have lost its ability to in- by guest on September 24, 2021 teract with inflammasome components. Instead, CARD8-FS en- Other

Symptoms Positive Family History codes a unique 25-aa polypeptide sequence of unknown function that is absent from all other known CARD8 isoforms. Therefore, we tested the functionality of CARD8-FS to regulate the inflam- masome by interrogating its ability to interact with the inflam- masome (22). We expressed Flag-tagged NLRP3, Flag-tagged CARD8, VSV-tagged CARD8, and VSV-tagged CARD8-FS in HEK293T cells. Lysates were immunoprecipitated with an anti- Symptoms Constitutional Flag Ab and analyzed for coimmunoprecipitating . As previously reported, we showed that, in the presence of Flag- NLRP3, VSV-CARD8 was coimmunoprecipitated (Fig. 2, lane 35 P, APH, AR, My, H, AbdP, V 7). Similarly VSV-CARD8 was coimmunoprecipitated with Flag- CARD8 (Fig. 2, lane 9), indicating the ability of full-length

PFAPA patients CARD8 to form dimers. In contrast, we found that VSV– + CARD8-FS, expressing a truncated protein with a m.w. ∼ 10 kDa, lost the ability to associate with NLRP3 (Fig. 2, lane 8), indicating Fever (d) Interval (d) Duration of that this variant is unable to modulate NLRP3 activity. The CARD8- FS variant also lost the ability to form dimers with full-length CARD8 via CARD–CARD interactions (Fig. 2, lane 10), which is in accordance with the lack of a CARD domain in the variant. Furthermore, we examined the effect of CARD8 and CARD8-FS

Age at on inflammasome activation by interrogating caspase-1 cleavage Onset (y) Sex after cotransfection of HEK293T cells with NLRP3 inflammasome components (100 ng of NLRP3, 50 ng of ASC, 20 ng of CASP1) in the presence of empty vector, CARD8, or CARD8-FS. We found that CARD8 inhibits inflammasome activation, but CARD8-FS has no effect on caspase-1 cleavage compared with full-length CARD8 Patient No. (Fig. 3). Response within 12 h. 1 (Idv_26389) 2 M 4 3 (Idv_27360)4 (Idv_27461) 8 0.257 F (Idv_28014) F8 (Idv_28152) 2.5 2 0.7510 (Idv_28852) 1 M11 42 (Idv_29769) 4 15 6 M P, AD, APH 1.92 P, AD, F APH 4 F 15 3 H, AbdP H, AbdP 5 35 P, AD 28 Mother P, and 35 APH aunt: P, AD, APH AbdP, D None P AbdP H Father, mother, and uncle: NA Father: recurrent pharyngitis AbdP None Aunt: recurrent pharyngitis NLRP3: Q705K het Yes NA APH MVK: c.769-7T 2 (Idv_26813) 8.17 M 5 30 P, APH AR, AbdP None Yes 5 (Idv_27466) 2.83 M 3 30 P, AD, APH Sister: PFAPA; mother: 9 (Idv_28294) 0.83 F 5 21 P, AD, APH AbdP Father: recurrent fever Yes 6 (Idv_27770) 1.67 F 7 30 P, AD, APH Mother and father: a AbdP, abdominal pain; AD, cervical adenitis; APH, aphthous stomatitis; AR, arthralgia; D, diarrhea; F, female; H, headache; het, heterozygous; M, male; My, myalgia; NA,These not available, patients never treated with steroids; P, pharyngitis; V, results demonstrate that the CARD8-FS polymorphism vomiting. Table I. Clinical characteristics of the CARD8-FS generates a loss-of-function allele. The Journal of Immunology 2067

Table II. Comparison of clinical characteristics between CARD8-FS+ and CARD8-FS+ PFAPA patients

Characteristics CARD8-FS+ (n = 11) CARD8-FS2 (n = 68) p Value Median age at onset (y) 1.92 1.4 0.89 Median duration (d) 4 3.5 0.49 Median interval (d) 30 30 0.73 Positive familial history (n [%]) 7 (64) 29 (43) 0.19 Pharyngitis (n [%]) 11 (100) 65 (96) 0.48 Oral aphthosis (n [%]) 9 (82) 30 (44) 0.02* Adenitis (n [%]) 8 (73) 39 (57) 0.34 Symptoms out of flares (n [%]) 4 (36) 7 (10) 0.02* Complete cluster (n [%]) 6 (55) 21 (31) 0.12 Onset after 5 y of age (n [%]) 2 (18) 11 (16) 0.87 Good response to steroids (n [%]) 7/7 (100) 56/57 (98) 0.72 *p , 0.05.

Discussion could explain the fact that, in this study, PFAPA was not found to be In recent years, several studies showed that mutations in inflammasome significantly associated with the C10X variant. genes are key players in the onset of classical autoinflammatory In contrast, the CARD8-FS variant described in this article is Downloaded from syndromes with penetrant inheritance patterns. Additionally, some located in exon 7, which is conserved among most reported CARD8 studies demonstrated that more complex autoinflammatory dis- isoforms (23). Therefore, the premature stop codon resulting from eases are affected by relatively frequent and less-penetrant poly- this variant is unlikely to be rescued by alternative splicing that morphisms, such as Q705K and C10X in NLRP3 and CARD8 restores the FIIND and CARD domains. The premature termina- genes, respectively (25). These polymorphisms are believed to tion codon introduced by CARD8-FS is likely to direct the mutant enhance inflammasome activity, leading to increased IL-1b pro- CARD8 mRNAs for degradation via nonsense-mediated decay, duction. In this study, we identified a new CARD8 polymorphism leading to decreased CARD8 expression in these patients. More- http://www.jimmunol.org/ (CARD8-FS) that is significantly associated with classical PFAPA. over, we showed that the protein translated from a CARD8-FS CARD8 is known to interact with NLRPs proteins that assemble mRNA was unstable compared with CARD8. However, in an into inflammasome complexes; however, its physiological function experimental set-up with forced expression of the variant, we has not been identified with certitude. Like most negative regulators of found that CARD8-FS lost its ability to interact with NLRP3, the inflammasome, CARD8 is not present in the mouse genome (26), indicating that, beyond the fact that CARD8-FS is probably precluding any relevant functional investigations in animal models. expressed at very low levels, remaining protein products are un- Studies in humans and the identification of disease-associated vari- able to regulate inflammasome assembly. We also tested the

ants were instrumental in the characterization of CARD8 (12, 25). In ability of this variant to dimerize with CARD8. No residual in- by guest on September 24, 2021 particular, identification of the C10X mutant was linked to various teraction between CARD8 and CARD8-FS was detected, sug- inflammatory diseases, including Crohn’s disease, rheumatoid ar- gesting that CARD8-FS does not interfere with the normal thritis, cardiovascular diseases, and ankylosing spondylitis (24, 27, function of the wild-type CARD8 allele in heterozygous PFAPA 28). However, some findings have not been replicated robustly, patients. Moreover, we show that CARD8-FS has no effect on suggesting that the effect of the relatively frequent C10X polymor- caspase-1 cleavage compared with full-length CARD8, which phism might be modest or requires association with additional fac- could explain the excess inflammasome activation in CARD8-FS tors to significantly drive disease outcome (25). Another explanation patients. Altogether, these data suggest that PFAPA patients carry- is that the C10X mutation is located in the region of CARD8 that is ing a CARD8-FS allele have an overall decreased CARD8 activity highly variable and can be bypassed by alternative splicing that re- but are likely to retain one functional CARD8 allele. The absence of stores FIIND and CARD functionality (23). This could present an homozygous CARD8-FS PFAPA patients in our cohort might adaptation mechanism to compensate for CARD8 deficiency and suggest the possibility of a much more severe inflammatory phe- notype than PFAPA phenotype in homozygous individuals. PFAPA syndrome is not a sporadic disease, as shown by in- creasingly reported familial susceptibility that emphasizes a probable genetic cause for the disease. A recent genetic study of patients from 14 families indicated that PFAPAwas unlikely to be a monogenic condition and suggested an oligogenic or complex inheritance of variants (14). The role of low-penetrant mutations in genes that are involved in other monogenic autoinflammatory diseases was suggested by the significantly higher frequencies of three NLRP3 variants (R488K, V198M, Q705K) in PFAPA co- horts (6). The pathogenic role of the Q705K variant is a matter of debate because it is present in ∼5% of the healthy population. Interestingly, carriers of the Q705K variant with a typical CAPS phenotype were described (29), suggesting that additional genetic FIGURE 2. Evaluation of the interaction among NLRP3, full-length and/or environmental modifiers are involved. CARD8, and CARD8-FS. HEK293T cells were cotransfected with ex- Although IL-1 dysregulation in PFAPA is clearly supported by pression plasmids for Flag-NLRP3, Flag-CARD8, VSV-CARD8, and the in vitro data on IL-1 secretion by PBMCs from PFAPA patients and VSV–CARD8-FS variant. Whole-cell lysates were immunoprecipitated the positive clinical responses observed in patients treated with with anti-Flag Ab and analyzed by Western blot 24 h after transfection. anti–IL-1 biologics (7), a question that remains to be addressed is 2068 CARD8 VARIANT WITH IMPAIRED NLRP3 INTERACTION IN PFAPA

FIGURE 3. CARD8-FS has no effect on caspase-1 cleavage compared with full-length CARD8. HEK293T cells cotransfected with NLRP3 inflamma- some components (100 ng of NLRP3, 50 ng of ASC, 20 ng of CASP1) in the presence of empty vector, CARD8, or CARD8-FS. Whole-cell lysates were ana- lyzed for the cleavage of caspase-1 48 h after trans- fection. Representative immunoblot of caspase-1 cleavage (A) and quantifications of three independent immunoblot experiments (B). xxp , 0.01 versus lane 3 (without NLRP3), *p , 0.05 versus lane 5 (NLRP3 + CARD8).

whether inflammasome abnormality is the main trigger of in- for sample management; and Tiziana Valensise and Thomas Peters for flammatory flares in these patients. A decreased ability of CARD8 Sanger sequencing support. to bind NLRP3 is consistent with increased inflammasome activity Downloaded from and IL-1b production. This is further supported by the fact that Disclosures mutant NLRP3 proteins that cause inherited CAPS have lost their M.H. received consultancy fees from Novartis and Abbvie. The other au- ability to bind CARD8 (12). Moreover, a previous study showed thors have no financial conflicts of interest. that reduced CARD8 expression by small interfering RNA increases b the production of active IL-1 in the presence of inflammasome References agonists (12). Consistent with its role as a negative regulator of the

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Table SI – List of 32 genes of interest for targeted sequencing

Gene Start End CARD8 chr19 48706352 48759253 CASP1 chr11 104896119 104972208 CIITA chr16 10970988 11026129 IL1F5 chr2 113816164 113822375 MEFV chr16 3291976 3306677 MVK chr12 110011009 110035121 NAIP chr5 70264259 70320991 NLRC3 chr16 3588982 3627451 NLRC4 chr2 32449467 32490973 NLRC5 chr16 57023346 57117493 NLRP1 chr17 5402696 5522794 NLRP10 chr11 7980920 7987023 NLRP11 chr19 56296712 56348178 NLRP12 chr19 54296804 54327698 NLRP13 chr19 56407260 56443752 NLRP14 chr11 7041626 7092807 NLRP2 chr19 55476387 55512560 NLRP3 chr1 247579407 247612460 NLRP4 chr19 56347893 56393270 NLRP5 chr19 56511041 56573229 NLRP6 chr11 278314 285409 NLRP7 chr19 55434826 55477661 NLRP8 chr19 56459147 56500045 NLRP9 chr19 56219747 56249818 NLRX1 chr11 119037226 119054775 NOD1 chr7 30464092 30518450 NOD2 chr16 50727463 50767038 PLCG2 chr16 81772651 81991949 PSTPIP1 chr15 77285649 77329723 TNFRSF1A chr12 6437872 6451333 TNFRSF8 chr1 12123383 12204314

1 Table SII – List of 82 PFAPA subjects and their CARD8 and NLRP3 variants (genotypes for the CARD8 frameshift variant (rs140826611; determined by NGS and Sanger) and the C10X variant (rs2043211; determined by NGS). The state of NLRP3 Q705K mutation is also indicated (blank space represents homozygous reference).

CARD8-FS (rs140826611) CARD8 C10X Subject ID NGS Sanger (rs2043211) NLRP3 Q705K idv_26389 C,CTT C,CTT A,T idv_26813 C,CTT C,CTT A,T idv_27360 C,CTT C,CTT A,T heterozygous idv_27461 C,CTT C,CTT A,T idv_27466 C,CTT C,CTT A,T heterozygous idv_27770 C,CTT C,CTT A,A idv_28014 C,CTT C,CTT A,A idv_28152 C,CTT C,CTT A,A idv_28294 C,CTT C,CTT A,T idv_28531 C,CTT no call A,A idv_28852 C,CTT C,CTT A,T idv_29769 C,CTT C,CTT A,A fam_10109 C,C C,C A,T fam_10206 C,C C,C A,A idv_22694 C,C C,C A,T idv_24414 C,C C,C A,A idv_24521 C,C C,C T,T idv_25143 C,C C,C A,A idv_25770 C,C C,C A,T idv_25771 C,C C,C A,T idv_25777 C,C C,C A,A idv_25783 C,C C,C A,A idv_26082 C,C C,C A,T idv_26083 C,C C,C A,A heterozygous idv_26084 C,C C,C A,T idv_26085 C,C C,C A,T idv_26383 C,C C,C T,T idv_26384 C,C C,C A,A idv_26385 C,C C,C A,A idv_26386 C,C C,C A,T idv_26387 C,C C,C A,A idv_26502 C,C C,C A,A idv_26603 C,C C,C A,T idv_26604 C,C C,C A,T idv_26605 C,C C,C A,T idv_26606 C,C C,C A,T heterozygous idv_26607 C,C C,C T,T heterozygous idv_26608 C,C C,C T,T

2 idv_26689 C,C C,C A,T idv_26708 C,C C,C A,A idv_26812 C,C C,C A,A idv_26814 C,C C,C A,A idv_26816 C,C C,C A,T heterozygous idv_26817 C,C C,C A,T idv_26942 C,C C,C A,A idv_27134 C,C C,C A,T idv_27282 C,C C,C A,T idv_27359 C,C C,C A,T idv_27363 C,C C,C A,A idv_27364 C,C C,C A,A idv_27365 C,C C,C A,A idv_27491 C,C C,C A,T idv_27515 C,C C,C T,T heterozygous idv_27607 C,C no call A,T idv_27700 C,C C,C T,T idv_27709 C,C no call A,T no call idv_27710 C,C C,C A,A idv_27711 C,C C,C A,A idv_27769 C,C C,C A,T idv_28013 C,C C,C T,T idv_28015 C,C C,C A,A heterozygous idv_28060 C,C C,C A,A idv_28224 C,C C,C A,A idv_28465 C,C C,C A,T idv_28536 C,C C,C A,A idv_28582 C,C C,C A,A idv_28664 C,C C,C A,A idv_28702 C,C C,C A,A idv_28721 C,C C,C T,T idv_28722 C,C C,C A,A heterozygous idv_28770 C,C C,C T,T idv_28801 C,C C,C A,T idv_28841 C,C C,C A,A idv_28885 C,C C,C T,T idv_28980 C,C C,C A,T idv_29007 C,C C,C A,A idv_29061 C,C C,C A,T idv_29085 C,C C,C A,A idv_29306 C,C C,C A,T idv_29956 C,C C,C T,T idv_30096 C,C C,C A,A idv_30325 C,C C,C T,T

3

Table SIII – 2×2 contingency table with genotype frequencies of the CARD8 frameshift polymorphism (rs140826611) determined by Sanger sequencing in PFAPA patients and controls

PFAPA (%) Control (%) n=79 n=95 Carriers 11 (13.9) 3 (3.2) Non-carriers 68 (86.1) 92 (96.8)

4