Isolated chronic mucocutaneous candidiasis due to a novel duplication variant of IL17RC

Purpose Inborn errors of the IL-17A/F-responsive pathway lead to chronic mucocutaneous candidiasis (CMC) as a predominant clinical phenotype, without other significant clinical manifestations apart from mucocutaneous staphylococcal diseases. Amongst inborn errors affecting IL-17-dependent immunity, autosomal recessive (AR) IL-17RC deficiency is a rare disease with only three kindreds described to date. The lack of an in vitro functional evaluation system of IL17RC variants renders its diagnosis difficult. We sought to characterize a seven-year-old Japanese girl with CMC carrying a novel homozygous duplication variant of IL17RC and establish a simple in vitro system to evaluate the impact of this variant. Methods Flow cytometry, qPCR, RNA-sequencing, and immunoblotting were conducted, and an IL17RC-knockout cell line was established for functional evaluation. Results The patient presented with oral and mucocutaneous candidiasis without staphylococcal diseases since the age of three months. Genetic analysis showed that the novel duplication variant (Chr3: 9,971,476–9,971,606 dup (+ 131bp)) involving exon 13 of IL17RC results in a premature stop codon (p.D457Afs*16 or p.D457Afs*17). Our functional evaluation system revealed this duplication to be loss-of-function and enabled discrimination between loss-of-function and neutral IL17RC variants. The lack of response to IL-17A by the patient’s SV40-immortalized fibroblasts was restored by introducing WT-IL17RC, suggesting that the genotype identified is responsible for her clinical phenotype. Conclusions The clinical and cellular phenotype of the current case of AR IL-17RC deficiency supports a previous report on this rare disorder. Our newly established evaluation system will be useful for diagnosis of AR IL-17RC deficiency, providing accurate validation of unknown IL17RC variants.

Isolated CMC refers to patients with CMC as the predominant clinical phenotype without other signi cant clinical manifestations (1,4,5,27). However, some patients with isolated CMC may develop mucocutaneous Staphylococcus aureus infections (27)(28)(29). The discovery of patients with isolated CMC and inborn errors of immunity (IEI) has greatly contributed to our understanding of both the molecular and cellular bases of CMC. Indeed, mono-or biallelic deleterious variants of IL17F, IL17RA, IL17RC, and TRAF3IP2 (encoding ACT1) have been detected in patients with isolated CMC, and MAPK8 has been identi ed in patients with both CMC and a connective tissue disorder (27)(28)(29)(30)(31)(32)(33)(34). These ve genes are all directly involved in the IL-17A/IL-17F-dependent IL-17RA/IL-17RC-mediated signaling pathway. Clinical and molecular investigations of these patients suggest that host mucocutaneous immunity against Candida critically depends on IL-17 immunity. IL-17RA is ubiquitously expressed, whereas IL-17RC is predominantly expressed on epithelial and mesenchymal cells (35)(36)(37). The two chains form a dimeric receptor for IL-17A and IL-17F homo-or heterodimers, which are mainly produced by Th17 cells (38). IL-17A and IL-17F (IL-17A/F) binding to their receptors results in recruitment of ACT1, an adapter molecule activating several downstream signaling pathways (35,38), to the cytoplasmic domains of the receptors, leading to release of antimicrobial peptides and cytokines/chemokines such as IL-6, G-CSF, CXCL1 and CXCL8 to control Candida infection.
AR IL-17RC de ciency was reported in 2015 in three unrelated patients from three families originating from Argentina and Turkey with isolated CMC and no staphylococcal mucocutaneous manifestations (30). However, additional patients have not yet been reported. Those three patients carried different homozygous nonsense variants, i.e., p.Q138*, p.R376*, and p.Q378*, inherited from heterozygous asymptomatic parents. Fibroblasts from the patients did not exhibit responses to IL-17A/F homo-and heterodimers. We herein report a patient with isolated CMC and a homozygous novel duplication variant of IL17RC. In addition, we successfully established a simple in vitro system that can accurately assess the functional impact of IL17RC variants.

Molecular Genetics
Genetic testing was performed after obtaining written informed consent from the participants or their guardians. Genomic DNA was extracted from peripheral blood leukocytes and subjected to NGS based gene panel testing for IEI and Sanger sequencing. The detailed method of gene panel testing was described previously (39). Brie y, paired-end sequence libraries were constructed using the TruSeq DNA PCR Free Library Kit (Illumina). The libraries were subjected to 150-bp paired-end sequencing on the HiSeq X Ten system (Illumina). Reads from the fastq les were aligned to the human reference genome GRCh37/hg19 using Burrows-Wheeler Aligner Software. Duplicate reads were removed using Picard Mark Duplicates. The mapped reads around insertions/deletions (Indels) were realigned by the Genome Analysis Toolkit (GATK) Version 4.0. Variant calling was performed using GATK HaplotypeCaller. The called single-nucleotide variants (SNVs) and Indels were annotated using the SnpEff software 4.3.

Quantitative PCR
Total RNA was extracted from the cell lines with Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany) or SingleShot Cell Lysis Kit (Bio-Rad, Hercules, CA) and reverse transcribed by Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) or iScript Adv cDNA synthesis kit (Bio-Rad, Hercules, CA). All procedures were performed according to the manufacturer's instructions. Quantitative real-time PCR was performed with Taqman Gene Expression Master Mix (Applied Biosystems) and probes (Table. S1) on StepOnePlus Real-Time PCR system (Applied Biosystems). The expression levels of each transcript were determined in triplicate and normalized to the level of GAPDH. Data analysis was performed with the comparative threshold cycle method (also known as the 2 −ΔΔCt method).

IL17RC knockout HeLa cell preparation
The CRISPR/Cas9 system was used to generate IL17RC knockout HeLa cells. Two sgRNA sequences (GGTGCGTAGGCGCCAGCACG and CTGTGACCTCTGTCTGCGTG) targeting the IL17RC gene were designed in exon 3 using CRISPRdirect (40). Cloning of the respective sgRNA tandem constructs into pSpCas9(BB)-2A-GFP (PX458) (Addgene plasmid #48138) was performed according to previous reports and con rmed by sequencing (41). Transfection of sgRNA-Cas9 Plasmid into WT HeLa cells was performed using the Neon™ transfection system (Invitrogen/Thermo Fisher Scienti c Inc.) according to the manufacturer's instructions. After 48 hours of incubation from transfection, GFP expressing cells were sorted with FACSAria™ III (Becton Dickinson, Franklin Lakes, USA) into single, and expanded under conditions at 37°C, 5% CO2. Successful knockout of IL17RC was con rmed by direct sequencing of the genomic DNA and quantitative PCR for IL17RC of the candidate clones (Fig.S2). CXCL1 quantitative PCR using the IL17RC -targeted HeLa cell line IL17RC-knockout HeLa cells were generated using the CRISPR-Cas9 system. Brie y, plasmids expressing both pSpCas9 and gRNA designed using exon 13 of IL17RC were transfected into WT HeLa cells by electroporation and subsequently sorted into single cells. The IL17RC-de cient HeLa cells were plated in 96-well plates at a density of 1.6 x 10 4 cells/well and incubated for 24 hours and then transfected with mock, pUNO1 vector, WT or IL17RC mutant plasmid (20 ng). After 24 hours, the transfected cells were left unstimulated or stimulated with 100 ng/mL recombinant human IL-17A for 4 hours. cDNA was synthesized using total RNA extracted from the cells, and quantitative PCR was performed for CXCL1. Expression level of each transcript was determined in triplicate and normalized to the level of GAPDH.
Data analysis was performed with the comparative threshold cycle method (also known as the 2 −ΔΔCt method).

Isolation and functional characterization of human T cells
Naïve and memory CD4 + T cells were isolated by excluding Tregs (CD4 + CD25 hi CD127 lo ) then sorting CD45RA + CCR7 + cells and CD45RA − CCR7 +/− cells, respectively. Purity of the recovered populations was >
Other materials and methods are described in the "Supplementary Materials".

A sporadic case of CMC
We examined a seven-year-old girl born to Japanese parents from the same city but without any known consanguinity (Fig. 1A). The prenatal history was normal, and there was no family history of IEI. From the age of three months onward, the patient developed oral and cutaneous candidiasis, especially at the external eye angle. (Fig. 1B). Mucositis of the nasal cavity and vulva was also observed. These symptoms of oral and cutaneous candidiasis responded well to uconazole treatment, and are occurred with discontinuation of treatment. Endoscopy examination at the age of seven years revealed that she had esophageal candidiasis (Fig. 1B). Antifungal drugs had never been used continuously due to the concern of emerging resistant strains. Her oral and cutaneous candidiasis therefore persisted, though she never developed invasive candidiasis. She had sensitive skin and recurrent sweating rashes, which especially grew worse in summer. Mosquito bites were abnormally swollen, sometimes resembling impetigo. Her growth and development were normal, including hair, nails, teeth, and sweating. She also developed herpes zoster of the left shoulder and right side of the head at the age of six years. Otherwise, she had no episodes indicating susceptibility to severe bacterial or viral diseases. Immunological investigations were unremarkable (Table. S2). She was thus given a diagnosis of isolated CMC based on the absence of any phenotypes other than CMC.

Identi cation of a homozygous novel duplication variant of IL17RC
We performed next-generation sequencing (NGS) gene panel testing for IEI using genomic DNA from the patient's leukocytes, revealing an increased number of reads in a region including the entire exon 13 of IL17RC (Fig. 1C). This nding was not detected by the variant calling system but was identi ed manually when we directly analyzed the data aligned to the reference genome. Subsequent Sanger sequencing of the patient's DNA identi ed a homozygous novel duplication variant (Chr3: 9,971,476-9,971,606 dup(+ 131bp), c.1324_1372dup) of IL17RC (Fig. 1D). A genetic test of the patient's healthy mother and siblings revealed them to be heterozygous for the variant or wildtype (Fig. 1D). A genetic test was not available for the father. This duplication variant was not found in various public databases, such as gnomAD (https://gnomad.broadinstitute.org, v2.1.1) or TOPMED/BRAVO ( https://bravo.sph.umich.edu), suggesting that it is a novel and private variant. Analysis of mRNA extracted from the patient's broblasts revealed that this variant results in a duplication of 46 (exon13dup_1) or 49 (exon13dup_2) bases IL17RC of exon 13 (Figs. 1E, S1). Both the 46-and 49-base duplications in exon 13 cause a frameshift resulting in a premature stop codon (p.D457Afs*16 or p.D457Afs*17) (Fig. 1E). This variant affects a conserved SEFIR (similar expression to broblast growth factor genes and IL-17R) domain of IL-17RC, which is essential for signaling to ACT1 (42) (Fig. 1F). Additionally, the variant has a high CADD (combined annotation-dependent depletion) score of 33 (Fig. 1G). Thus, the identi ed variant is predicted to be lossof-function. Collectively, these data suggest that this novel homozygous duplication variant of IL17RC is causal for the patient's isolated CMC.
Evolutionary and epidemiological genetics of AR IL-17RC de ciency Among IEI causing isolated CMC, AR IL-17RA de ciency has been reported thus far in 28 cases from 16 kindreds, but AR IL-17RC de ciency has been reported in only four cases, including the current case (Case summary: Tables S3, S4) (27,29,30,(43)(44)(45). We therefore focused on causes of the difference in the frequency of AR IL-17RC de ciency and AR IL-17RA de ciency. We rst investigated genomic selection indices to estimate the strength of negative selection at the IL17RA and IL17RC loci. The consensus score for negative selection (CoNeS), as developed with the combination of several intraspeci c and interspeci c statistics, was calculated to be 0.73 (top 76.7%) and 1.09 (top 86.9%) for IL17RA and IL17RC, respectively (Table S5) (46). Furthermore, the gene damage index (GDI) values were 11.0 (top 92.0%) and 6.62 (top 78.2%) for IL17RA and IL17RC, respectively (47). These results indicate that both IL17RA and IL17RC are not under strong negative selection, with no obvious difference in their selection pressure. Next, we analyzed the frequency of predicted loss-of-function (pLOF) variants in gnomAD. The cumulative frequency of pLOF variants is lower in IL17RA than in IL17RC, at 1.70×10 − 4 and 1.22×10 − 3 , respectively, even after ltering out a likely benign variant (Fig. 2B). Intriguingly, an individual harboring homozygous p.Q378* in IL17RC, a variant previously reported to cause isolated CMC, is listed in gnomAD (n = 1, allele frequency = 4.03×10 − 4 ), whereas no homozygous individual for pLOF variants in IL17RA was found ( Fig. 2A).

Abnormal IL17RC mRNA and protein expression in patient broblasts
We rst assessed levels of IL17RC mRNA by RT-qPCR in the patient's SV40-immortalized broblasts. We found decreased IL17RC but not IL17RA mRNA expression in her broblasts compared to cells from healthy controls or from a patient with AR IL-17RA de ciency (27) (Fig. 3A, 3B). These results are comparable to ndings for broblasts from previously reported patients with homozygous variants (p.Q138*, p.Q378*) of IL17RC (30). We next assessed the level of IL-17RC protein expression in the patient's broblasts. As in a previous report, immunoblot analysis failed to identify a band speci c for IL-17RC in SV40-immortalized broblasts from healthy controls and the above-mentioned patients, possibly due to low expression levels of IL-17RC (30). Therefore, we transiently transfected the broblasts with an empty vector or expression plasmid containing wildtype (WT) or exon13dup IL17RC and then assessed protein expression. Cells transfected with the vector plasmid WT IL-17RC showed a band at the expected molecular weight (MW) of 86 kDa corresponding to IL-17RC isoform 1 when using a polyclonal antibody directed against amino acids 113-258 of IL-17RC (Fig. 3C). In contrast, a truncated band of approximately 52 kDa was detected in cells transfected with the plasmid encoding exon13dup IL17RC (Fig. 3C, top panel). Similar ndings were obtained using an antibody against the N-terminal V5-tag (Fig.  3C, bottom panel). These results suggest that the duplication variant of IL17RC identi ed in the present patient impacts both mRNA and protein IL-17RC levels.
Establishment of a functional validation system using CXCL1 quantitative PCR for IL17RC variants Together with the development of NGS approaches, establishing appropriate evaluation systems for variants of unknown signi cance has become imperative. However, to date, there is no simple in vitro system to validate the pathogenicity of IL17RC variants. Currently, patient-derived broblasts are used to functionally assess the impact of IL17RC variants (30). We thus established an in vitro system to functionally assess the impact of IL17RC variants by measuring induction of CXCL1 expression by RT-qPCR in an IL17RC-knockout HeLa cell line after 4 hours of stimulation with IL-17A (100 ng/mL). We selected HeLa cells because they are immortalized epithelial tissue cell lines expressing IL-17RA and IL-17RC. The IL17RC gene was knocked out using the CRISPR/Cas9 system (for details, see Methods and Materials, Fig. S2). In this IL17RC-knockout HeLa cell system, no CXCL1 expression was induced after IL-17A stimulation of mock cells, whereas strong induction was observed upon after IL-17A stimulation of cells transfected with WT IL17RC. Similarly, cells transfected with any of the gnomAD variants with an MAF > 0.001 or found at a low frequency (MAF ≤ 0.001) but with a CADD score > 24 induced high levels of CXCL1 expression after IL-17A stimulation (Fig. 1G, 4). In marked contrast, no CXCL1 expression was induced when cells transfected with any of the previously or currently identi ed IL17RC mutant alleles were stimulated with IL-17A (Fig. 4), further demonstrating the deleteriousness of these alleles. These results show the value of such a system in assessing the impact of novel IL17RC variants, clearly discriminating between loss-of-function and neutral IL17RC variants. Collectively, these ndings demonstrate that the novel duplication variant of IL17RC identi ed in the current study is functionally deleterious.

Patient broblasts showed abolished responses to IL-17A
We next performed RT-qPCR to assess the response of patient broblasts to IL-17A. IL-17A stimulation (100 ng/mL for 2 or 8 hours) induced expression of both CXCL1 and IL6 mRNA in broblasts from healthy controls but not from the patient carrying the homozygous duplication variant of IL17RC (Fig. 5A,  B). These results are similar to those for broblasts from previously reported patients with homozygous IL17RC (p.Q138*, p.Q378*) (30) or IL17RA (p.Q284*) (27) variants (Fig. 5A, 5B). Furthermore, transfecting patient broblasts with a plasmid encoding WT but not mutant IL-17RC (isoform 1) restored the response to IL-17A (Fig. 5C). Conversely, the response to IL-1β was normal in all of these cells (Fig. S3A). Taken together, the lack of response to IL-17A in the patient's SV40-immortalized broblasts was rescued by WT IL-17RC expression, con rming this variant to be the genetic cause of her isolated CMC. We then used 3'-RNA sequencing to further investigate the molecular and functional impact of AR IL-17RC de ciency in response to IL-17A. IL-17A stimulation of healthy control SV40-immortalized broblasts upregulated IL-17 signaling pathway-related genes, such as CXCL1, CXCL8, and NFKBIZ (Fig. 5D). In contrast, no changes in expression of these genes were observed in the patient's broblasts or in broblasts from an IL-17RAde cient patient following stimulation with IL-17A (27) (Fig. 5D). IL-1β stimulation upregulated the same pattern of cytokine response-related genes among broblasts from healthy subjects and IL-17RC-de cient and IL-17RA-de cient patient (Fig. 5E). Enrichment analysis of these upregulated genes showed that the same Gene Ontology terms were enriched in both the control and patient cells, indicating no molecular differences in response to IL-1β (Fig. S3B). These data suggest that AR IL-17RC de ciency, similar to AR IL-17RA de ciency, abolishes IL-17A signaling but that these defects do not affect the IL-1β signaling pathway.

Normal differentiation of IL-17-producing T cells and secretion of IL-17 cytokines
We next performed deep immunophenotyping and in vitro functional experiments to investigate the detailed immune cell biology of our patient. The numbers and frequencies of her total B cells and B-cell subsets were similar to those in healthy donors (Fig. S4A-E). Consistent with intact in vitro B-cell maturation in vivo, the ability of naïve B cells from the patient to undergo proliferation, Ig class switching, and plasma cell generation in response to T-dependent and T-independent stimuli in vitro were also similar to those in healthy controls (Fig. S4F-H). Furthermore, frequencies of CD4 + T-cell subsets, such as Th1, Th17, T follicular helper (Tfh), and regulatory T (Treg) cells, were normal, except for a higher frequency of Th17-phenotype cells among all Tfh cells compared to healthy controls ( Fig. S5A-C, S5E-G).
The frequencies of CD8 + T-cell subsets were also similar to those in healthy donors (Fig. S5B, S5D). Under in vitro differentiation conditions, the proportion of IL-17A-producing memory CD4 + T cells and levels of IL-17A production by these cells were similar between the patient and healthy controls (Fig. 6C, 6D).
Nevertheless, production of IFN-γ and TNF-a by memory CD4 + T cells was lower in the patient than in healthy donors (Fig. 6A, S6A). Finally, although proportions of Th2 phenotype cells in the patient were similar to those in healthy controls, production of Th2 cytokines secreted by memory T cells were higher (Fig. 6B, S6B). Overall, distribution of leukocyte subsets and CD4 + T-cell differentiation and function in vivo and ex vivo in our patient were largely within the dynamic ranges observed for CD4 + T cells from healthy donors, suggesting only a very modest, if any, effect of IL-17RC de ciency on the behavior and function of these cells.

Discussion
We report the fourth patient and the rst Asian patient with AR IL-17RC de ciency due to a homozygous novel duplication variant of IL17RC. Consistent with previously reported IL-17RC-de cient patients (30), this patient had early-onset mucocutaneous candidiasis but without any other severe infectious diseases, including mucocutaneous S. aureus disease or autoimmune clinical signs. This novel variant causes duplication of 46 or 49 bp within exon 13, leading to a frameshift and a premature stop codon at position 457. The patient's broblasts exhibited a complete lack of CXCL1 or IL6 upregulation after IL-17A stimulation, though this defect was restored by introduction of the WT IL17RC allele. We therefore conclude that the homozygous IL17RC variant identi ed in this patient with isolated CMC is a novel pathogenic variant leading to AR IL-17RC de ciency. These results support that IL-17A/F signaling mediated by IL-17RC is essential for mucocutaneous defense against Candida but is otherwise redundant for immunity against other pathogens.
Patients with AR IL-17RA or AR ACT1 de ciency frequently develop staphylococcal skin and mucosal infections. Hence, IL-17 immunity is essential for mucocutaneous defense not only against Candida but also against S. aureus (27)(28)(29). On the other hand, mucocutaneous staphylococcal infections were not detected in three previously identi ed patients (30) or the present patient with AR IL-17RC de ciency. The mechanism for this phenotypic difference remains unclear. Abnormal IL-17E immunity, which is affected in AR IL-17RA de ciency but preserved in AR IL-17RC de ciency, has been suggested but not demonstrated as a plausible mechanism to explain this phenotypic difference (30). Three patients from three generations with CMC, mucocutaneous S. aureus infection, and a connective tissue disorder due to AD de ciency of c-Jun N-terminal kinase 1 (JNK1), a component of the MAPK signaling pathway, were recently reported (18). These patients displayed impaired responses to IL-17A and IL-17F and low proportions of Th17 cells but intact responses to IL-17E. A recent study suggested that heterodimers of IL-17RA and IL-17RD function as alternative receptor subunits of IL-17A in primary mouse and human keratinocytes (48). IL-17A signaling is mediated by both IL-17RC and IL-17RD, and the IL-17RA/IL-17RD pathway mainly activates p38 MAPK and JNK (48). These ndings suggest that the IL-17RC-independent IL-17RA/IL-17RD pathway may provide the IL-17 immune activity required for protection against infection by S. aureus in IL-17RC-de cient patients. Regardless, additional research is needed to fully understand the precise role of IL-17 immunity in mucocutaneous defense against S. aureus.
To date, no system has been developed for qualitative evaluation of IL17RC variants in vitro. In this study, we successfully established a precise in vitro assay system using a CRISPR-generated IL17RC-knockout HeLa cell line. The system was able to accurately and unambiguously distinguish loss-of-function variants from neutral variants found in public databases and predicted to be functionally intact. In general, increased identi cation of rare variants through comprehensive approaches in genomic research has emphasized the importance of arti cial evaluation systems that accurately assess the impact of variants in the absence of patient-derived samples. In particular, evaluation of IL17RC variants requires patient-derived broblasts, making an arti cial system even more important. Diagnosis based on accurate evaluation of pathogenicity for identi ed variants allows for appropriate management and/or treatment of the patient.
AR IL-17RA de ciency is the major genetic cause in patients with isolated CMC due to impaired IL-17 immunity, with 28 cases reported to date. However, only four cases of AR IL-17RC de ciency, including the current case, have been reported thus far. Evolutional and epidemiological genetics analyses revealed no obvious difference in the strength of negative selection at the IL17RA and IL17RC loci. However, in the general population (gnomAD), the cumulative frequency of pLOF variants is higher for IL17RC than IL17RA, inconsistent with the fact that the number of patients with AR IL-17RA de ciency identi ed thus far is higher than that with AR IL-17RC de ciency. These results thus do not explain the observed difference in the incidence of IL-17RA and IL-17RC de ciency. Given that a homozygous p.Q378* variant of IL17RC is included in a public database, the presence of undiagnosed mild cases or cases with incomplete penetrance may be expected for AR IL-17RC de ciency. In contrast, no homozygous deleterious variant of IL17RA was found in the public database, suggesting higher penetrance for AR IL-17RA de ciency. If this hypothesis is true, some cases of AR IL-17RC de ciency may not be diagnosed.
Further study is needed to fully characterize the cellular, clinical, and frequency differences between AR IL-17RA and IL-17RC de ciencies.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.