A Novel IL17RA Mutation In A Male Child With Chronic Mucocutaneous Candidiasis

Nalan Yakici (  nalanyakici@hotmail.com ) Karadeniz Technical University Faculty of Medicine https://orcid.org/0000-0003-0738-4679 Sevil Oskay Halaçli Hacettepe University Medical School Cagman Tan Hacettepe University Medical School Pinar Gur Cetinkaya Hacettepe University Medical School Halil Tuna Akar Hacettepe University Medical School Busra Cavdarli Ankara City Hospital Begum Ozbek Hacettepe University Medical School Deniz Cagdas Hacettepe University Medical School Ilhan Tezcan Hacettepe University Medical School


Introduction
Chronic mucocutaneous candidiasis (CMC) is characterized with persistent and/or recurrent fungal infections involving the nails, skin, and oral and genital mucosa caused by candida species, mainly Candida albicans [1]. It is an infectious phenotype in patients with inherited or acquired T-cell de ciency [1][2][3]. Studies to explain the etiology of CMC have shown that Th17 cells and IL17 are particularly important in mucocutaneous host defense against Candida species [4][5][6].
Isolated CMC or CMC disease (CMCD) is de ned as a condition that is characterized by persistent or recurrent infections of the nails, skin, and oral and genital mucosa, caused typically by C. albicans, and less often by Staphylococcus aureus, without other severe infectious or autoimmune ndings [1].
The rst genetic etiologies of the isolated CMC form were deciphered by identifying de ciencies in autosomal recessive (AR) IL17RA and autosomal dominant (AD) IL17F [9].
The IL17R family, includes ve receptor subunits; IL17RA, IL17RB, IL17RC, IL17RD and IL17RE. These receptors form various heterodimers, through which different IL17 cytokines signal in an ACT1dependent manner and IL17RA forms the most common subunit of all these receptor complexes. IL17A exerts its function through the heterodimeric structure created by IL17RA and IL17RC receptors [12].
This paper presents a case of a 6-year-old male patient with a clinical picture of CMCD who was found to carry a homozygous [(c.787C> T) (p.Arg263Ter)] mutation in IL17RA gene that has not been reported previously.

Genetic Analysis
Genomic DNA was isolated from the peripheral blood sample of the patient (EasyOne-Qiagen). A total of 266 PID-associated genes were sequenced so as to include exon-intron and splice-site regions using targeted next-generation sequencing on the ION Torrent platform [14]. The sequencing process detected 923 nucleotide changes in the patient, and these variants were analyzed using the Ionreporter 5.10 software (ThermoFischer). Filtering was carried out to identify the pathogenic gene mutation among the variations detected by the analysis. Alleles with an allele frequency of <1% were included in the analysis for ltering.
For the detection of IL17RA expression, peripheral blood mononuclear cells (PBMCs) were seperated by density gradient method. Then, cells were washed and stained with anti-IL17RA antibody (Biolegend, USA). For the determination of CD4+ IL17A+ and CD4+ IL17F Th17 cells, PBMCs were stained with anti-CD4, anti-IL17A and anti-IL17F antibodies (BD, USA). Stained cells were analysed using FACS CANTO II ow cytometer (BD, USA).
To determine lymphocyte proliferation, following seperation of PBMCs, cells were washed with PBS (Phosphate -Buffer-Saline) and stained with CFSE for 5 min. After washing steps cells were feeded with RPMI including L-Glutamine (1 %) and penicillin/streptomycin (1%) and incubated in 37 C o incubator including 5% CO 2 for 96 hours. Analysis was performed by using FACS CANTO II ow cytometer (BD, USA).

Results
Patient Report: A 3.5-year-old male patient with consanguineous ( Figure 1A) parents presented to the Pediatric Immunology Clinic of Hacettepe University School of Medicine with eczema, and recurrent oral and genital candida infections.
His symptoms started at the age of 8 months as watery, itchy and eczematous lesions on the neck, back ear and inner side of the arms and legs, oral candida plaques and candida infection in the genital area.
Skin lesions were alleviated by local corticosteroid and oral antifungal therapy, but no improvement in oral candida plaques or genital fungal infection was observed.
Upon physical examination, oral and genital candida infections and dry skin were observed, and there was no failure to thrive in the patient.
Scalp and nails were not affected by candida infection. In addition, there was no history of bacterial respiratory tract infections such as pneumonia, sinusitis or otitis at the time of diagnosis, and the patient did not have bacterial skin infections such as skin abscess, psoriasis or folliculitis.
The whole blood count, immunoglobulin and lymphocyte subgroup levels were normal. T and B cell subgroups and lymphocyte proliferation values were also normal ( Table 1).
Prophylaxis with uconazole was initiated. Oral and genital candida infections resolved after uconazole treatment.
The patient was followed up for 2.5 years with uconazole prophylaxis without symptoms. However, in the last 6 months, purulent bullous lesions developed especially on the ngertips, and sometimes around the mouth ( Figure 2). These lesions recurred 5-6 times within 6 months. Staphylococcus aureus grew in the microbial sample taken from the lesion.

Mutation in IL17RA
We detected a homozygous mutation [(c.787C> T (p.Arg263Ter)] in the exon 8 of the IL17RA gene, being identi ed as a mutation occurring in the extracellular domain of the protein, and resulting a premature stop codon using NGS panel ( Figure 1B). Then we con rm the mutation by Sanger sequencing ( Figure  1C). This variant was not identi ed in 400 individuals examined for an NGS PID panel study in the Pediatric Immunology Laboratory of Hacettepe University and 1000 healthy Turkish individuals.
Detection of IL17RA expression and Th17 cell percentage IL17RA expression was observed to be severely reduced in patients' PBMCs. Furthermore we detected decreased CD4+ IL17A+ Th17 cell percentage as well as reduced IL17F expression in patients' CD4+ T lymphocytes. T lymphocyte proliferation was evaluated as normal compared to healthy control samples ( Figure 3).

Discussion
Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infection of the nails, skin, and oral and genital mucosa by Candida species, mainly Candida albicans [1].
The rst genetic cause of isolated CMC was reported in 2011. Puel et al. identi ed IL17RA and ILF de ciency, two genetic etiological causes of isolated CMC [9].
IL17F de ciency was rst described in 2011 by Puel et al in several members of an Argentine family suffering from mucocutaneous candidiasis [9]. The index patient, a ve-year-old boy, was the second of a family of three children. His family was unrelated and of Spanish descent living in Argentina. His oldest sister, mother, grandmother and uncle also suffered from CMC from the rst year of her life, her grandmother had cutaneous and recurrent vaginal candidiasis and her uncle showed interdigital intertrigo. His older sister died at the age of six of sudden encephalopathy of uncertain etiology associated with diffuse oral candidiasis. The index patient does not have growth disorders and developmental abnormalities with normal hair, nails, teeth and sweating. He had persistent oral candidiasis with thrush of the soft palate, cheek, and tongue from the rst year of his life.
IL17RC was described by Ling et al in 2015 in 3 patients, one of Argentina and two of them Turkish origin [10]. The 37-year-old female patient of Argentine origin suffered from chronic intertrigo and pustules on her face since the age of 4 year, but with a normal scalp, nails and teeth. She developed recurrent oral and esophageal thrush. However, she had never suffered from any severe bacterial, viral or other fungal infection. Other patient was a 12-year-old boy born to Turkish parents, with no known parental consanguinity. He has suffered from CMC since the age of 2 mo, with chronic intertrigo, aphthous stomatitis, oral thrush, and onychomycosis, but no reported serious bacterial, viral or other fungal infections. The third patient was an 8-year-old boy from a third-degree relative Turkish family. He displayed persistent oral candidiasis, with white plaques all over the buccal mucosa and dorsal surface of the tongue since early infancy and pustules on the skin and scalp. He had no history of any other signi cant bacterial, viral or other fungal infectious disease or signi cant developmental defects, with normal hair, nails, teeth.
De ciency of activator 1 of nuclear factor kappa-beta (ACT1) has been described in 7 patients to date. ACT1 de ciency was rst reported in 2013 in two siblings born to consanguineous Algerian parents [11].
One of the patients was a 29-year-old male and the other a 26-year-old female. Both had seborrheic dermatitis severe enough to require hospitalization in infancy. At the age of eight, the brother developed macrokeilitis with frequent Candida albicans infections of the tongue and mouth. He had never had cutaneous disease due to Candida or any other fungus. He had no cutaneous staphylococcal disease. Bilateral staphylococcal blepharitis occurred at the age of seven years, with many recurrences. She developed folliculitis decalvans at the age of 17 years. She had cicatricial alopecia due to Staphylococcus aureus and several episodes of Candida albicans infection in the buccal and genital areas. There was also nails infection due to several episodes of Candida parapsilosis. The third case described in 2019 was an 18-year-old Indian boy born to consanguineous parents. Genetic testing showed a novel homozygous mutation in the TRAF3IP2 gene, which was not previously reported. This patient had recurrent attacks of thrush and pneumonia since the rst year of his life. There were dental abnormalities, esophageal stricture possibly due to previous esophageal candidiasis, and hypergammaglobulinaemia due to recurrent infections [16].
In 2020, a novel missense mutation in the TRAF3IP2 gene was detected in 4 siblings (two sisters and two brothers) from Lebanon whose parents are related [17]. There were similar clinical ndings in all siblings.
They suffer from pustular lesions on the scalp especially in the second year of life. These lesions were progressive and resulted in alopecia that scarred over the following months and years. Later pustular lesions appeared over the entire body and resulted in scarring hair loss at certain areas of the body, particularly the lower extremities. These histological ndings were suggestive of discoid lupus erythematosus. Swab cultures taken from pustules grew Staphylococcus aureus.
IL17RA de ciency, which is the genetic etiologic cause of CMCD, was rst identi ed by Puel et al. in 2011 in a French child of Morocco origin, whose parents were consanguineous [9]. The index patient, a sevenyear-old boy, was the youngest of a family of ve siblings. Neither his siblings nor his father had any particular medical history, except for his eldest brother who died at birth. His mother suffered from severe atopic dermatitis and chronic allergic rhinitis. The index patient had no failure to thrive and no developmental abnormalities. He had normal hair, nails, teeth, and sweating. Beginning in the rst month of life, the patient suffered from recurrent mucocutaneous candidiasis (buttocks and thrush) resistant to local antifungal therapy. He had also developed a chronic candidal interdigital intertrigo at the age of six. At the age of 5 months, the patient also developed S. aureus skin abscess and folliculitis on the hip. He was also treated for several attacks of conjunctivitis, acute media otitis, bronchitis and folliculitis in the rst four years of his life. He had developed severe atopic dermatitis from the age of 1.5 years and was diagnosed with chronic allergic rhinitis at the age of 5. A gradual spontaneous improvement in eczematous dermatitis had been observed over the years, and at the age of seven only retroauricular ssures were persistent.
Then, in 2015, Fellmann et al. in two siblings from Sri Lanka who presented with recurrent mucocutaneous candidiasis, chronic skin infections, respiratory tract infections, and vasculitis since childhood, detected a large chromosomal deletion, including entire regions of the genes IL17RA, CECR1 (encoding adenosine deaminase 2) and XKR3 (X Kell blood group 3) [16]. The rst of the siblings presented with growth retardation, recurrent orovaginal candida infection and skin lesions. At the age of 6 years, splenomegaly, scaly ichthyosiform skin lesions on the extremities, neck and chest, and psoriasiform skin lesions on the scalp developed. Candida albicans and Staphylococcus aureus were grown in the cultures taken from the lesioned skin areas. When she was 10 years old, progressive central neutropenia, recurrent lung infections and lung abscess developed. She died from septic shock when she was 16 years old. The second of the siblings had no failure to thrive and no developmental abnormalities.
She had orovaginal candidiasis and staphylococcal skin infections that could not be controlled with antifungal and antibiotic treatments from the age of 2 years. She also developed splenomegaly, nonscathrical alopecia, progressive psoriasiform scalp lesions, scaly ichthyosiform skin lesions appearing on the extremity, neck, and chest. At the age of 18, signs of retinal vasculitis, venous occlusion and bleeding occurred. It has been reported that chronic mucocutaneous infections seen in these patients are due to IL17RA de ciency and vasculitis and chronic in ammation due to ADA2 de ciency.
Later in 2016, Levy et al. [15] reported the largest case series with an autosomal recessive mutation in the IL17RA gene in 21 patients from 12 families, 11 of whom were relatives, including the initial case. In all of these patients, the rst episode of CMC occurred in the rst 6 months of life, and 14 patients had various staphylococcal skin infections (skin abscesses, folliculitis, furuncles, crusted pustular lesions on the face and scalp) at the same age. CMC affected skin (intertrigo), scalp, mucosal areas (oral thrush, anogenital candidiasis) or nails. In addition to these skin infections, 8 patients had recurrent respiratory tract infections (sinusitis, otitis, lobar pneumonia, bronchitis). One patient was treated with a diagnosis of suspected pulmonary tuberculosis and one with a diagnosis of tuberculous meningitis. Three patients had only oral mucosal candidiasis. Eczema was detected in 4 patients.
IL17RA gene consists of extracellular (EC), transmembrane (TM), intracellular (IC) and SEFIR (SEFIR (similar expression to broblast growth factor genes) and IL17R) domains [12]. We found a novel homozygous mutation that cause premature stop codon. Detecting severely decreased IL17RA protein expression in patients' PBMCs in parallel with decreased CD4+ IL17A+ cells and reduced expression of IL17F supported the pathogenicity of the mutation. IL17A and IL17F produced by Th17 cells bind to IL17RA and activate cellular signaling to start in ammatory response. In the absence and reduced expression of IL17RA, decreased Th17 cell numbers were seen in the reported patients with IL17RA defects as in our study.
Staphylococal infections have been reported so far only in IL17RA and ACT1 de ciencies among isolated causes of CMC, but has not been reported in patients with IL17F and IL17RC de ciencies [9][10][11]. Although the mechanism is not clear, it is thought that staphylococcal infections may result from the inability of IL17E [15]. In the studies unlike patients with IL17RC and IL17F de ciency, PBMCs of patients with IL17RA and ACT1 de ciency did not respond to IL17E [9][10][11]. IL17E exerts its function through the heterodimeric structure created by IL17RA and IL17RB receptors [12]. ACT1 is an essential adapter protein in the signaling of all IL17 cytokines [11]. This mechanism may also explain the staphylococcal skin abscess that developed in our patient.
In conclusion, immunologic studies should be carried out for IL17-associated mechanisms, including AR IL17RA mutations, in patients with chronic moniliasis, genital candidiasis, recurrent bacterial and/or fungal infections of the skin and nails, psoriatic and eczematous skin lesions and recurrent bacterial respiratory tract infections in the absence of other systemic and laboratory ndings.

Declarations Acknowledgment
This study received grants number TSA-2018-17339 and 315S125 from Hacettepe University and TUBITAK, respectively. We thank Ekim Taşkiran for his help in the genetic analysis. The authors would like to thank participants for being a part of this study.
Funding: This study received grants number TSA-2018-17339 and 315S125 from Hacettepe Universty and TUBITAK, respectively.
Con icts of interest/Competing interests: The authors declare that there are no con icts of interest.