The Extended Clinical Phenotype of 26 Patients with Chronic Mucocutaneous Candidiasis due to Gain-of-Function Mutations in STAT1

Gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) result in unbalanced STAT signaling and cause immune dysregulation and immunodeficiency. The latter is often characterized by the susceptibility to recurrent Candida infections, resulting in the clinical picture of chronic mucocutaneous candidiasis (CMC). This study aims to assess the frequency of GOF STAT1 mutations in a large international cohort of CMC patients. STAT1 was sequenced in genomic DNA from 57 CMC patients and 35 healthy family members. The functional relevance of nine different STAT1 variants was shown by flow cytometric analysis of STAT1 phosphorylation in patients’ peripheral blood cells (PBMC) after stimulation with interferon (IFN)-α, IFN-γ or interleukin-27 respectively. Extended clinical data sets were collected and summarized for 26 patients. Heterozygous mutations within STAT1 were identified in 35 of 57 CMC patients (61 %). Out of 39 familial cases from 11 families, 26 patients (67 %) from 9 families and out of 18 sporadic cases, 9 patients (50 %) were shown to have heterozygous mutations within STAT1. Thirteen distinct STAT1 mutations are reported in this paper. Eight of these mutations are known to cause CMC (p.M202V, p.A267V, p.R274W, p.R274Q, p.T385M, p.K388E, p.N397D, and p.F404Y). However, five STAT1 variants (p.F172L, p.Y287D, p.P293S, p.T385K and p.S466R) have not been reported before in CMC patients. STAT1 mutations are frequently observed in patients suffering from CMC. Thus, sequence analysis of STAT1 in CMC patients is advised. Measurement of IFN- or IL-induced STAT1 phosphorylation in PBMC provides a fast and reliable diagnostic tool and should be carried out in addition to genetic testing.

infections, resulting in the clinical picture of chronic mucocutaneous candidiasis (CMC). This study aims to assess the frequency of GOF STAT1 mutations in a large international cohort of CMC patients. Methods STAT1 was sequenced in genomic DNA from 57 CMC patients and 35 healthy family members. The functional relevance of nine different STAT1 variants was shown by flow cytometric analysis of STAT1 phosphorylation in patients' peripheral blood cells (PBMC) after stimulation with interferon (IFN)-α, IFN-γ or interleukin-27 respectively. Extended clinical data sets were collected and summarized for 26 patients.
Results Heterozygous mutations within STAT1 were identified in 35 of 57 CMC patients (61 %). Out of 39 familial cases from 11 families, 26 patients (67 %) from 9 families and out of 18 sporadic cases, 9 patients (50 %) were shown to have heterozygous mutations within STAT1. Thirteen distinct STAT1 mutations are reported in this paper. Eight of these mutations are known to cause CMC (p.M202V, p.A267V, p.R274W, p.R274Q, p.T385M, p.K388E, p.N397D, and p.F404Y). However, five STAT1 variants (p.F172L, p.Y287D, p.P293S, p.T385K and p.S466R) have not been reported before in CMC patients. Conclusion STAT1 mutations are frequently observed in patients suffering from CMC. Thus, sequence analysis of STAT1 in CMC patients is advised. Measurement of IFN-or ILinduced STAT1 phosphorylation in PBMC provides a fast and reliable diagnostic tool and should be carried out in addi-

Introduction
Chronic mucocutaneous candidiasis (CMC) constitutes a collective term for a heterogeneous group of syndromes with the common feature of chronic non-invasive Candida infections of the skin, nails and mucous membranes, primarily with Candida albicans. Reasons behind the susceptibility to Candida infections are manifold. Infection with the human immunodeficiency virus as well as the prolonged use of glucocorticoids or antibiotics predispose to fungal infections, but the disease may also manifest as part of rare primary immunodeficiencies caused by monogenic Mendelian traits affecting the cellmediated immunity necessary for fighting Candida infections [1][2][3][4]. CMC presents heterogeneously both in clinical manifestations and genetic background, however, studies conducted so far emphasize the key role of T helper 17 (Th17) cells and the impaired effector function of their cytokines interleukin 17 (IL-17) and interleukin 22 . These cytokines have been shown to be essential for mucocutaneous anti-fungal host defense [5][6][7]. Indeed, patients with the autosomal dominant form of hyper IgE syndrome (HIES) have severely reduced numbers of IL-17 producing circulating T cells due to dominant-negative mutations of the signal transducer and activator of transcription 3 (STAT3), and often suffer from CMC [8][9][10][11]. CMC has also been reported in a large multiplex kindred segregating an autosomal recessive mutation in the caspase recruitment domain-containing protein 9 (CARD9). Other important infectious phenotypes reported in this family were dermatophytosis and Candida meningitis [12]. Patients with autoimmune polyendocrinopathy candidiasis and ectodermal dystrophy (APECED)-syndrome bearing biallelic mutations in the autoimmune regulator (AIRE) gene are as well susceptible to Candida infections but no other pathogens. These patients have high titers of neutralizing autoantibodies against IL-17A, IL-17F and IL-22 [13][14][15]. Patients with heterozygous IL17F mutations and homozygous IL17RA or IL17RC mutations have impaired IL-17 signaling and suffer from CMC [16,17]. Furthermore, a biallelic ACT1 deficiency has been shown to underlie one consanguineous CMC family [18]. Finally, gain-of-function (GOF) missense mutations in the signal transducer and activator of transcription 1 (STAT1) were shown to cause autosomal dominant familial CMC, often associated with thyroid disease, and represent the most common genetic etiology of CMC [19,20]. The majority of GOF-STAT1 mutations are confined to the coiledcoil domain (CCD) of STAT1, however, several other GOF mutations have been found in the DNA-binding domain (DBD) [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]. Besides increasing the susceptibility to candidiasis, several STAT1 GOF mutations are also associated with other fungal infections such as coccidioidomycosis or histoplasmosis [34]. Analysis of mutated STAT1 proteins revealed a prolonged phosphorylation leading to prolonged transcription factor activity. Increased STAT1 responses to the interferons α/β and γ as well as IL-27 were shown to repress the differentiation of IL-17 producing T cells through mechanisms that are not yet completely understood [20,24,26]. The identification of genetic defects in patients with CMC offers the opportunity to confirm the diagnosis in both familial and sporadic CMC patients, to provide genetic counseling, and to enable a more precise classification of CMC. In this study, we have explored the frequency of STAT1 mutations in a large cohort of 92 individuals consisting of 57 patients with CMC (39 familial cases and 18 sporadic cases) and 35 healthy relatives from 11 unrelated families. We analyzed patients and healthy family members in order to elucidate the underlying genetic defect and tested interferon (IFN)-and IL-stimulated STAT1 phosphorylation in patients' peripheral blood cells by flow cytometric analysis. We compiled an in depth description of the STAT1 clinical CMC phenotype in order to provide a clear clinical picture for this condition.

Patients and Controls
Inclusion criteria for this study were i) the clinical diagnosis of CMC according to the referring immunologist, ii) the availability of genomic DNA, and iii) a signed consent form for genetic research. Candidiasis was proven by swab, biopsy, or the combination of both in at least one affected family member and in every sporadic patient. The study was approved by the local ethics review boards of the University College London and the University Medical Center Freiburg. Three of the 11 families originated from Germany and three other families from Norway. One family each came from Brazil, Canada, Colombia, Slovakia and the USA. Additionally, we included six sporadic patients from the UK, three from Germany, two respectively from Colombia and Belgium and one patient each from Brazil, Canada, Denmark, India and the USA (Table 1). Due to unavailability of previous clinical data and lossto-follow up of several patients, the clinical analysis was limited to 26 of 35 patients carrying heterozygous STAT1 mutations.

PCR and Sequencing Analysis
Genomic DNA of patients and family members was isolated from whole blood (Gentra Puregene purification kit, Qiagen, Crawley, United Kingdom). To assess the presence of STAT1 mutations, all coding exons of STAT1 were amplified by PCR according to standard protocols with Taq polymerase (PeqLab, Fareham, United Kingdom). PCR products were purified using shrimp alkaline phosphatase (Promega, Madison, Mich) and Exonuclease I (Thermo Scientific, Waltham, Mass). Primer sequences and PCR amplification conditions are available on request. The amplified DNA fragments were subsequently sequenced with the ABI PRISM BigDye Terminator kit v3.1 (Applied Biosystems, Foster City, Calif). Sequencing was performed using the 3130xl Applied Biosystems Genetic Analyzer, DNA Sequencing Analysis software, version 5.2 (Applied Biosystems), and Sequencher, version 4.8 (Gene Codes Corp, Ann Arbor, Mich).

Identification of STAT1 Mutations in 61 % of all CMC Patients
Of the 57 patients with a clinical diagnosis of CMC, a total of 35 (61 %) had heterozygous mutations in STAT1 (Fig. 1, Table 1); 26 of these cases were familial, affecting 9 of 11 families (82 %), whereas 9 of 18 sporadic cases (50 %) had STAT1 mutations. In one sporadic patient, we identified the variation p.V266I, which has a minor allele frequency of 0.1 % and is listed on dbSNP as Brs41473544^. We classified this variant as not associated with CMC, as its functional testing did not show STAT1 hyper-phosphorylation (see below). In contrast, 22 patients (13 familial and 9 sporadic) as well as 34 of 35 healthy family members did not show any mutation in the coding regions of STAT1 or their flanking intronic sequences. Interestingly, one so far healthy 77-year-old female family member was identified as a carrier of the mutation p.R274Q, passing the mutation on to at least three offspring, all affected by CMC. Ten CMC patients from families Fam10 and Fam11 did not harbor a mutation in STAT1. Moreover, three patients from Fam02 in which the mutation p.A267V segregated in four other family members (a father and his three children) did not have a STAT1 mutation. These three patients were subsequently classified as phenocopies. Patients without STAT1 mutation were analyzed by next generation sequencing for CARD9, IL17F, IL17RA, IL17RB, AIRE and ACT1, but no mutation was found in these genes. Overall, we found thirteen distinct heterozygous STAT1 mutations ( Table 1, Fig. 2). Eight of these have been described previously: p.M202V, p.A267V, p.R274W, p.R274Q, p.T385M, p.K388E, p.N397D and p.F404Y [19-26, 28, 30, 31]; six patients had five distinct, novel mutations: p.F172L, p.Y287D, p.T385K, p.P293S and p.S466R, that have not been reported in CMC patients before. While we reported the mutation p.F404Y recently [25], we demonstrate the functional proof of the GOF activity of this mutation below. None of the newly identified mutations is present in the population-variation data sets 1000 Genomes    Fig. 2).
We documented a mixed picture of skin infections in the CMC cohort under study. Altogether, 50 % (13/26) of patients reported intertrigo, 46 % (12/26) pustules and 44 % (11/25) infections of the scalp; and 50 % (9/18) of affected patients reported chronic skin infections. More chronic than recurrent Nine female patients of reproductive age provided information about vulvovaginal candidiasis. In 67 % (6/9) of these female patients Candida infection of the vulva was documented and candidiasis affected both vagina and perineal region in the two-thirds of cases (67 %, 4/6). Complete response after antifungal therapy was likely (80 %, 4/5), but infections of the vulvovaginal area often reoccurred (83 %, 5/6) when antifungal treatment was stopped.
The majority of patients received both topical and systemic antifungal treatment. Oral therapy with fluconazole and other triazoles was often used to provide a long-lasting prophylaxis. Oral preparations of amphotericin B were administered in severe cases. In case of topical treatment, nystatin proved to be a good alternative to triazoles. Duration of topical antifungal treatment ranged from 2 weeks to several months while medical prescription was reported to continue for about 4 weeks per episode in most cases.
Although candidiasis presented in many patients as the main or only type of infection, other infections were also observed: Dermatophytosis was reported in 14 % (3/22) of patients (Table 3). In the respective cases, Trichophyton species were isolated from affected skin lesions. If prevalent, bacterial infections primarily affected the respiratory tract. One or more episodes of bronchitis were observed in 41 % (9/22). Two-fifths of patients reported a history of pneumonia (41 %, 9/22). Sinusitis was also common, with 48 % (12/25) of patients giving a positive history. Furthermore, bacteria caused skin infections, mainly presenting as folliculitis (32 %, 7/22). Recurrent viral infections were rarely observed. Herpes simplex virus infections (18 %, 4/22) and papillomavirus infections (14 %, 3/22) were the most prevalent.  CMC was also associated with auto-immune conditions in our cohort. With 44 % (11/25) of patients being affected, hypothyroidism was by far the most common autoimmune condition reported. Alopecia and vitiligo were observed in one patient each (5 %, 1/21) (Table 3).
With regard to serious complications, no patient reported systemic fungal infection, and no patient had Candida meningitis. Other severe complications, such as cerebral aneurysm, cerebral vasculitis, and squamous cell cancer of the esophagus, have been described in other CMC cohorts [20,37,38]. Two unrelated patients from our cohort were reported with cerebral aneurysms. One of them developed cerebral vasculitis. Aneurysms were detected in both patients through MRI at the age of 8 and 10 years, respectively.
We further analyzed lymphocyte subpopulations, serum immunoglobulin (Ig) levels, autoantibodies, and thyroid markers in all patients from our cohort ( Table 4) (19 %, 3/16). No thyroid carcinoma was detected in the cohort under study. Detection of other autoantibodies such as antimicrosomal, anti-insulin or anti-GAD65 antibodies occurred rarely (Table 4).

Discussion
In the present study we found that in a group of 57 CMC patients, 35 cases (61 %) had a heterozygous GOF mutation in STAT1. Twenty-three patients had a STAT1 mutation in the CCD, underlining the importance of this domain as a mutation locus, while 12 had a DBD mutation in STAT1. The majority of families (82 %, 9/11) as well as half of the sporadic cases (50 %, 9/18) harbored a heterozygous STAT1 mutation. Several mutations we identified in the cohort under study have been described previously (Fig. 2). Van de Veerdonk et al. published that patients of a Dutch family carrying the mutation p.R274W in the CCD had severe dermatophytosis and autoimmune phenomena (autoimmune hemolysis, pernicious anemia, autoimmune hepatitis), in addition to severe, chronic oropharyngeal candidiasis [19]. These severe autoimmune phenomena were not observed in our patients. However, the association between the STAT1 mutation p.A267V and hypothyroidism, which has been reported [19], was also seen in two of our families.
During this study, we were able to clarify the genetic background of a large family (Fam02) with an autosomal dominant CMC type associated with hypothyroidism previously published by Atkinson et al. in 2001 [35]. The family mapped to a candidate linkage region on chromosome 2p; however, no mutation could be identified at that time. In this kindred, we detected the heterozygous STAT1 mutation p.A267V in four of the affected family members. However, STAT1 is located on chromosome 2q and not 2p. Only the most severely affected members with both CMC and thyroid disease harbored this STAT1 mutation, while other family members, solely suffering from hypothyroidism, did not carry it. A likely explanation for the incorrect linkage analysis may be the misclassification of these patients due to the misleading influence of the thyroid disease, since thyroid autoimmunity is among the most common autoimmune conditions.
The high prevalence of thyroiditis which we observed in the STAT1 mutated CMC patients in this study is an interesting aspect. One possibility for STAT1 mutations causing hypothyroidism might be the formation of thyroid autoantibodies [20,22]. Thyroglobulin autoantibodies (TgAb) were detected in 13 % (2/16) of patients in our cohort. TgAb are often found in patients with Hashimoto's thyroiditis, which constitutes a common cause for hypothyroidism [39]. Furthermore, we speculate that a second mechanism may be causally related to hypothyroidism in this cohort of patients. Staab et al. assessed expression of cytokine-regulated STAT proteins in patients diagnosed as having Hashimoto's disease or focal lymphocytic thyroiditis. In these patients, activated STAT1 dimers were detected in numerous infiltrating lymphocytes, macrophages as well as in oncocytes, while STAT3 expression was restricted to epithelial cells and showed a clear association with low levels of stromal fibrosis, suggesting that STAT3 serves as a protective factor in the remodeling of the inflamed thyroid gland [40]. The imbalance between STAT1-dependent signaling and STAT3-dependent signaling might further amplify degenerative processes in the thyroid gland. Moreover, mutated STAT1 may contribute to hypothyroidism by interfering with thyrotropin signaling pathway. The production of suppressor of cytokine signaling 1 (SOCS1) is induced by thyrotropin, which then alters STAT1 phosphorylation [41]. Thyrotropin may act as a cytokine inhibitor in thyroid tissue in order to rescue thyroid cells. This process might be impeded by GOF STAT1 mutations [19,41].
We were not able to identify STAT1 mutations in all of the CMC patients under study. Other candidate genes tested (CARD9, IL17F, IL17RA, IL17RB, AIRE and ACT1) did not reveal a molecular cause for CMC in our cohort. The cause(s) for CMC in the remainder of the cohort described here (39 %, 22/57) still needs to be identified. However, due to the high frequency of mutations, we propose that sequencing of STAT1 should be performed in every patient suspected of having a genetic form of autosomal dominant CMC. Identification of a mutation in the CCD or the DBD in the context of clinical diagnosed CMC makes STAT1 a likely candidate for the underlying genetic defect. Functional immunological analysis should be performed, especially when mutations are found, which have not been formally proven to result in GOF. We suggest that the analysis of the frequency of Th17 cells in peripheral blood may be performed as well as testing for IFN-and/or IL-stimulated STAT1 phosphorylation by flow cytometry. While we were able to show an increased phosphorylation of STAT1 for several mutations, however, not all STAT1 variants seem to be disease causing, even when amino acid changes occur next to known disease causing mutations. Our functional assay indicated that e.g., p.V266I does not cause a GOF in STAT1. The variant V266I was previously published by Uzel et al., after being observed in an infant with an IPEX-like phenotype [28]. Uzel et al. described an enhanced DNA binding and transactivation of STAT1 for the mutation. However, this variant is also listed on dbSNP as rs41473544, the minor allele frequency is recorded to be 0.0006 in the 1000 Genomes database. When performing functional testing, we failed to observe a GOF for this variant.
In summary, the relevance of this variant remains unclear. These findings emphasize the importance of complementing genetic analysis through functional investigation. Taken together, STAT1 is the most probable candidate gene in CMC patients but not the only gene for autosomal dominant inherited CMC. Finding the missing genetic defects still remains an interesting challenge for future research.