STAT1 Mutations in Autosomal Dominant Chronic Mucocutaneous Candidiasis · pathway leads to...

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The new england journal of medicine 10.1056/nejmoa1100102 nejm.org 1 original article STAT1 Mutations in Autosomal Dominant Chronic Mucocutaneous Candidiasis Frank L. van de Veerdonk, M.D., Ph.D., Theo S. Plantinga, Ph.D., Alexander Hoischen, Ph.D., Sanne P. Smeekens, M.Sc., Leo A.B. Joosten, Ph.D., Christian Gilissen, Ph.D., Peer Arts, Ph.D., Diana C. Rosentul, M.Sc., Andrew J. Carmichael, M.D., Chantal A.A. Smits-van der Graaf, M.D., Ph.D., Bart Jan Kullberg, M.D., Ph.D., Jos W.M. van der Meer, M.D., Ph.D., Desa Lilic, M.D., Ph.D., Joris A. Veltman, Ph.D., and Mihai G. Netea, M.D., Ph.D. From the Departments of Medicine (F.L.V., T.S.P., S.P.S., L.A.B.J., D.C.R., C.A.A.S.G., B.J.K., J.W.M.M., M.G.N.), Human Genet- ics (A.H., C.G., P.A., J.A.V.), and Pulmonary Diseases (C.A.A.S.G.), Radboud University Nijmegen Medical Center, and the Nijme- gen Institute for Infection, Inflammation, and Immunity (F.L.V., T.S.P., S.P.S., L.A.B.J., D.C.R., C.A.A.S.G., B.J.K., J.W.M.M., M.G.N.) — both in Nijmegen, the Netherlands; and the Department of Dermatology, James Cook University Hospital, Middlesbrough (A.J.C.), and the Institute for Cellular Medi- cine, Newcastle University, Newcastle upon Tyne (D.L.) — both in the United Kingdom. Address reprint requests to Dr. van der Meer at the Department of Medicine, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, the Nether- lands, or at [email protected]. The following two groups of authors con- tributed equally to this article: Drs. van de Veerdonk, Plantinga, and Hoischen; and Drs. Lilic, Veltman, and Netea. This article (10.1056/NEJMoa1100102) was published on June 29, 2011, at NEJM.org. N Engl J Med 2011. Copyright © 2011 Massachusetts Medical Society. Abstract Background Chronic mucocutaneous candidiasis (CMC) is characterized by susceptibility to can- dida infection of skin, nails, and mucous membranes. Patients with recessive CMC and autoimmunity have mutations in the autoimmune regulator AIRE. The cause of autosomal dominant CMC is unknown. Methods We evaluated 14 patients from five families with autosomal dominant CMC. We incubated their peripheral-blood mononuclear cells with different combinations of stimuli to test the integrity of pathways that mediate immunity, which led to the selection of 100 genes that were most likely to contain the genetic defect. We used an array-based sequence-capture assay, followed by next-generation sequencing, to iden- tify mutations. Results The mononuclear cells from the affected patients were characterized by poor pro- duction of interferon-γ, interleukin-17, and interleukin-22, suggesting that the defect lay within the interleukin-12 receptor and interleukin-23 receptor signaling pathways. We identified heterozygous missense mutations in the DNA sequence encoding the coiled-coil (CC) domain of signal transducer and activator of transcription 1 ( STAT1) in the patients. These mutations lead to defective responses in type 1 and type 17 helper T cells (Th1 and Th17). The interferon-γ receptor pathway was intact in these patients. Conclusions Mutations in the CC domain of STAT1 underlie autosomal dominant CMC and lead to defective Th1 and Th17 responses, which may explain the increased susceptibility to fungal infection. (Funded by the Netherlands Organization for Scientific Research and others.) The New England Journal of Medicine Downloaded from nejm.org on June 30, 2011. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved.

Transcript of STAT1 Mutations in Autosomal Dominant Chronic Mucocutaneous Candidiasis · pathway leads to...

T h e n e w e ngl a nd j o u r na l o f m e dic i n e

10.1056/nejmoa1100102  nejm.org 1

original article

STAT1 Mutations in Autosomal Dominant Chronic Mucocutaneous CandidiasisFrank L. van de Veerdonk, M.D., Ph.D., Theo S. Plantinga, Ph.D.,

Alexander Hoischen, Ph.D., Sanne P. Smeekens, M.Sc., Leo A.B. Joosten, Ph.D., Christian Gilissen, Ph.D., Peer Arts, Ph.D.,

Diana C. Rosentul, M.Sc., Andrew J. Carmichael, M.D., Chantal A.A. Smits-van der Graaf, M.D., Ph.D., Bart Jan Kullberg, M.D., Ph.D.,

Jos W.M. van der Meer, M.D., Ph.D., Desa Lilic, M.D., Ph.D., Joris A. Veltman, Ph.D., and Mihai G. Netea, M.D., Ph.D.

From the Departments of Medicine (F.L.V., T.S.P., S.P.S., L.A.B.J., D.C.R., C.A.A.S.G., B.J.K., J.W.M.M., M.G.N.), Human Genet-ics (A.H., C.G., P.A., J.A.V.), and Pulmonary Diseases (C.A.A.S.G.), Radboud University Nijmegen Medical Center, and the Nijme-gen Institute for Infection, Inflammation, and Immunity (F.L.V., T.S.P., S.P.S., L.A.B.J., D.C.R., C.A.A.S.G., B.J.K., J.W.M.M., M.G.N.) — both in Nijmegen, the Netherlands; and the Department of Dermatology, James Cook University Hospital, Middlesbrough (A.J.C.), and the Institute for Cellular Medi-cine, Newcastle University, Newcastle upon Tyne (D.L.) — both in the United Kingdom. Address reprint requests to Dr. van der Meer at the Department of Medicine, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, the Nether-lands, or at [email protected].

The following two groups of authors con-tributed equally to this article: Drs. van de Veerdonk, Plantinga, and Hoischen; and Drs. Lilic, Veltman, and Netea.

This article (10.1056/NEJMoa1100102) was published on June 29, 2011, at NEJM.org.

N Engl J Med 2011.Copyright © 2011 Massachusetts Medical Society.

A bs tr ac t

Background

Chronic mucocutaneous candidiasis (CMC) is characterized by susceptibility to can-dida infection of skin, nails, and mucous membranes. Patients with recessive CMC and autoimmunity have mutations in the autoimmune regulator AIRE. The cause of autosomal dominant CMC is unknown.

Methods

We evaluated 14 patients from five families with autosomal dominant CMC. We incubated their peripheral-blood mononuclear cells with different combinations of stimuli to test the integrity of pathways that mediate immunity, which led to the selection of 100 genes that were most likely to contain the genetic defect. We used an array-based sequence-capture assay, followed by next-generation sequencing, to iden-tify mutations.

Results

The mononuclear cells from the affected patients were characterized by poor pro-duction of interferon-γ, interleukin-17, and interleukin-22, suggesting that the defect lay within the interleukin-12 receptor and interleukin-23 receptor signaling pathways. We identified heterozygous missense mutations in the DNA sequence encoding the coiled-coil (CC) domain of signal transducer and activator of transcription 1 (STAT1) in the patients. These mutations lead to defective responses in type 1 and type 17 helper T cells (Th1 and Th17). The interferon-γ receptor pathway was intact in these patients.

Conclusions

Mutations in the CC domain of STAT1 underlie autosomal dominant CMC and lead to defective Th1 and Th17 responses, which may explain the increased susceptibility to fungal infection. (Funded by the Netherlands Organization for Scientific Research and others.)

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Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency dis-order that is characterized by susceptibility

to infection of the skin, nails, and mucous mem-branes by candida species and dermatophytes.1 There are several CMC subtypes: autosomal reces-sive autoimmune polyendocrinopathy candidiasis with ectodermal dystrophy (APECED), autosomal dominant CMC with or without thyroid disease, and autosomal recessive, isolated CMC.

The defect in APECED resides in the autoim-mune regulator AIRE, which has a key role in im-munotolerance.2 The susceptibility to candida in patients with APECED is attributed to autoantibod-ies to interleukin-17 and interleukin-22,3 since type 17 helper T cells (Th17) are crucial for mucosal antifungal immunity.4 Little is known about the defects underlying susceptibility to candida in pa-tients with autosomal dominant CMC. In a pre-vious study, we found that Th1–interferon-γ re-sponses were defective in patients with autosomal dominant CMC.5 In a more recent study, investiga-tors found defective Th17 responses in patients with this disorder.6 Defective recognition of can-dida because of mutations in the dectin-1–CARD9 pathway leads to increased susceptibility to fun-gi,7,8 but the clinical picture is less severe than in autosomal dominant CMC. We sought the ge-netic cause of susceptibility to mucocutaneous fungal infection in families with autosomal dom-inant CMC.

Me thods

Study Design

The study was approved by the ethics committee at Radboud University Nijmegen Medical Center and the Newcastle and North Tyneside research ethics committee. Written informed consent was obtained from all family members and healthy control sub-jects who were evaluated.

Family 1

Family 1 was a nonconsanguineous family of Dutch descent in which the father, daughter, and son have had severe CMC since early childhood (Fig. 1A, and Tables S1 and S2 in the Supplementary Appendix, available with the full text of this article at NEJM .org). The patients have severe oropharyngeal chronic candidiasis and severe dermatophytosis and candidiasis of the feet (Fig. 1B). The father has

autoimmune hepatitis, and the daughter has auto-immune hemolysis and pernicious anemia, along with antiphospholipid antibodies. The daughter has had pulmonary embolism and Pneumocystis jirovecii pneumonia with symptomatic cytomega-lovirus infection. The son has extensive dermato-phytosis (Trichophyton rubrum) of the feet but no autoimmune phenomena. We characterized the im-mune responses of peripheral-blood mononuclear cells (PBMCs) from the three affected family mem-bers, the unaffected mother, and three healthy con-trol subjects. The father has nine unaffected sib-lings (Fig. 1A). We carried out genetic analyses of all affected and unaffected family members.

Families 2, 3, and 4

We performed immunologic and genetic analyses of samples from nine members of three unrelat-ed families of European descent from the United Kingdom (Families 2, 3, and 4). All nine patients have autosomal dominant CMC with hypothyroid-ism. Their characteristics have been reported previ-ously9,10 (Table S1 in the Supplementary Appendix). One patient in Family 2 has oral squamous-cell carcinoma; the father in Family 4 died of esopha-geal cancer.9 All patients were screened for AIRE mutations to rule out the APECED syndrome.11 We also analyzed samples from unaffected members of Family 4.

Family 5

We analyzed two members with CMC and three unaffected members of a Dutch family (Family 5). Both Patient 1 and her mother had esophageal can-cer (Table S1 in the Supplementary Appendix).

Unaffected Subjects

We assessed 301 unrelated healthy Dutch control subjects and 56 healthy British control subjects of European ancestry for the genetic mutations of CMC. We also analyzed an in-house Nijmegen da-tabase of 100 exome data sets that were derived from healthy subjects of European ancestry with-out signs of CMC. A questionnaire concerning the ethnic origin of parents and grandparents of the healthy subjects confirmed their ancestry in the Netherlands and the United Kingdom.

Immunologic Studies

We incubated PBMCs (5×106 per milliliter), ob-tained by density centrifugation, at 37°C in 96-well

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plates (Greiner),12 with or without heat-killed Can-dida albicans (1×106 microorganisms per milliliter [strain UC820]), Escherichia coli lipopolysaccharide (1 ng per milliliter) (Sigma-Aldrich), interleukin-1β (10 ng per milliliter), interleukin-12 (10 ng per mil-liliter), interleukin-18 (50 ng per milliliter), inter-leukin-23 (10 ng per milliliter) (R&D Systems), or interferon-γ (1 μg per milliliter) (Boehringer). We used an enzyme-linked immunosorbent assay to measure levels of interleukin-1β, tumor necrosis factor α (TNF-α), interleukin-17, interleukin-22 (R&D Systems), interferon-γ, and interleukin-6 (PeliKine) after 24 and 48 hours of incubation (in the absence of serum) and after 5 days of incuba-tion (in the presence of 10% serum). All experi-ments were performed at least three times.

Sequence Capture and DNA Sequencing

We applied array-based sequence capture followed by next-generation sequencing (454 Life Sciences) to analyze 100 genes from known immunologic pathways (Table S3 in the Supplementary Appen-dix). Details regarding coverage statistics are also provided in Table S4 in the Supplementary Appendix.

Validation of Mutations and Haplotype Analysis

To validate the presence of mutations in signal transducer and activator of transcription 1 (STAT1) in affected patients, we amplified their DNA, using a polymerase-chain-reaction (PCR) assay, and se-quenced the amplified DNA fragments by Sanger’s method. All coding exons of the coiled-coil (CC) domain of STAT1, including exon 10, were ampli-fied and analyzed. AIRE mutations were excluded by sequencing the gene as described previously.13 We used 250K single-nucleotide-polymorphism (SNP) arrays (Affymetrix)14 and Sanger sequencing to de-termine haplotypes associated with the STAT1mutations.

R esult s

Immunologic Defects in Family 1

Among unaffected members of Family 1, stimula-tion of PBMCs with candida produced normal amounts of the cytokine proteins interleukin-1β, interleukin-6, and TNF-α (Fig. 2A, and Fig. S1A in the Supplementary Appendix), along with normal activation of toll-like receptor 4 (TLR4), toll-like

receptor 2 (TLR2), and dectin-1–receptor signaling pathways (Fig. 2B).15 In contrast, family mem bers with autosomal dominant CMC had low levels of helper T-cell cytokines interferon-γ, interleukin-17, and interleukin-22 in PBMCs in response to can-dida stimulation (Fig. 2C, and Fig S1 in the Sup-plementary Appendix).

We subsequently investigated the pathways leading to the production of interferon-γ, interleu-kin-17, and interleukin-22 by the patients’ T cells.

B Clinical Manifestations

A Family 1

P1

Patient 1 Patient 2

Patient 1 Patient 3

P3P2

Figure 1. Pedigree of Family 1 with Autosomal Dominant Chronic Mucocu-taneous Candidiasis (CMC) and Clinical Signs in Affected Family Members.

Panel A shows the pedigree of a Dutch family in which three members of two generations have clinical symptoms characteristic of CMC (black sym-bols). Squares indicate male family members, and circles female family members. The affected family members have severe dermatophytosis and candidiasis of the feet and severe oropharyngeal chronic candidiasis (Panel B). Patients 1 and 2 also have other autoimmune disorders.

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The induction of interferon-γ on exposure of these cells to a combination of interleukin-12 and in-terleukin-18 was partially impaired (Fig. 2D). Ex-posure to interleukin-12 alone resulted in unde-

tectable interferon-γ (Fig. 2D), and exposure to interleukin-23 and interleukin-1β resulted in no detectable interleukin-17 or interleukin-22 pro-duction (Fig. 2E, and Fig S1B in the Supplemen-

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Figure 2. Immunologic Defects in the Affected Members of Family 1.

Peripheral-blood mononuclear cells (PBMCs) from two unrelated healthy control subjects, the unaffected spouse of Patient 1 (Control 3), the unaffected mother of Patients 2 and 3, and the three affected family members (Patients 1, 2, and 3) were stimulated with heat-killed Candida albicans (1×106 microorganisms per milliliter) for 24 hours (Panel A); with the toll-like receptor 4 ligand Escherichia coli lipopoly-saccharide (LPS) (1 ng per milliliter), the toll-like receptor 2 ligand tripalmitoyl-S-glycerylcysteine (Pam3Cys) (10 μg per milliliter), the dectin-1 ligand β-glucan (10 μg per milliliter), or a combination of β-glucan and Pam3Cys for 24 hours (Panel B); with heat-killed C. albi-cans (1×106 microorganisms per milliliter) for 5 days (for interleukin-17) and for 48 hours (for interferon-γ) (Panel C); with interleukin-12 (10 ng per milliliter) with or without interleukin-18 (50 ng per milliliter) for 48 hours (Panel D); and with a combination of interleukin-1β (10 ng per milliliter) and interleukin-23 (10 ng per milliliter) for 5 days (for interleukin-22) and with interleukin-1β (10 ng per milliliter) for 24 hours (for interleukin-6) (Panel E). All cytokines were measured by means of an enzyme-linked immunosorbent assay.

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STAT1 Mutations in Chronic Mucocutaneous Candidiasis

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tary Appendix). Mitogenic stimulation, however, induced normal interferon-γ production in these patients (data not shown). Normal production of interleukin-6 in response to interleukin-1β stim-ulation proved that the pathway that is dependent on interleukin-1 receptor type I was intact (Fig. 2E). We concluded that both the interleukin-12 and interleukin-23 pathways are perturbed in affected family members (Fig. 3).

Missense Mutation in STAT1

STAT3 mutations, which are known to cause the hyperimmunoglobulin E syndrome and are in-volved in the interleukin-12 receptor and interleu-kin-23 receptor signaling pathways, were not pres-ent in the three affected members of Family 1. Neither did we find mutations in STAT4, which en-codes a protein that is also involved in these sig-naling pathways, or in AIRE. On the basis of the observed cytokine defects, we selected 100 genes encoding proteins that are relevant in interleu-kin-12 and interleukin-23 signaling and in Th1 and Th17 responses (Table S3 in the Supplementary Appendix).

Sequence Analysis of 100 Candidate Genes

Using array-based sequence capture followed by next-generation sequencing, we observed an aver-age of 723 variants per sample. Considering the rare nature of the disease and the autosomal dom-inant pattern of inheritance, we decided to filter these variants against known SNP variants. Of the 723 identified variants, 651 corresponded with known SNPs or were located in a known polymor-phic region. After the exclusion of variants that were detected by exome-sequencing projects,16,17

in an in-house database, or through the 1000 Ge-nomes Project, we were left with an average of 38 novel variants per patient (Table S8 in the Supple-mentary Appendix). Of these variants, only 4 per patient were nonsynonymous coding variants, and of these, no more than 4 were called in at least 20% of all reads and were therefore consid-ered to be heterozygous candidate variants.

Of the 11 candidate variants, only 7 were ob-served in affected family members. Of these 7 vari-ants, 3 cosegregated in the three affected members of Family 1 (Table S9 in the Supplementary Ap-pendix). These patients each carried a heterozy-gous variant of the STAT1 gene in exon 10 mapping to chromosome 2 (c.820C→T; p.Arg274Trp). The mutation predicts an amino acid change (arginine

to tryptophan) in the CC domain of STAT1 (Fig. S2 and S3 in the Supplementary Appendix). In each affected patient, STAT1 was the only gene with a heterozygous, nonsynonymous coding variant, mak ing it the most likely candidate gene. One unaffected member of the family (the wife of Pa-tient 1 and the mother of Patients 2 and 3) and

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Figure 3. Molecules Shared by the Interleukin-12–Interleukin-23 Pathway.

The interleukin-12 and interleukin-23 receptor intracellular pathways share the adapter molecules Tyk2 and Jak2, leading to downstream activation of the STAT, SOCS, and PIAS proteins. The unresponsiveness to cytokines inter-leukin-12 and interleukin-23 in patients with autosomal dominant chronic mucocutaneous candidiasis most likely suggests a defect in one of these adapter proteins.

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two unrelated healthy subjects did not carry any mutation in STAT1. Of all novel, heterozygous, nonsynonymous coding variants, those in STAT1 affected amino acid residues that showed the stron-gest conservation over 44 vertebrate species.

STAT1 Mutations in Other Affected Persons

Next we investigated three British families of Eu-ropean descent with autosomal dominant CMC (Fig. 4, and Tables S1 and S10 in the Supplementary Appendix). Using the same technique, we observed a different STAT1 variant (c.800C→T; p.Ala267Val), which also affected exon 10 in an affected patient. An affected member of another family (Patient 5 in Family 2) had very poor coverage for exon 10 of STAT1. However, manual read inspection showed the same variant in two of five reads. In each of the affected patients, we confirmed the presence of this mutation on PCR amplification of the DNA se-quence encoding the CC domain of STAT1, followed by Sanger sequencing (Fig. 4, and Table S10 in the Supplementary Appendix).

Sequence analysis of STAT1 in additional af-fected patients revealed the Arg274Trp variant in a member of a third family (Patient 1 in Family 4) and the Ala267Val variant in two patients from a second Dutch family with autosomal dominant CMC (Patients 1 and 2 in Family 5) (Fig. 4). We did not identify the Ala267Val or Arg274Trp variant in 301 unrelated healthy Dutch control subjects or in 56 healthy British control subjects. Nor did we observe such variants in 100 exome data sets from subjects of Dutch ancestry or in the 179 subjects of European descent with DNA samples that were sequenced as part of the 1000 Genomes Project.18 Further analysis of the 100 in-house exomes iden-tified only a single novel missense change in the STAT1 gene (c.314T→C), which we attribute to a mapping artifact caused by an underlying repeat element in exon 5.

Haplotype Analysis

To determine whether the STAT1 mutations are founder mutations, we performed haplotype anal-ysis using high-density SNP arrays and Sanger se-quencing of genomic DNA from affected and un-affected members of all the families in our study. We deduced that the Arg274Trp and Ala267Val mu-tations lie on different haplotypes (Table S11 in the Supplementary Appendix). Furthermore, each mu-tation lies on a distinct haplotype that is common to all families bearing the mutation, suggesting

that there is a founder effect for each mutation, al-though it is possible that the same mutation oc-curred more than once on a common haplotype by chance. The latter is corroborated by the fact that in Family 1, only Patient 1 is affected and nine siblings are unaffected (suggesting but not proving the ex-istence of a de novo mutation).

Functional Studies

Cytokine analysis of PBMCs obtained from patients with CMC in the confirmation study revealed de-fects similar to those observed in Family 1: de-fective interferon-γ production in response to interleukin-12 stimulation and no interleukin-22 production in response to interleukin-1β and in-terleukin-23 stimulation (Fig. S6A in the Supple-mentary Appendix). In the affected members of Family 1, we found that the addition of interferon-γ to E. coli lipopolysaccharide resulted in increased TNF-α production, which showed that the in ter fer-on-γ signaling pathway was intact (Fig. S6B in the Supplementary Appendix).

Discussion

We found that mutations affecting the CC domain of STAT1 in patients with autosomal dominant CMC lead to defective Th1 and Th17 responses, char-acterized by reduced production of interferon-γ, interleukin-17, and interleukin-22; these cytokines are crucial for the antifungal defense of skin and mucosa. Affected members of Family 1 had normal monocyte-dependent cytokine responses but se-verely impaired Th1 and Th17 responses. These findings are in line with both studies showing de-fective production of interferon-γ, interleukin-17, and interleukin-22 in patients with CMC5,6 or the hyperimmunoglobulin E syndrome19 and with the observation that interleukin-17–defective mice are susceptible to oral candidiasis.4

STAT1 mutations have been described in pa-tients with increased susceptibility to viruses and mycobacteria.20-23 These mutations are located in regions of STAT1 that encode the Src homology 2 (SH2) or DNA-binding domains of the protein and result in defective signaling of interferon-γ and type I interferon–receptor pathways.20-23 A mutation in the CC domain of STAT1 (Phe172Ser) resulted in decreased expression of STAT1 pro-tein,24 whereas other mutations in the CC domain blocked dimerization of nonphosphorylated mol-ecules.25 The mutations affecting the CC domain

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that we observed in patients with autosomal dominant CMC exclusively affect Th1 and Th17 responses, possibly by modifying the interaction of STAT1 with its binding partners STAT3 and STAT4.26 STAT1/STAT1 homodimers mediate sig-naling by the interferon-γ receptor, which induces resistance to intracellular microorganisms.26 In-ter fer on-γ–receptor signaling was preserved in the patients with autosomal dominant CMC, which may explain their normal susceptibility to myco-bacteria and viruses. However, infections other than those caused by candida and dermatophytes, ranging from bacterial chest infections to pneu-mocystis and cytomegalovirus infections, occurred in the Dutch and British patients.

Affected members in two of the families with the Arg274Trp mutation had autoimmune disor-ders. STAT1 and interleukin-17 have been reported to be involved in the pathogenesis of autoimmune diseases.27 Interleukin-17 seems unlikely to be the cause of these diseases, since it is produced in low levels by PBMCs from affected patients. Three affected patients have hypothyroidism, with two

of them carrying the c.800C→T mutation and one carrying the c.820C→T mutation. It is known that thyrotropin induces the production of suppressor of cytokine signaling 1 (SOCS1), which in turn can alter STAT1 phosphorylation.28 Thyrotropin may act as a cytokine inhibitor in thyroid tissue, and mutated STAT1 may hamper the rescue of thyroid cells by thyrotropin and contribute to hypothyroid-ism. In addition, there may be decreased iodine accumulation, as is the case with Stat1-deficient mice.29 Finally, one of the affected Dutch patients (and her deceased mother) and two members of the British families, all of whom carried the Ala267Val mutation, had esophageal or oral car-cinoma.9 The loss of function of STAT1 has been linked to esophageal carcinoma.30,31

In conclusion, we found that mutations af-fecting the CC domain of STAT1 cause autosomal dominant CMC. Mutant STAT1 probably affects the host defense against candida species through abnormal Th1 and Th17 responses. These find-ings should facilitate the diagnosis of CMC in pa-tients with chronic candidiasis.

Family 2

Family 3 Family 5

Male Female

CMC with candida

CMC with candida and hypothyroidism

NT

NTNT

P1

Family 4

NT NT

P1

P1 P1P3

P2P2

P2

P5

P3 P4

Figure 4. Confirmation of STAT1 Mutations in Patients with Chronic Mucocutaneous Candidiasis (CMC).

Shown are the pedigrees of four additional families with autosomal dominant CMC: three families from the United Kingdom with CMC and thyroid disease (Families 2, 3, and 4) and one Dutch family with CMC and esophageal carci-noma (Family 5). In these families, 11 patients were tested for STAT1 mutations. Patients in Families 2, 3, and 5 were found to have mutation Ala267Val, and those in Family 4 were found to have mutation Arg274Trp. NT denotes not tested.

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10.1056/nejmoa1100102  nejm.org8

STAT1 Mutations in Chronic Mucocutaneous Candidiasis

Supported by grants from the Netherlands Organization for Scientific Research (to Dr. Netea), the Primary Immunodefi-ciency Association UK (to Dr. Lilic), the Techgene Project funded by the European Union (Health-F5-2009-223143, to Drs. Veltman and Arts), the Aneuploidy Project (LSHG-CT-2006-37627, to Drs. Hoischen and Veltman), and the Netherlands Organization for

Health Research and Development (ZonMW grant 917.66.36, to Dr. Veltman).

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

We thank our patients and their families; and Joanne Sedgwick, Nienke Wieskamp, and Dr. Jolien Tol for their contributions.

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