Gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1): chronic mucocutaneous candidiasis accompanied by enamel defects and delayed dental shedding.

To the Editor:

Heterozygous gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have recently been identified as a cause of chronic mucocutaneous candidiasis (CMC). Uzel et al described “STAT1 gain-of-function mutations in patients with FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked syndrome.” They briefly mentioned the presence of poor enamel in 1 patient and structural and functional gastrointestinal defects in another patient. Here we present a patient with CMC associated with dental anomalies, diaphragmatic hernia, and esophageal dysmotility in whom the phenotype led to a broad differential diagnosis ranging from severe combined immunodeficiency inspired by the neonatal onset of infections to humoral immune deficiency, immune dysregulation–polyendocrinopathy–enteropathy–X-linked syndrome, nuclear factor κB essential modulator (NEMO) deficiency, Shwachman-Diamond syndrome, autosomal dominant hyper-IgE syndrome, and, finally, CMC caused by gain-of-function mutation in STAT1. We stress the early onset of respiratory tract infections, as well as the dental and gastrointestinal defects, in this patient.

The patient was born at term small for gestational age (−2 SD) as the third son of unrelated parents. He presented with recurrent lower respiratory tract infections from birth, intractable diarrhea, failure to thrive, seborrheic dermatitis, and CMC. Small-bowel biopsy showed villous atrophy interpreted as celiac-like disease, yet diarrhea was unresponsive to a gluten-free diet. Primary dentition showed enamel defects. Recurrent lower respiratory tract infections with Haemophilus influenzae and Streptococcus pneumoniae led to the development of bronchiectasis. Partial IgG2 deficiency (0.34 g/L; normal range, 0.72-3.4 g/L) was found at 3 years of age. Anti-tetanus antibody levels were protective (1.2 mg/L; protective level, >1 mg/L), suggesting an intact anti-protein antibody response. Intravenous immunoglobulin (IVIG) substitution was initiated. Diaphragmatic hernia, gastroesophageal reflux disease, and disturbed esophageal motility were demonstrated and led to a Nissen fundoplication.

At 13 years of age, the patient presented at the immunology clinic with severe growth retardation (Fig 1, A), pubertal delay, bronchiectasis, atonic esophagus with multiple diverticula (Fig 1, B and C), atrophic duodenal mucosa (Fig 1, D) corresponding to villous blunting or atrophy, joint hyperlaxity, osteopenia with recurrent fractures, delayed dental development with retention of primary teeth necessitating dental extractions (Fig 1, E and F), severe erosive tooth wear suggestive of enamel hypoplasia (Fig 1, E and F), seborrheic dermatitis, aphthous stomatitis, and CMC (see Table E1 in this article's Online Repository at www.jacionline.org). The autoimmune regulator (AIRE) and caspase recruitment domain family, member 9 (CARD9), genes were sequenced, but no mutations were found. After withdrawal of IVIG, antibody response to unconjugated pneumococcal vaccine was tested and showed protective titers for 9 of 14 serotypes tested (see Table E2 in this article's Online Repository at www.jacionline.org). Increased IgG levels (21 g/L; normal range, 5.76-12.65 g/L) and persistent partial IgG2 deficiency were noted (0.80 g/L; normal range, 1.06-6.10 g/L). An extended autoantibody screening panel was performed, including thyroid-related, adrenal gland–related, and anti–IFN-α and anti–IFN-ω antibodies. Only anti–salivary gland antibodies were demonstrated (for the entire panel, see the Methods section in this article's Online Repository at www.jacionline.org). Immunophenotyping showed a low percentage of switched memory B cells (1.3%; normal range, 5% to 10%; see Table E3 in this article's Online Repository at www.jacionline.org). Because withdrawal of IVIG was associated with an increased incidence of pneumonia, treatment was optimized by restarting IVIG and initiating azithromycin (both for antibacterial prophylaxis and its anti-inflammatory actions) and fluconazole prophylaxis, as well as overnight tube feeding. Growth hormone therapy and puberty induction led to a correction of the growth deficit (Fig 1, A). Finally, at the age of 18 years, hypothyroidism was diagnosed almost simultaneously with the initial reports on STAT1 coiled-coil domain gain-of-function mutations.3, 4 A mutation in the DNA-binding domain of STAT1 was detected (c.1154C>T, p.T385M; Fig 2, A; for information on the analysis, see the Results section in this article's Online Repository at www.jacionline.org). The mutation was not found in the parents or the 2 male siblings of the index patient. As described previously, T385M is a gain-of-function mutation.1, 5, 6, 7 Likewise, we showed increased STAT1 phosphorylation in response to IFN-α and IFN-γ in the patient compared with control values (Fig 2, B). Also, the electrophoretic mobility shift assay (EMSA) showed increased gamma-activated sequence (GAS) binding activity on stimulation with IFN-γ (Fig 2, C).

Fig 1

Clinical characteristics. A, Growth charts showing severe growth retardation that only picks up after growth hormone therapy and puberty induction with tube feeding overnight. Arrows indicate onset of tube feeding and growth hormone therapy, respectively. B, Computed tomography showing an atonic esophagus (arrow) with air containing paraesophageal diverticula. C, Computed tomography of the chest showing multiple saccular bronchiectases and bronchial wall thickening. D, Atrophy in the duodenum, as seen on endoscopy. E and F, Severe erosive tooth wear, caries, and retained primary teeth.

Table E1

Clinical characteristics

Present age	20 y
Ethnicity	White
Initial presentation	At birth
CMC	Oral, esophageal, skin
Teeth	Retained primary dentition, severe caries and erosive tooth wear
Skin	Severe seborrheic dermatitis
Pulmonary infections	From birth
	Haemophilus influenzae, Streptococcus pneumoniae, Pseudomonas aeruginosa
	Aspergillus fumigatus
	Bronchiectasis at age 3 y, chronic obstructive pulmonary disease
Other infections	Molluscum contagiosum, Pseudomonas folliculitis, herpes zoster (2×)
Cardiovascular	−
Central nervous system	−
Endocrine	At birth: small for gestational age
	Growth retardation
	Delayed puberty
	Hypothyroidism (antibodies −) at age 18 y
Gastrointestinal	Oral aphthous ulcers, recurrent ulcerative gastritis, esophagitis
	Duodenal atrophic mucosa, villous blunting
	Diaphragmatic hernia
Bone	Osteopenia, multiple fractures
Other	Cystic fibrosis and primary ciliary dyskinesia were excluded.

Table E2

Specific anti-pneumococcal antibody concentrations (in milligrams per miter) in the patient 3 weeks after vaccination with unconjugated pneumococcal vaccine

	PS1	PS3	PS4	PS8	PS9N	PS12F	PS14	PS19F	PS23F	PS6B	PS7F	PS18C	PS19A	PS9V
Patient	0.28	0.37	2.33	0.20	1.31	0.38	6.37	2.71	5.44	4.11	1.35	2.52	0.91	2.81
Cutoff	0.53	0.64	0.49	1.02	0.63	0.46	0.57	0.74	0.24	0.80	1.45	0.34	0.96	0.69

The postvaccination serotype-specific fifth percentile (Cutoff) values obtained in 75 healthy subjects are provided as well. PS, Pneumococcal serotype.

Table E3

Immunologic characteristics

	Age 1 y	Age 3 y	Age 13 y	Age 20 y∗
Lymphocyte subsets
CD19+ B cells			622/μL (82-476/μL)
Naive CD19+CD27− cells			94% of CD19+ (60-80)
IgM memory CD27+IgM+ cells			1.3% of CD19+ (1-5)
Switched memory CD27+IgM− cells			1% of CD19+ (>5)
CD3+CD4+T cells			886/μL (455-1885 μL)
CD3+CD8+ T cells			459/μL (219-1124/μL)
Naive CD4+ cells			75% of CD3 (30% to 65%)
TH17 cells				TH17 cells 0% (0.03-0.67)
Lymphocyte proliferation
PHA	Normal		Normal
Concanavalin A	Normal		Normal
Tetanus	Normal		Normal
Candida	Normal		Normal
Immunoglobulin levels (g/L)
IgG		16.7 (4.8-11.3)	21.4 (5.8-12.7)	22.8 (7.51-15.60)
IgG2		0.34 (0.72-3.40)	0.80 (1.06-6.10)	1.8 (1.5-6.4)
IgA		0.78 (0.35-1.9)	1 (0.81-2.32)	0.28 (0.82-4.53)
IgM		1.11 (0.34-1.34)	1.2 (0.30-1.59)	0.72 (0.43-3.04)

Normal values are shown in parentheses.

During IVIG treatment.

Fig 2

The mutant T385M STAT1 allele is a gain-of-phosphorylation and gain-of-function mutation. A, Direct sequence analysis of exon 14 of STAT1 (forward sequence) in a control subject and the patient with a c.1153C>T resulting in p.T385M. B, Intracellular staining of phosphorylated tyrosine 701 STAT1 (STAT1p) in lymphocytes after stimulation with IFN-γ (2000 IU/mL, left panel) or IFN-α (105 IU/mL, right panel) for 15 minutes. STAT1 and STATp are shown in a control subject (red) and in the T385M patient (blue). Unstimulated conditions are represented as dashed lines. Results shown are representative of 2 independent experiments. MFI, Mean fluorescence intensity. C, Evaluation of STAT1, STAT1 phosphorylation, and STAT1p GAS DNA-binding capacity. Fibroblasts derived from wild-type (WT)/WT control subjects (C1 and C2), p.T385M/WT (patient P1), and p.K388E/WT (patient P2) were stimulated with 100 U/mL IFN-α (α) or 100 U/mL IFN-γ (Υ) or left unstimulated (−) for 60 minutes. a, Western blotting was carried out for detection of STAT1 and STAT1p levels in nuclear extracts (5 μg per sample). Heterogeneous nuclear ribonucleoprotein I (hnRNP I) was used as a loading control reference. b, STAT1 GAS DNA-binding capacity was evaluated by using EMSA. One microgram of nuclear extract was preincubated with 20,000 cpm of GAS probe at room temperature before nondenaturing PAGE separating free from STAT-bound probe.

The mechanism that leads to gain of function in the T385M mutation is not entirely clear. Takezaki et al suggested an impaired dephosphorylation of STAT1. However, it is also possible that there is impaired dissociation from the DNA or a problem with the reciprocal association of the DNA-binding domain with the coiled-coil domain. After diagnosis, extended immunophenotyping of PBMCs was performed and showed absence of TH17 cells, as described by Liu et al.

The dental anomalies in the patient were impressive. Both primary and permanent teeth showed rapid loss of tooth substance, with severe caries and erosive tooth wear reminiscent of the dental anomalies encountered in patients with Shwachman-Diamond syndrome or Ora1/Stromal interaction molecule 1 deficiency. Moreover, deciduous teeth had to be extracted because of delayed shedding. The latter feature resembles autosomal dominant hyper-IgE syndrome.

Several hypotheses to explain the dental anomalies were put forward. First, antibiotic and antimycotic therapy and acidic hypercaloric nutrition were blamed. Second, malabsorption of calcium and vitamin D was investigated. Third, in the context of recurrent aphthous stomatitis, a sicca syndrome was suspected. Although salivary flow was low, treatment with oral saliva analogues did not improve the dental condition. There were no biochemical or clinical signs of hypothyroidism until age 18 years in our patient, excluding this as a cause for the delayed shedding of deciduous teeth. Although a role for STAT1 signaling has been demonstrated in amelogenesis and dentinogenesis in rats, further research is needed to investigate the potential causal relationship between STAT1 gain-of-function mutation and abnormal dental development.8, 9

Aside from the persistent villous blunting, diaphragmatic hernia and esophageal dysmotility are remarkable gastrointestinal features. On computed tomographic (CT) scanning, as well as endoscopy, the esophagus appeared wide open and atonic, with multiple diverticula present. Thus defects in the development of the upper gastrointestinal tract seem to be a noteworthy feature of this syndrome, as hypothesized by Uzel et al. Whether the gastrointestinal manifestations are all secondary to CMC or a primary manifestation of disturbed STAT1 signaling is yet to be determined.1, 6

In conclusion, we report extensive dental anomalies, as well as diaphragmatic hernia and esophageal dysmotility, in a patient with early onset of lower respiratory tract infections in the context of a gain-of-function mutation in the DNA-binding domain of STAT1. These features add to the complexity of the phenotype observed in patients with a gain-of-function mutation in STAT1.