Expanding the Phenotypic and Genotypic Landscape of Autoimmune Polyendocrine Syndrome Type 1

Abstract

Context

Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare monogenic autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene and characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and primary adrenal insufficiency. Comprehensive characterizations of large patient cohorts are rare.

Objective

To perform an extensive clinical, immunological, and genetic characterization of a large nationwide Russian APS-1 cohort.

Subjects and Methods

Clinical components were mapped by systematic investigations, sera were screened for autoantibodies associated with APS-1, and AIRE mutations were characterized by Sanger sequencing.

Results

We identified 112 patients with APS-1, which is, to the best of our knowledge, the largest cohort described to date. Careful phenotyping revealed several additional and uncommon phenotypes such as cerebellar ataxia with pseudotumor, ptosis, and retinitis pigmentosa. Neutralizing autoantibodies to interferon-ω were found in all patients except for one. The major Finnish mutation c.769C>T (p.R257*) was the most frequent and was present in 72% of the alleles. Altogether, 19 different mutations were found, of which 9 were unknown: c.38T>C (p.L13P), c.173C>T (p.A58V), c.280C>T (p.Q94*), c.554C>G (p.S185*), c.661A>T (p.K221*), c.821del (p.Gly274Afs*104), c.1195G>C (p.A399P), c.1302C>A (p.C434*), and c.1497del (p.A500Pfs*21).

Conclusions

The spectrum of phenotypes and AIRE mutation in APS-1 has been expanded. The Finnish major mutation is the most common mutation in Russia and is almost as common as in Finland. Assay of interferon antibodies is a robust screening tool for APS-1.

Autoimmune polyendocrine syndrome type 1 (APS-1), also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), is a rare inherited monogenic autoimmune disease characterized by three major clinical components: chronic mucocutaneous candidiasis (CMC), hypoparathyroidism (HPT), and adrenal insufficiency (AI) (1–3). In addition to the three major components, these patients can develop >20 organ-specific so-called minor components. The clinical situation is diverse in terms of the disease course and components, even within the same family, indicating that other genes, environmental factors, or stochastic events modify the phenotype (2, 4, 5). These factors also contribute to missed and delayed diagnoses.

A typical feature of APS-1 is the presence of a wide range of autoantibodies, which can be divided into two groups: organ-specific and cytokine and interferon (IFN) antibodies (Abs) (6, 7). The former are typically directed against intracellular enzymes with very restricted tissue distribution and associate with organ-specific disease in the organ in which the autoantigen is expressed (8). The latter typically appear at a very early age (6, 9) and are specific for APS-1 and suitable for diagnostic screening.

APS-1 is caused by mutations in the autoimmune regulator (AIRE) gene (10). AIRE has a key role in regulating the expression of tissue-specific antigens in thymic medullary epithelial cells, thereby facilitating negative selection (11), and, possibly, a similar role for peripheral tolerance in the lymph nodes (12). Recently, AIRE was shown to induce the development of early thymic regulatory T cells (11), with a role in harnessing autoreactive cells. More than 100 different mutations have been described to date (Human Gene Mutation Database; available at: www.hgmd.org), spread throughout the gene. Inheritance is autosomal recessive, requiring mutations of both alleles; however, a nonclassic milder form with dominant negative mutations in the first plant homeodomain zinc finger (13) and SAND domains has been described (14).

Current knowledge on the natural course of APS-1 is largely from small case series and reports. Only a few large series of comprehensively characterized APS-1 cases have been reported from Finland (1), Italy (15, 16), the United States (17, 18), Israel (19), and Norway (5, 20), some of which were biased to the selection by the presence of AI (15, 17). In the present study, to the best of our knowledge, we report one of the largest cohorts described to date with longitudinal follow-up data revealing previously unknown clinical components and AIRE mutations.

Subjects and Methods

Patients

The patients were recruited from throughout Russia; most of these patients were investigated by ourselves, except for 22 who were investigated at local centers across Russia. The diagnosis was made clinically by either the presence of two of the three major components (one component if a sibling had established APS-1) or two disease-causing AIRE mutations and at least one clinical component. CMC was diagnosed by the presence of recurrent mucous membrane, nail, and skin infections. The diagnosis of AI was based on low levels of serum cortisol (<100 nmol/L) combined with an adrenocorticotropic hormone value greater than two times the upper reference limit. Alternatively, AI was diagnosed by failure to reach a serum cortisol of 500 nmol/L during a standard synacthen test. HPT was diagnosed by a combination of hypocalcemia, hyperphosphatemia, low parathyroid hormone (PTH), and normal renal function. Alopecia, vitiligo, and hair depigmentation were diagnosed by clinical examination. The clinical features of hepatic failure and/or chronic elevation of hepatic transaminases (more than three times the upper reference limit) without evidence of virus- or drug-induced hepatitis was defined as chronic autoimmune hepatitis. The diagnosis was established by biopsy examination in a few cases. Recurrent diarrhea and/or constipation was considered to indicate malabsorption. Primary ovarian insufficiency (POI) was diagnosed by amenorrhea for >6 months and elevated luteinizing hormone and follicle-stimulating hormone. Enamel hypoplasia was recognized by typical visible enamel defects and confirmed by a dentist. The diagnosis of diabetes mellitus (DM) was determined according to the World Health Organization criteria (21, 22). Pernicious anemia was indicated by the presence of macrocytic anemia and low serum vitamin B₁₂ levels and either autoantibodies against parietal cells or intrinsic factor or confirmed by biopsy-verified autoimmune gastritis. Hypothyroidism was diagnosed by the combination of elevated thyrotropin and low free thyroxine levels. Ophthalmic components were diagnosed by an ophthalmologist. The examination included ophthalmoscopy and Schirmer’s test. Rare components such as pure red cell aplasia, spleen aplasia, cerebellar ataxia, and metaphyseal dysplasia required additional diagnostic tools, which have been described in detail for each case, as appropriate.

Clinical examination and follow-up period

The patients were followed up every 6 to 12 months for 8.5 years (range 0.8 to 17). The standard protocol included full clinical examination, screening blood chemistry and hormonal status, and ultrasonography of the abdominal organs and thyroid. Computed tomography of the brain was performed as indicated for patients with HPT. Ophthalmologists, hepatologists, gastroenterologists, hematologists, neurologists, and dentists were consulted as required.

Assay of autoantibodies

IFN-ω and IFN-α2 Abs were assayed using a cell-based Ab assays and the HEK-blue IFN-α/β cell line (Invitrogen, Carlsbad, CA), as described previously (23). Abs against interleukin (IL)-17F and IL-22, 21-hydroxylase (21OH), side-chain cleavage enzyme (SCC), aromatic l-amino acid decarboxylase (AADC), CYP1A2, tryptophan hydroxylase, NACHT leucine-rich-repeat protein 5 (NALP5), putative potassium channel regulator (KCNRG), and glutamic acid decarboxylase 65 (GAD65) were assayed using radioligand binding assays, as described by Ekwall et al. (24). Anti-acetylcholine receptor Abs were measured using a commercially available radioimmunoassay kit (IBL International, Hamburg, Germany).

Mutational analysis of AIRE mutations

DNA was extracted from patient or control EDTA blood samples in accordance with standard protocols. The 14 exons of AIRE were amplified by polymerase chain reaction and sequenced (5). Copy number analysis was performed using a duplex TaqMan real-time polymerase chain reaction assay (25) using primers and probes for AIRE exon 1 and 9.

Statistical analysis

The data were analyzed using Statistica (StatSoft/TIBCO Software Inc., Carlsbad, CA), version 10, statistical software. Comparisons of categorical variables were performed using the χ² test. Correlations between the components of the disease and autoantibodies titer were calculated using the Spearman r test. A P value of <0.05 was considered to indicate statistical significance.

Ethical approval

The local ethics committee at Endocrinology Research Center of Russia approved the study, and all patients or their parents or guardians provided written informed consent.

Results

Spectrum of clinical manifestations

Altogether, 112 individuals from 102 families (63 females) with an age range of 2.7 to 44.6 years and a mean age of 19.4 years fulfilled the diagnostic criteria of APS-1. The mean age at diagnosis was 9 years. Some minor clinical components (e.g., angular erythema, periodical fever with rash, pigmented retinitis, autoimmune hepatitis, ptosis) manifested before the diagnosis was established (Fig. 1) and were retrospectively recognized as disease components. Seven patients were lost to follow-up during the study period of 4 years and nine died. The three major components (HPT, CMC, AI) were present in 78%, 75%, and 67%, respectively. POI was diagnosed in 48% of the female patients aged >15 years. Alopecia, enamel hypoplasia, and malabsorption were found in approximately one third of patients and were the most frequent minor components (26, 27) (Table 1). Overall, 23 different components were identified, and each patient had a mean of 5 components (range, 1 to 11).

Figure 1.

Mean age at manifestation of all components of APS-1. The mean age at diagnosis of APS-1 is indicated (9 years) to show that some minor clinical manifestations are present before the diagnosis (light gray bars).

Table 1.

Prevalence of Disease Components in 112 Patients With APS-1^a

Component	% (n)	Females/Males	Age at Onset, y (Range)
Main components
HPT	78 (88)	56/32	7.0 (1.5–16.3)
Mucocutaneous candidiasis	75 (84)	47/37	4.8 (0.1–21.5)
Adrenal failure	67 (75)	39/36	9.9 (3.3–28.1)
Other endocrine components
Primary ovarian failure	48b	16/0	15.1 (13–44.2)
Hypothyroidism/hyperthyroidism	13 (15)	11/4	9.4 (1.5–16.5)
Type 1 DM	9 (12)	5/7	12.1 (1.7–21.2)
Ectodermal components
Enamel hypoplasia	28 (32)	20/30	15.6 (5–29.0)
Alopecia	34 (38)	24/14	10.5 (4.3–23.0)
Vitiligo and/or hair depigmentation	9 (11)	5/5	12.1 (6.3–19.1)
Gastrointestinal components
Malabsorption/chronic diarrhea or obstipation	25 (28)	20/9	11.1 (2.0–40.0)
Autoimmune hepatitis	11 (12)	7/5	4.5 (1.5–13.5)
Pernicious anemia	8 (10)	3/7	11.4 (5.0–25.0)
Other/rare components
Ptosis	8 (9)	5/4	6.9 (0–8.0)
Retinitis pigmentosa	3.6 (4)	2/2	1.7 (0.2–25)
Chronic blepharitis, dry eyes	3.6 (4)	2/2	11.0 (4–29)
Angular erythema or periodical fever with rash	4.5 (5)	2/2	3 (0.7–5)
Metaphyseal dysplasia	3.6 (4)	2/2	10.5 (4–17)
Asplenism	2 (2)	2/0	22 (16–40)
Pure red cell aplasiac	1 (1)	1/0	21
Renal failure	1 (1)	0/1	14
CA	1 (1)	0/1	20.8
Thyrotropinomac	1 (1)	0/1	11.1

Abbreviation: CA, cerebellar ataxia.

^a

Demographic data: female/male ratio, 1.1 (64/58); family cases, 10; age, 19.4 years (range, 2.7 to 44.6); deceased, 9 patients; classic triad, 46 patients; and classic dyad, 92 patients.

^b

Among female patients aged >15 years.

^c

Reported previously (26, 27).

The classic dyad was present in 92 patients and the triad in 46; 16 patients remained oligosymptomatic with only one major component (Table 2). Of these 16 patients, 6 had CMC, 7 had HPT, and 2 had AI. Also, two brothers, who were homozygous for c.769C>T (p.R257*), had CMC at 2 and 3 years of age, with alopecia areata diagnosed at 14 and 13 years of age, respectively. At 34 and 39 years of age, they had not developed any other APS-1 component. Both were well-educated, social, married men and had healthy children. They lacked any of the organ-specific autoantibodies tested for; however, both displayed Abs against INF-ω, INF-α2, and IL-22, and one had IL-17F autoantibodies.

Table 2.

Clinical Findings and AIRE Mutations in Patients With APS-1 With One Major Component

Major Component	Age of Onset, y	Age at Last Examination, y	Other Components (Age of Onset, y)	AIRE Mutation	INF-ω Abs
CMCa	2	31	Alopecia (14)	p.R257*/p.R257*	Positive
CMCa	3	36	Alopecia (13)	p.R257*/p.R257*	Positive
CMC	0.8	18	Alopecia (14)	p.R257*/NF	Positive
CMC	7	8	Autoimmune hepatitis (5), fever with rash and arthralgia (5), enamel hypoplasia (7)	p.R257*/p.Q94*	Positive
CMC	1.5	6	Fever with rash and arthralgia (3), malabsorption with diarrhea (3)	p.R257*/p.R257*	Positive
CMC	0.3	2.5	Autoimmune annular erythema (1.5)	p.R257*/p.R257*	Positive
HPT	8	12	None	p.R257*/p.R257*	Positive
HPT	2.5	10	None	p.R257*/NF	Positive
HPT	2.0	5	None	p.R257*/p.R257*	Positive
HPT	8	10	None	p.R257*/p.R257*	ND
HPT	12	14	None	ND	Positive
HPT	7	10	Malabsorption (7)	p.R257*/p.R257*	Positive
HPT	12.5	20	Enamel hypoplasia	p.R257*/p.R471C	Negative
HPT	3	4	None	p.C302Y/NF	Positive
AI	12	15	Hearing loss (3), pernicious anemia (5), vitiligo (9), enamel hypoplasia, ptosis, glucose intolerance (15)	p.Leu323sefs*51/p.P326L	Positive
AI	5	8	None	ND	Positive

Abbreviation: ND, not determined.

^a

Siblings.

Chronic mucocutaneous candidiasis

CMC was present in 84 of 112 patients (75%), with a median age at the first episode of 4.8 years (range, 0.1 to 21.5). The clinical course of CMC varied from insignificant (affecting one or two nails in three patients) to severe antifungal drug-resistant cases with recurrent stomatitis and gingivitis. Candidiasis of the skin (forearm, foot) was seen in two cases (Fig. 2). We observed long periods of remission (≤10 years) in some patients. Esophageal candidiasis was found in 12 cases by endoscopy. In all 12 patients, candidiasis was evident at other locations such as the nails or skin. In five patients (aged 25 to 35 years), clinically important symptoms of dysphagia were present, and two had undergone balloon dilatation because of stricture. Squamous cell carcinoma or other malignancies were not seen.

Figure 2.

Variable phenotype and rare manifestations in APS-1. (A) MRI scan of the brain of a patient with cerebellar ataxia. Clinical photographs showing (B) enamel hypoplasia; candidiasis of the (C) skin, (D) nails, and (E) oral mucosa; (F) angular erythema; (G) retinitis pigmentosa; (H) ptosis and vitiligo; and (I–L) metaphyseal dysplasia.

Hypoparathyroidism

HPT was diagnosed in 78% of all patients (56 females and 32 males) at a median age of 7.0 years (range, 1.5 to 16.3). We found no difference in the age of onset between the males and females (age, 5.25 years; range, 1.5 to 16, and 6.25 years; range, 1.6 to 16, respectively). All were treated with alfacalcidol or calcitriol and calcium. One patient had had malabsorption since 20 years of age, which had led to clinically important hypocalcemia, despite alfacalcidol doses of ≤20 µg/d. Gastroscopy showed atrophic autoimmune gastritis and autoimmune enteritis evidenced by duodenal atrophy and a lack of enterochromaffin cells. The patient had anti-tryptophan hydroxylase Abs but no transglutaminase autoantibodies, arguing against the presence of celiac disease. Recombinant PTH 1-34 was then given, with some effect, but only after continuous administration (~40 µg/d) did the calcium level normalize without additional vitamin D and calcium supplementation. Urinary calcium excretion measured during PTH therapy at 8 and 12 months was in the normal range (2.8 mmol/d).

Adrenal insufficiency

AI was diagnosed in 67% of patients (39 females and 36 males) at a median age of 9.9 years (range, 3.3 to 28.1). Most of these patients received replacement therapy with hydrocortisone and fludrocortisone; a few patients took prednisone. Most of the patients experienced one or more adrenal crisis during the follow-up period. One patient died of an adrenal crisis.

Alopecia

Alopecia was the most frequent minor component, present in 38 of 112 patients (34%; 24 females). The median age at the onset of alopecia was 10.5 years (range, 4.3 to 23.0). Most patients had severe alopecia. Hair loss manifested with one patch of hair loss; however, it progressed to the universal form in 24 patients. Ten patients had one or several patches of hair loss with preserved eyebrows and eyelashes. Two patients had partial loss of their eyebrows and eyelashes and one patch without progression during long-term follow-up. We observed spontaneous resolution of the patches in four patients with mild alopecia areata and partial regrowth in six patients with alopecia universalis. This periodic hair loss and regrowth illustrates the “wax and wane” course of alopecia. In one patient, regrowth of the hair was secondary to immunosuppressive therapy for red cell aplasia. Alopecia was a substantial complaint expressed, especially by the women.

Vitiligo

Of the 112 patients, 11 had vitiligo (10%; six females; Fig. 2). The median age at onset was 12.1 years (range, 6.3 to 19.1). Three of the vitiligo patients experienced depigmentation of their hair, eyebrows, and eyelashes. Three patients had areas of depigmentation on both the face and the body; the others had depigmentation areas only on the body. Vitiligo was the first component in one patient with AIRE mutations p.R257* and p.A58V.

Malabsorption

Gastrointestinal components included chronic diarrhea or obstipation (which can lead to severe malabsorption and treatment difficulties), candida esophagitis with evidence of esophagus stricture and dysphagia, chronic atrophic gastritis, pernicious anemia, and autoimmune hepatitis. Every fourth patient (28 of 112) complained of chronic diarrhea and/or obstipation, which significantly decreased their quality of life and interfered with replacement therapy. In some patients, it was the first component of the disease, presenting alone or together with candidiasis many years before HPT and AI. In one patient homozygous for p.R257*, APS-1 was suspected because of the combination of chronic diarrhea, onychomycosis, and fever with rash at 3 years of age. In most patients, the significance of chronic diarrhea as a first component was not considered by pediatricians and was only later considered as an APS-1 component in retrospect. Chronic diarrhea interchanged with chronic obstipation and periods of normal gastrointestinal motility, possibly reflecting autoimmune attacks and relapses. The serum calcium levels in patients with constipation were checked and were not a major cause of malabsorption. When malabsorption was severe, it interfered with drug absorption, and intravenous replacement of calcium and potassium was required for many patients. One female patient with severe malabsorption and HPT required daily intravenous calcium injections (described earlier).

Autoimmune hepatitis

Autoimmune hepatitis (AH) was diagnosed in 12 patients (11.0%; seven females). The age of onset of AH ranged from 1.5 to 13.5 years (mean, 4.5). The clinical presentation at onset varied from severe in eight patients (jaundice, hepatomegaly, splenomegaly, arthralgia, cytopenia) to mild (fatigue, mild hepatomegaly) or asymptomatic in three patients, who had chronic elevation of transaminases on annual testing. Liver biopsy findings were available for six patients. In five patients, histological signs of cirrhosis were seen, and in one patient, a 5-year-old girl, mild histological activity (Knodell score, 2 to 5) and moderate fibrosis (Desmet score, 2) were present. Autoantibodies against CYP1A2 were present in 10 of 11 patients with AH with available sera compared with 25 of 76 without hepatitis (P < 0.05). All six patients with biopsy-verified AH had positive findings. Autoantibodies against AADC were found in 10 of the 11 patients with AH compared with 65 of 79 patients without AH (P = 0.065). Of the biopsy-verified patients, five of six had positive findings.

Seven of 12 patients were treated with prednisolone and/or azathioprine. One woman was weaned off immunosuppressive therapy after 13 years of treatment at 22 years of age. A boy with AH since 2 years of age was able to discontinue prednisolone 15 years later; however, subsequent elevation of liver enzymes led to renewed prednisolone treatment.

Ocular manifestations

Ocular components included dry eyes, keratitis, chronic blepharitis, loss of the eyelashes, and ptosis (Fig. 2). None of the nine patients with ptosis had Abs to anti-acetylcholine receptor. HPT was present in six of the nine patients with ptosis. The mean age of diagnosis was 8 years (range, 1 to 15). The severity of ptosis varied but did not lead to surgical correction in any patient, and we did not see progression of ptosis during the follow-up period. Ptosis had not resolved in any of the patients at the last follow-up examination and also was not congenital in any patient. The mean observation period since the onset of ptosis was 8 years (range, 3 to 31). Pigmented retinal dystrophy was identified in four patients; the cases of three patients were described previously (4). We found acquired retinal dystrophy in an additional 26-year-old female patient (Fig. 2). Retinal dystrophy became apparent after cataract extraction and supports the idea that retinopathy in APS-1 is acquired owing to autoimmune mechanisms (patient 35; Supplemental Table 1).

Rare components

Metaphyseal dysplasia was observed in four patients (Fig. 2), with the severe case of one patient described previously (4). A female patient was diagnosed with autoimmune pure red cell aplasia at the age of 22 years (26). She initially responded to mycophenolate mofetil therapy but then developed a relapse and died of sepsis at 26 years of age despite renewal of cytostatic treatment. Cerebellar ataxia was diagnosed in one patient homozygous for p.R257* with “classic” disease development, including HPT since he was 8 years old, hypothyroidism since 11 years, CMC since 13 years, and AI since he was 16 years old. At age, 20 diplopia and progressive gait ataxia developed. Magnetic resonance imaging (MRI) revealed an irregular enhanced mass (12 × 15 mm) in the left cerebellar hemisphere (Fig. 2). Partial surgical resection was performed. Histological and immunohistochemistry analyses could not distinguish between an atypical lymphoproliferative syndrome and inflammatory pseudotumor. The test results for cytomegalovirus, human herpes virus, and Epstein-Barr virus in the serum and cerebrovascular fluid were negative. Vitamin B₁₂ deficiency was excluded. He had anti-GAD Abs (196 U/mL) but not Abs against cerebellar Purkinje cells. The bone marrow aspirate findings were normal. We concluded that autoimmune encephalitis with pseudotumor was the most probable diagnosis. Chemotherapy with rituximab, cyclophosphamide, and intravenous immunoglobulins was without effect. Brain MRI every 6 months revealed a stable situation for the next 3 years; however, he developed additional manifestations such as enuresis and echolalia and became completely dependent on help. The cases with rare components are summarized in Supplemental Table 2.

Disease course

The first manifestation was one of the three major components in 100 of the 112 patients (89%). In 58 patients (52%), the first manifestation was CMC, in 31 (28%) it was HPT, and in 11 (9%) it was AI. Other first components were pernicious anemia (n = 2), and retinitis pigmentosa leading to blindness (n = 2); in one of these patients, ptosis and ophthalmoplegia developed in the first year of life, leading initially to the erroneous diagnosis of Kerns-Sayer syndrome. AH was the first component in one patient and manifested as early as 1.5 years of age. Arthralgia and fever with rash were the first components in another patient, followed by AH and CMC. Finally, vitiligo and DM was the first component in one patient each. Disease progression varied significantly among the patients. The detailed sequential development of components is shown in Supplemental Fig. 1 and Supplemental Tables 1 and 3. The order of CMC-HPT-AI was frequently observed, but the disease course was extremely varied.

Complications or coincidences?

We also found patients with cataracts (n = 22), renal stones (n = 3), gallbladder stones (n = 5), calcification of the brain (n = 27), and nephrocalcinosis (n = 2). All but one of these patients had HPT, one of the patients with gallbladder stones. Epilepsy was diagnosed in one patient. He experienced generalized seizures despite normal calcium and glucose levels. Brain imaging revealed multiple small-size calcifications in the lateral ventricles and epiphysis. However, none of the other patients with brain calcifications and normal calcium levels experienced seizures. Multiple venous thromboses were seen in one patient with asplenia who required anticoagulant therapy. Three patients developed pulmonary disorders such as frequent episodes of bronchitis, more than two cases of pneumonia annually, and asthma. One of these patients died of pneumonia and cardiac-respiratory failure at 9 years of age. Signs of bronchiolitis or interstitial lung disease were not observed. None of these patients had KCNRG Abs (28).

Fertility and pregnancy

Seven women gave birth to nine healthy children (maternal age at pregnancy range, 19 to 34 years; median, 21). All the pregnancies were normal without severe complications. The female patient who gave birth to a child at 34 years of age developed menopause at 44 years of age. POI was diagnosed in 48% of the females aged >15 years. Substantial elevation of luteinizing hormone and follicle-stimulating hormone were the first markers of POI and were seen in patients with normal menstrual cycles. Amenorrhea developed within the next 6 months to 2 years after the initial elevation of gonadotropins. Anti-Müllerian hormone was less than the reference range in all female patients with POI.

Mortality

Nine patients (8%) died during the follow-up period (Supplemental Table 4), two of unknown causes. The age at death ranged from 9 to 32 years of age and the disease duration from 7 to 27 years.

Autoantibodies

Of the 112 patients, 93 were tested for Abs against INF-ω and 82 were tested for Abs against INF-α2. INF-ω Abs were positive in 92 of 93 patients (99%; sensitivity, 99%), and INF-α2 Abs were positive in 75 of 82 patients (92%; sensitivity, 91.4%). Seven patients had negative findings for INF-α2 Abs; however, the finding was borderline in two patients. All seven patients had HPT, and in six patients, HPT was the first manifestation. Only one APS-1 patient was negative for INF-ω Abs. He was also negative for INF-α2, IL-22, and IL17F Abs on two different samples taken 5 years apart. NALP5 Abs, GAD Abs, and SCC Abs were also negative, and 21-OH Abs and AADC Abs were elevated. HPT had been diagnosed at 14 years of age. At the latest examination at 22 years old, he did not have any other components except for enamel hypoplasia. He was compound heterozygous for p.R257* and p.R471C. Both parents were healthy; his father was heterozygous for p.R257* and the mother for p.R471C.

IL-22 Abs were detected in 95% (73 of 77) of the patients with CMC and in 80% (12 of 15) of patients without CMC [P < 0.001; specificity, 20%; sensitivity, 94.8%; positive predictive value (PPV), 85.9%; negative predictive value (NPV), 42.9%]. A statistically significant association with CMC was observed (P < 0.05). Of the 77 patients with and the 16 patients without CMC, 32 (41.6%) and 5 (31.3%) had Abs to IL-17F, respectively. These Abs were not associated with CMC (P > 0.05).

Abs against 21OH and AADC were investigated in 93 patients, Abs against NALP5 in 92, Abs against CYP1A1 in 90, and Abs against SCC in 89 patients (Fig. 3). In general, an association was found between the presence of Abs and disease manifestations in the organ in which the autoantigen is expressed. For example, 21OH Abs correlated with AI (P < 0.001; specificity, 61.5%; sensitivity, 83.6%; PPV, 84.9%; NPV 59.3%). In addition, the correlation between SCC Abs and POI was significant (P < 0.05; specificity, 52.6%; sensitivity, 91.7%; PPV, 55%; NPV, 90.9%). The correlation between Abs to SCC and AI was also statistically significant (P < 0.001) and similar to that of 21OH Abs (specificity, 80%; sensitivity, 70.3%; PPV, 90%; NPV, 51.3%). Finally, the correlation between Abs to CYP1A2 and AH was significant (P < 0.05). However, no correlation between Abs to GAD and DM or malabsorption was found.

Figure 3.

Organ-specific antibodies in patients with APS-1. Y-axis shows Ab indexes. Dashed lines represent the cutoff levels; solid lines, the median levels. (A) ABs against 21OH in APS-1 patients with (AI+) and without AI (AI−). (B) ABs against SCC in AI+ and AI− APS-1 patients. (C) ABs against SCC in APS-1 patients with (POI+) and without POI (POI−). (D) ABs against AADC in APS-1 patients with (AH+) and without AH (AH−). (E) ABs against CYP1A2 in AH+ and AH− APS-1 patients. (F) ABs against NALP5 in APS-1 patients with (HPT+) and without HPT (HPT−). (G) ABs against IL-22 in APS-1 patients with (CMC+) and without CMC (CMC−).

AIRE mutations

DNA from 106 patients was available for AIRE sequencing. Altogether, 19 different AIRE mutations were identified, 9 of which were previously unknown (Table 3). The Finnish major mutation p.R257* was the most frequent, found in 153 of 212 alleles (72%). Of these 106 patients, 63 were homozygous for p.R257*, and 30 were heterozygous. The mutation p.A58V was the second most frequently found of the nine alleles (4.2%). Two patients were homozygous and five were compound heterozygous with p.R257*. p.A58V was not confined to any particular area of Russia. p.Т16M was found in six alleles and p.Leu323Serfs*51 in four. p.A399P was found in one patient of Belarussian origin. We did not find the second mutation in seven patients. However, they all had at least one major manifestation and all those tested (6 of 7) had IFNω Abs. Copy number analysis performed in six of the seven patients did not reveal deletions in AIRE. c.821delG was found in one patient with the diagnostic dyad combined with previously described mutation p.T16M. A heterozygous p.Q94* mutation combined with p.R257* was found in a 7-year-old female patient with AH, CMC, and periodic fever with rash without any endocrine disorders at her last examination.

Table 3.

Previously Unknown Mutations in AIRE Among Russian Patients With APS-1

Alleles, n	Nucleotide Changea	Amino Acid Change	Domain	Exon
1	c.38T>C	p.L13P	CARD	1
7	c.173C>T	p.A58V	CARD	2
1	c.280C>T	p.Q94*	CARD	2
1	c.554C>G	p.S185*	SAND	5
1	c.661A>T	p.K221*	SAND	6
1	c.821delG	p.Gly274Alafs*104	SAND	7
1	c.1195G>C	p.A399P	Between PHD1 and PHD2	10
1	c.1302C>A	p.C434*	PHD2	11
1	c.1497del	p.A500Profs*21	C-terminal	12

^a

NM_000383.3.

Discussion

APECED is a very rare inherited organ-specific autoimmune disease. Approximately 400 cases have been reported worldwide since the first patient was described in 1929 by Thorpe and Handley (29). To the best of our knowledge, we have described the largest cohort of patients with clinically characterized APS-1 to date. Long-term follow-up data allowed us to expand our knowledge of clinical phenotypes, including the description of additional disease components and mutations in AIRE.

Reports from various European countries have estimated the prevalence of APS-1 at ~80,000 to 130,000, with the exceptions of Finland and Sardinia, where founder mutations are present. A similar calculation for Russia gave a prevalence of ~1/1 million, pointing to the likely possibility that only ~1 in 10 patients have been identified and APS-1 diagnosed. Lack of knowledge about APS-1 by general health care practitioners, the long intervals between the appearance of additional components, the unavailability of diagnostic tests, and because some patients present with milder disease as adults (2, 5) could, at least in part, explain the low prevalence.

The spectrum of clinical manifestations in our patients was broad, ranging from 1 to 11 components. In addition to the previously described ocular manifestations (keratitis, blepharitis, retinitis), we observed ptosis in nine patients. Ptosis has not been reported earlier as an APS-1 component, but it is a typical component of other autoimmune disorders associated with the thymus, in particular, myasthenia gravis (30). The muscle weakness typical of myasthenia gravis was not diagnosed in any of our patients, and none of the patients tested had acetylcholine receptor Abs. We speculate that the ptosis in our patients could have also been related to an unknown autoimmune mechanism.

Cerebellar ataxia (CA) was present in one of our patients. CA was previously reported in only one case of APS-1 (31); however, the association of CA with other types of polyendocrine syndromes and type 1 DM is well known and suggests an autoimmune pathogenesis for this manifestation (32–34). High levels of Abs to GAD were found in our patient, similar the levels found in other patients with CA, stiff-person syndrome, and type 1 DM (35). We also found an enhanced mass in the cerebellum on brain MRI, in contrast to the usual cerebellar atrophy seen in patients with autoimmune CA. Histologic examination revealed heavy lymphocyte infiltration that could have represented autoimmune inflammation.

Abs against type I IFNs in APECED patients were discovered in 2006 (6) and were recently suggested as an additional diagnostic criterion for APECED (7). The diagnostic value has since been demonstrated in several studies (5, 23, 36) and was also confirmed in the present study. Abs against IFN-ω were present in 99% and against IFN-α in 92% of all investigated patients; however, the role of INFs in APS-1 pathogenesis is still poorly understood. Some cases without INF Abs have been reported (6), although we found only one patient (patient 93; Supplemental Table 1). That patient had HPT and enamel dysplasia as his only manifestations (from age 12) and only revealed a low positive index of AADC, CYP1A2, and 21OH Abs. This patient had p.R257* and p.R471С, with the latter sequence variant of unknown pathogenicity. Using thymic 4D6 cells to test the effect of various mutations on AIRE-regulated transcription, p.R471C did not show any inhibitory effect (13). However, we could not rule out other mutations in noncoding regions. A similar female Italian case with isolated AI, homozygous for p.R471C and without Abs to INFs developed chronic vaginal candidiasis during the follow-up period (37). Taken together, the combination of p.R257* and p.R471С could explain the mild phenotype. Further follow-up is needed to clarify the pathogenicity of p.R471C.

We also confirmed earlier findings of a high frequency of IL-22 Abs and an association with CMC (38–40). In addition, we found these Abs in patients without APS-1, including one patient with AI secondary to a DAX1 mutation, two healthy heterozygous p.R257* carriers, and one female patient with autoimmune AI (data not shown). All the patients were investigated for CMC, other clinical components of APS-1, and other Abs, with negative results. To the best of our knowledge, IL-22 Abs are specific for APECED and have not been reported in patients without APECED or healthy individuals (39, 40). We did not find a strong association between IL-17F Abs and CMC, although it has been reported by others (38).

The presence of 21OH Abs correlated with and predicted for AI in our patients, which could be crucial for patient survival. Anti-CYP1A2 Abs correlated with AH and were positive in most of the affected individuals. We found a relatively high percentage of positive results among those without AH; however, in many cases, the index was only slightly elevated. Autoantibodies against AADC had a similar sensitivity but lower specificity. NALP5 Abs were also very informative, with a clear correlation to HPT, confirming earlier results (41). In contrast to our expectations, no correlation was found between NALP5 Abs and POI (42). Also, GAD Abs did not correlate with DM or malabsorption, such as was shown by others (8).

AIRE mutations were found in all the patients; seven had only one mutation. p.R257* was the most frequent in our Russian population, with a frequency similar to that in Finland and Poland (10, 43). In addition, we found nine unknown mutations. The most frequent of these, p.A58V, is located in the CARD domain in the same area as most missense mutations in AIRE. Even if the alanine to valine change is not a major shift, it was predicted to be pathogenic by PolyPhen testing. Another CARD mutation (p.L13P) was found in one allele. Furthermore, a number of additional nonsense mutations were identified, including p.Q94*, p.S185*, p.L221*, and p.C434*; all clearly pathogenic. Finally, two small deletions leading to early truncations were found in exons 7 and 12. These results underpin previous findings that the Finnish major mutation is the dominant mutation in Eastern European populations (Slovenia, Poland). No apparent genotype to phenotype correlations were discovered, in line with previous reports.

In conclusion, systematic studies of the largest APS-1 cohort from Russia revealed large phenotypic variability, although p.R257* was by far the most common APS-1 mutation. Ptosis was found in several patients and could be considered an APS-1 manifestation. APS-1 is underdiagnosed, difficult to manage, and requires a multidisciplinary medical team to provide appropriate care. Assay of IFN-ω Abs is a robust diagnostic marker. We found nine additional AIRE mutations, adding to the large spectrum of mutations previously described.

Abbreviations:

 
• 21OH

  21-hydroxylase

 
• Ab

  antibody

 
• AADC

  aromatic l-amino acid decarboxylase

 
• AH

  autoimmune hepatitis

 
• AI

  adrenal insufficiency

 
• AIRE

  autoimmune regulator

 
• APECED

  autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy

 
• APS-1

  autoimmune polyendocrine syndrome type 1

 
• CA

  cerebellar ataxia

 
• CMC

  chronic mucocutaneous candidiasis

 
• DM

  diabetes mellitus

 
• GAD

  glutamic acid decarboxylase

 
• HPT

  hypoparathyroidism

 
• IFN

  interferon

 
• IL

  interleukin

 
• MRI

  magnetic resonance imaging

 
• NALP5

  NACHT leucine-rich-repeat protein 5

 
• NPV

  negative predictive value

 
• POI

  primary ovarian insufficiency

 
• PPV

  positive predictive value

 
• PTH

  parathyroid hormone

 
• SCC

  side-chain cleavage enzyme.

Acknowledgments

We thank all the patients and their families for their participation, and we thank the physicians who provided the clinical data and blood samples.

This study was supported by the Research Council of Norway, Novonordisk Foundation, Regional Health Authorities of Western Norway, the CAF Foundation for Philanthropy Support and Development, and the Alfa-Endo Program (Russia).

Author contributions: E.M.O., L.S.S., M.A.K., I.I.D., and V.A.P. provided samples and clinical data for the patients. E.Y.Z., O.N.I., B.E.O., A.S.B.W., and P.M.K. performed AIRE sequencing and copy number analyses. B.E.O., A.S.B.W., L.B., and O.K. provided autoantibody analyses. E.M.O. and E.S.H. planned and coordinated the study and wrote the manuscript. All the authors discussed the results and commented on the text.

Disclosure Summary: The authors have nothing to disclose.