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Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Syndrome: Time to Review Diagnostic Criteria?

Pediatric Department and Institute of Molecular Medicine A. Nocivelli (C.M., S.G.), University of Brescia, 25123 Brescia, Italy; and Pediatric Department, Regional Hospital of Bolzano (G.R.), Bolzano, Italy

Address all correspondence and requests for reprints to: Dr. Fabio Buzi, Clinica Pediatrica dell’Università di Brescia, Az. Osp. Spedali Civili, P.le Spedali Civili 1, 25123 Brescia, Italy. E-mail: mazzolar{at}master.cci.unibs.it.

	Abstract

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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an autosomal-recessive syndrome defined by two of the following conditions: chronic mucocutaneous candidiasis, hypoparathyroidism, or Addison’s disease. Other autoimmune conditions may be associated, such as hypothyroidism, hypogonadism, insulin-dependent diabetes mellitus, chronic active hepatitis, pernicious anemia, vitiligo, alopecia, biliary cirrhosis, and ectodermal dysplasia. APECED is caused by mutations in the autoimmune regulator gene, mapping to 21q22.3.

We report on three patients whose clinical and molecular features challenge the currently used diagnostic criteria for APECED. AR presented at 15 yr of age with a history of recurrent infections and mucocutaneous candidiasis. He is now 21 yr old, and no other signs or symptoms of APECED have appeared to date.

DR presented at 7 yr of age with hypocalcemia and a prolonged Q-T interval on the electrocardiogram. He also had minor facial dysmorphisms and mild mental retardation. Serum calcium levels were low, PTH levels were undetectable, and hypoparathyroidism was therefore diagnosed. All other biochemical, immunological, and endocrinological tests were normal. DR is now 8 yr old with no other signs or symptoms of APECED.

ST presented at 14 yr of age for alopecia aerata and pitted nail dystrophy and goiter. Thyroid function was normal in the presence of thyroid-specific antibodies. No other signs or symptoms of APECED have appeared to date.

Genetic analysis revealed a typical mutation (R257X) on a single allele in both AP and DR; in ST, heterozygosity for a novel mutation (V484M) involving one of the zinc fingers of the plant homeodomain of the protein was found. The finding of a typical APECED mutation in two patients presenting with one isolated major clinical APECED feature and of a novel mutation in a patient presenting with atypical features of APECED onset suggests that the time might have come for updating the diagnostic criteria of this syndrome.

	Introduction

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AUTOIMMUNE polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM 240300) is an autosomal-recessive disorder affecting many tissues, mainly endocrine glands (1). It is also known as autoimmune polyglandular syndrome type I (APS I) (2); to confirm this syndrome, at least two of the following conditions must be present: chronic mucocutaneous candidiasis, hypoparathyroidism, or Addison’s disease. Candidiasis is normally the first manifestation of the disease, usually appearing before the age of 5 yr. It is usually followed by hypoparathyroidism (more commonly before the age of 10 yr) and thereafter by adrenal insufficiency (before the age of 15 yr) (3). Other endocrine and nonendocrine less frequently associated conditions include thyroid autoimmune disease (autoimmune thyroiditis and Graves’ disease), chronic active hepatitis, malabsorption, juvenile-onset pernicious anemia, alopecia, and primary hypogonadism (3). The most characteristic ectodermal manifestations in APECED are dental enamel hypoplasia, pitted nail dystrophy, and alopecia; keratopathy, vitiligo, and calcifications of the tympanic membranes may also be present (4). The occurrence in affected subjects of tissue-specific autoantibodies (Abs) underlines the role of autoimmunity in the pathogenesis of APECED (4). APECED is caused by mutations in the autoimmune regulator (AIRE) gene, which maps to 21q22.3 (5, 6). Although this condition is rare, it shows segregation in some populations, where its prevalence is rather high: Finns, 1:25,000 (7); Iranian Jews, 1:9,000 (8); and Sardinians, 1:14,500 (9). It is also relatively common in Northern Italy (3) and Sweden (10). Many different mutations have been described in the AIRE gene (5, 6, 9, 11, 12, 13, 14, 15, 16, 17), including point mutations, insertions, and deletions spread throughout the whole coding region of the gene. Mutations affecting splicing sites have also been reported (14, 15). In a few cases only one mutant allele of the AIRE gene has been reported in typical APECED patients, suggesting that the second mutation might be located in the regulatory regions of the gene (4). We report here two cases in which a typical AIRE mutation was found on a single allele; both presented with only one clinical feature of the syndrome. A third subject had more than one sign of APS I at presentation, although not the characteristic ones, and showed a novel mutation in heterozygosity. This, in our opinion, poses some questions about the definition of this syndrome.

	Subjects and Methods

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Subject 1

AR presented at 15 yr of age with a history of recurrent infections and mucocutaneous candidiasis. All endocrine tests were normal; specific Ab were undetectable. AR is now 21 yr old, and no other signs or symptoms of APECED have appeared.

Subject 2

DR presented at 7 yr of age with hypocalcemia and prolonged Q-T interval on the electrocardiogram. He also had minor facial dysmorphisms and mild mental retardation. Chromosomal analysis showed a normal male karyotype. Serum calcium levels were 1.4 mmol/liter (normal laboratory range, 2.2–2.6 mmol/liter); serum phosphate levels were 4.1 mmol/liter (normal laboratory range, 1.0–1.4 mmol/liter). PTH was undetectable; an x-ray of the wrist showed a decreased bone mineral calcium content. However, he had no symptoms of hypocalcemia and had been suffering in the past from recurrent episodes of laryngitis, suggesting possible hypocalcemic spasms of the larynx. The patient was treated with calcium and vitamin D, with normalization of calcium and phosphate levels together with a gradual normalization of the Q-T interval. All other biochemical, immunological, and endocrine blood tests were normal. DR is now 8.6 yr, and no other signs or symptoms of APECED have appeared.

Subject 3

ST was seen at 14 yr of age for alopecia aerata, pitted nail dystrophy, and goiter. Sexual development was completed, and the girl had regular menses. Thyroid function was normal [TSH, 2.8 mU/liter (normal laboratory range, 0.2–3.5 mU/liter); free T₄, 9.2 pg/ml (normal laboratory range, 6–18 pg/ml); free T₃, 2.7 pg/ml (normal laboratory range, 2.2–3.5 pg/ml)] in the presence of thyroid-specific Ab [thyroglobulin Ab, 973 U/ml (normal laboratory range, <20 U/ml); thyroid peroxidase Ab, 1452 U/ml (normal laboratory range, <15 U/ml)]. Thyroid ultrasound scan showed a nodular image in the right thyroid lobe, with a total volume of 9.57 ml. Antiendomysium Ab were absent; PTH was in the normal range (28.6 pg/ml; normal laboratory range, 9–65 pg/ml), as was 25-hydroxyvitamin D₃ (13.4 pg/ml; normal laboratory range, 16–74 pg/ml). Routine biochemical and hematological findings were normal. Cortisol (0900 h) was 736 nmol/liter (normal laboratory range, 179–690 nmol/liter), and ACTH was 2.8 pmol/liter (normal laboratory range, 2.2–11.7 pmol/liter).

The patients and their parents were investigated for APECED, although the first two (AR and DR) had only one major feature of the syndrome, and the third (ST) had ectodermal dystrophy associated with thyroid autoimmunity, which is a rare presentation of this disorder. DNA analysis was carried out only in the mother of patient DR, because his father refused to be tested.

Genomic DNA was extracted from peripheral blood. All 14 exons of the AIRE gene were amplified with the use of primers located on the respective flanking introns (11) and were analyzed by direct sequencing using the ABI PRISM 310 sequencer (PE Applied Biosystems, Foster City, CA). The analysis included sequencing of the donor/acceptor sites of all of the introns. A putative promoter featuring a CAT box, a TATA box, and a GC box, localized immediately upstream of the first exon, and the polyadenylation signal (GenBank AB006684, TATAAA, position 16674) (18) were sequenced.

Informed consent for the analyses carried out was obtained from the subjects and their parents.

	Results

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Patients RD and AR were heterozygous for the mutation R257X; the second mutation was not identified, as previously reported in other cases (4). In patient DR’s mother no mutations were found (in his father the analysis was not carried out); in patient AR’s mother, who had no signs or symptoms of APECED, the same mutation R257X was found in heterozygosity, whereas no such a mutation was detected in his father.

Patient ST was heterozygous for a mutation leading to the substitution of amino acid valine at position 484 with methionine (V484M); her mother showed the same mutation in the absence of signs or symptoms of APECED; in her father no mutations were found. The mutation R257X is one of the most frequent mutations causing APECED. The alteration V484M has not been previously reported and resides in one of the zinc fingers of the plant homeodomain (PHD) of the protein (5, 19). The PHD domains are known to be involved in chromatin-mediated regulation of transcription. As this kind of mutation, to our knowledge, has not been previously described (20), the analysis was repeated on 50 DNA samples from normal unrelated donors, and no such a mutation was detected, thus making the possibility of V484M being a polymorphism very unlikely.

The sequences of the promoter regions and polyadenylation signal performed on the patients and normal donors DNA did not show any differences.

	Discussion

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In 1981 Neufeld et al. (21) described two types of the APS: APS I, represented by patients having at least two conditions of the triad including Addison’s disease, hypoparathyroidism, and chronic mucocutaneous candidiasis; and APS II, represented by patients affected with Addison’s disease and autoimmune thyroid disease and/or insulin-dependent diabetes mellitus (Schmidt’s syndrome), but no hypoparathyroidism or candidiasis. In both APS I and II other immune disorders may also be present. There is a third condition, APS III, that is represented by patients with autoimmune thyroid disease plus one or more other autoimmune disorders, but no Addison’s disease. APECED has its onset in childhood-adolescence; the order by which the three major conditions more frequently appear in this syndrome is: mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease (3).

APECED is an autosomal-recessive condition caused by mutations in the AIRE gene, mapping to 21q22.3 (5, 6). Many different mutations have been described in the AIRE gene that are believed to alter the function of the AIRE protein either by inducing the synthesis of a truncated product or by recognition of the nonsense transcript and degradation of the mRNA (4). The AIRE protein is thought to have a role in the regulation of transcription (11) and in the thymic process of induction of self-tolerance and its maintenance (4). The most frequent AIRE mutations described include a typical Finnish mutation (R257X) (5), the Sardinian mutation (R139X) (6), and the Jewish-Iranian mutation (Y85C) (7). However, these mutations are not restricted to certain ethnic groups; for example, the Finnish mutation is also the most frequently found in northern Italy (3).

We have described two patients showing a typical APECED presentation (mucocutaneous candidiasis and hypoparathyroidism, respectively) and a third patient referred for signs and symptoms not typically seen at the onset of this disease (nail dystrophy and thyroid autoimmunity). Molecular analysis revealed heterozygosity for a typical mutation (R257X) in the first two patients, whereas in the third patient (ST) a novel mutation was identified. This kind of mutation is unlikely to be a polymorphism, as it was not found in 100 unrelated chromosomes of healthy controls. Moreover, it resides in one of the zinc fingers of the AIRE PHD, which is known to be involved in chromatin-mediated regulation of transcription (19).

Mutations at one allele only have been reported in other patients with APECED (4); in these cases a further mutation at a regulatory site of the gene has been hypothesized, although no such defects have been described to date. The hypothesis of a second mutation on the other allele has been considered in our patients. However, no mutations have been identified in the coding regions and the flanking splice sites in the promoter or in the region encompassing the polyadenylation site. The possibility that mutations reside in other, as yet uncharacterized regions of the AIRE gene will require the availability of solid functional assays of the AIRE protein. Beside this, the occurrence of an incomplete clinical presentation of APECED together with typical APECED mutations at one allele only questions whether these patients should be considered affected by this condition. In our opinion the association of clinical signs or symptoms of APECED with a typical mutation, although in a heterozygous form, is unlikely to happen by chance. Furthermore, the onset of hypoparathyroidism in a 7-yr-old child is very likely to reflect autoimmunity, suggesting its possible association with an APECED syndrome. In such a case we think it appropriate to perform an analysis of the AIRE gene to diagnose APECED in the presence of a mutation. Furthermore, according to the original criteria the third patient would have been diagnosed as APS III; indeed, she had thyroid autoimmunity in the absence of Addison’s disease, associated with ectodermal dystrophy and alopecia aerata, which are minor findings in both APS I and II. Nevertheless, genetic analysis based upon the suspicion of APECED supported the diagnosis by detecting a mutation at a critical site of the AIRE gene. Very recently a case of AIRE mutation was described in a cohort of patients affected by sporadic Addison’s disease in the absence of other signs of the clinical triad (22). The researchers concluded that it is worth screening for AIRE mutations even in patients presenting with only a single disease of the APECED spectrum. Our data for three additional patients strongly support this view and suggest that the statement about the presence of at least two of the three typical features required to make a diagnosis of APECED needs to be revised in view of recent developments in the genetics of this condition.

	Footnotes

 
Abbreviations: Ab, Autoantibodies; AIRE, autoimmune regulator; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; APS, autoimmune polyglandular syndrome type I; PHD, plant homeodomain.

Received September 24, 2002.

Accepted March 27, 2003.

	References

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