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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 8 2922-2926
Copyright © 2000 by The Endocrine Society

Original Studies

Novel Mutations of the Autoimmune Regulator Gene in Two Siblings with Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy1

Tomohiro Ishii, Yoshimi Suzuki, Naoki Ando, Nobutake Matsuo and Tsutomu Ogata

Department of Pediatrics, Keio University School of Medicine (T.I., N.M., T.O.), Tokyo 160-8582; and Department of Pediatrics, Toyohashi Municipal Hospital (Y.S., M.A.), Toyohashi 441-8570, Japan

Address all correspondence and requests for reprints to: Tomohiro Ishii, M.D., Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: tomishii{at}mac.com


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is the first multiple autoimmune disease that has been shown to be caused by mutations of a single gene named autoimmune regulator (AIRE). Fourteen different mutations of the AIRE gene have been identified in 61 patients from 55 families with APECED. However, there has been no report documenting AIRE gene mutations in the Asian population.

We report on 2 siblings with variable manifestations of APECED who were born to a Japanese mother and a Korean father. The 11-yr-old girl had intractable thrush and ungual candidiasis, hypoparathyroidism, and occipital alopecia. The 9-yr-old boy had mild ungual candidiasis alone. Direct sequencing revealed novel frameshift mutations of the AIRE gene: an insertion of a cytosine at nucleotide 29635 at the exon 10 (29635insC), which should lead to a premature termination at the codon 371, producing a truncated protein missing the second plant homeodomain-type zinc finger motif and the third LXXLL motif, and a deletion of a guanine at nucleotide 33031 at the exon 13 (33031delG), which should result in a premature termination at the codon 520, yielding a truncated protein missing the third LXXLL motif. The mother was heterozygous for 29635insC, and the father was heterozygous for 33031delG. The frameshift mutations were undetected in 40 alleles of 20 Japanese control subjects. The results imply that the C-terminus of AIRE protein including the third LXXLL motif plays a critical role in the development of APECED, and that the phenotypic spectrum can vary between siblings with the same mutations.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
AUTOIMMUNE polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; MIM 240300), or autoimmune polyglandular syndrome type I, is a rare autosomal recessive disorder characterized by 1) chronic mucocutaneous candidiasis; 2) multiple endocrinopathies, including hypoparathyroidism, adrenocortical failure, hypergonadotropic hypogonadism, type I diabetes mellitus, panhypopituitarism, and hypothyroidism; and 3) ectodermal manifestations, such as enamel dysplasia, nail dystrophy, alopecia, vitiligo, and keratopathy (1, 2). The typical disease components are candidiasis, hypoparathyroidism, and adrenocortical failure, and APECED is clinically indicated in an individual with at least two of the three major features (1, 2). The first manifestation usually occurs in childhood, whereas other manifestations continue to appear until at least the fifth decade of life (1). Because of the slowly progressive nature of the disease, early and precise diagnosis is often difficult in patients with APECED.

APECED is the first multiple autoimmune disease that has been shown to be caused by mutations of a single gene, named autoimmune regulator (AIRE) (3, 4). The AIRE gene maps to 21q22.3 and consists of 14 exons (3, 4). It is expressed in immune-related organs such as thymus, lymph nodes, and fetal liver, implying that the AIRE gene plays a pivotal role in the immune function (3). The AIRE protein is predicted to function as a transcription factor, because the protein resides mainly in the nucleus (5) and contains two plant homeodomain-type zinc finger motifs in addition to a bipartite nuclear targeting signal and three transcriptional coactivator-binding LXXLL motifs (3, 4). To date, 14 different mutations of the AIRE gene have been identified in a total of 61 patients from 55 families with APECED: 14 Finnish (3, 4), 1 Swiss (3), 14 British (4, 6, 7), 10 Sardinian (8), 9 Northern Italian (6), 1 New Zealander (6), and 1 French-Canadian (9). The spectrum of AIRE gene mutations is not variable, and Arg139Stop, Arg257Stop and Cys322 frameshift mutations account for 86.4% of the Sardinian, 68.2% of the Finish, and 78.1% of the British mutant alleles, respectively. However, there has been no report documenting AIRE gene mutations in the Asian population. In this paper, we report on two Asian siblings with variable manifestations of APECED, in whom novel compound heterozygous mutations were identified in the AIRE gene.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Case 1

This girl was born at 40 weeks gestation after an uncomplicated pregnancy and delivery. At birth, her height was 50.0 cm (+0.69 SD), and weight was 2.92 kg (-0.47 SD). Her mother was Japanese, and her father was Korean. The parents had no features of APECED.

At 3 yr of age, she was referred to Toyohashi Municipal Hospital because of recurrent thrush since 1 month of age and ungual candidiasis since 1 yr of age (Fig. 1Go, A and B). The thrush was found over the buccal membrane, including the tongue. Ungual candidiasis was seen in most digits, except for the right fifth and the left second fingers and the left first toe. The throat and external ear canal cultures grew Candida albicans. The immunological data at that time are shown in Table 1Go (left part) together with the genotype of histocompatibility leukocyte antigen (HLA). Peripheral neutropil and lymphocyte counts were above the normal range, whereas CD4-positive and CD8-positive T cell counts were within the normal range. Serum IgG and IgA levels were elevated, but the IgM level was normal. The IgG antibodies were identified against Candida antigens. A delayed skin hypersensitivity test to Candida antigens was strongly positive, as was a lymphoblast stimulation test to Candida antigens. The Candida infections responded poorly to topical and systemic antifungal agents, although they did not progress to a systemic level.



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  		Figure 1. Chronic mucocutaneous candidiasis in the two siblings. Thrush (A) and ungual candidiasis (B) in case 1 at 11 yr of age. Ungual candidiasis (C) in case 2 at 9 yr of age.

 

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  		Table 1. Immunological and endocrinological data

 
At 8 yr of age, the patient had repeated syncopes and a generalized tonic-clonic seizure. Endocrine data at that time are shown in Table 1Go (right part). She had PTH deficiency, with hypocalcemia and hyperphosphatemia. Brain computed tomography revealed bilateral calcifications in the basal ganglia. She was successfully treated with oral 1{alpha}-hydroxyvitamin D (0.05 µg/kg·day).

At 10 yr of age, analysis of 24-h urinary steroid hormone metabolites by gas chromatography-mass spectrometry showed mildly decreased secretion of 17-hydroxycorticosteroids (1.0 mmol/mol creatinine; normal range, 1.5–3.0). However, plasma ACTH and serum cortisol were within the normal range, and a prolonged ACTH stimulation test (0.5 mg/day, im, for 3 days) revealed sufficient steroidogenic response with no evidence for steroidogenic enzyme deficiency (data not shown). Serum antiadrenal antigens were negative. She had no adrenal failure in the absence of treatment. She suffered from transient occipital alopecia for several months. There were no other features of ectodermal dystrophy, such as enamel hypoplasia, keratopathy, or vitiligo. The karyotype was 46,XX. On the basis of the above findings, she was diagnosed as having APECED with overt chronic mucocutaneous candidiasis and hypoparathyroidism.

On the latest examination at 11 years of age, her height was 144.1 cm (-0.34 SD), and weight was 32.6 kg (-0.84 SD). Her psychomotor development appeared age appropriate. Serum calcium was 1.9 mmol/L, inorganic phosphate was 2.0 mmol/L, cortisol was 146 nmol/L, aldosterone was 361 pmol/L, plasma ACTH was 3.3 pmol/L, and PRA was 4.4 µg/L/h. Reexamination of cellular immunity was refused by the patient and the parents.

Case 2

This boy, the younger brother of case 1, was born at 38 weeks gestation after an uncomplicated pregnancy and delivery. At birth, his height was 52.6 cm (+1.7 SD), and weight was 2.98 kg (-0.58 SD). At 7 yr of age, he presented with ungual candidiasis in the left second and third fingers (Fig. 1CGo). The candidiasis did not progress to a systemic level. On the latest examination at 9 yr of age, his height was 142.7 cm (+1.6 SD), and weight was 31.7 kg (+0.18 SD). Physical examination showed no features of APECED, except for the ungual candidiasis in two fingers. Laboratory data at that time are given in Table 1Go; there were no obvious abnormalities, except for a mild decrease in peripheral neutropil count and a mild increase in the serum IgA level. His HLA genotype was identical to that of his elder sister.

Methods

Genomic DNA was extracted from peripheral leukocytes of the siblings and their parents. All 14 exons and their flanking exon-intron boundaries of the AIRE gene were amplified by PCR, using 14 pairs of oligonucleotide primers (4) (Table 2Go). The PCR was performed with AmpliTaq Gold DNA polymerase (PE Applied Biosystems, Foster City, CA) for 35 cycles consisting of 1 min at 94 C, 1 min at 60 C, and 1 min at 72 C. The PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN) and sequenced by a Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems). The DNA sequences of both strands were determined on an autosequencer (ABI PRISM 310, Genetic Analyzer, PE Applied Biosystems). Twenty Japanese subjects were similarly analyzed as controls. The parents gave informed consent for their children to participate in the study.


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  		Table 2. Sequences of oligonucleotide primers

 

   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Heterozygous 1-base cytosine insertion at the exon 10 and heterozygous 1-base guanine deletion at the exon 13 were identified in cases 1 and 2 (Fig. 2Go). According to the AIRE genomic sequence deposited in GenBank (accession no. AJ009610), the insertion of cytosine occurred at nucleotide position 29635 (29635insC, codon 370 for lysine), leading to a premature termination at the codon 371. The deletion of guanine took place at nucleotide position 33031 (33031delG, codon 505 for alanine), resulting in a premature termination at the codon 520. The mother was heterozygous for 29635insC, and the father was heterozygous for 33031delG. 29635insC and 33031delG were undetected in 40 alleles of 20 Japanese control subjects. The experiments were repeatedly carried out, confirming the mutations.



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  		Figure 2. Direct sequencing of the AIRE gene. The upper panel indicates heterozygosity for 29635insC in the exon 10, and the lower panel shows heterozygosity for 33031delG in the exon 13. The arrows represent the positions of mutations.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Two compound heterozygous frameshift mutations, 29635insC and 33031delG, were identified in the AIRE gene of the two siblings with APECED. Neither of the mutations has been reported previously. 29635insC occurred at the position of six consecutive cytosines in the proline-rich region of the exon 10. The run of the same base may predispose to slipped mispairing at the replication fork, resulting in an insertion or a deletion at a high frequency (10). This frameshift mutation is predicted to produce a truncated protein missing the second plant homeodomain-type zinc finger motif and the third LXXLL motif. The 33031delG occurred in exon 13, where a mutation has not been identified previously. This frameshift mutation is predicted to yield a truncated protein missing the third LXXLL motif. Thus, it is inferred that the novel 29635insC and 33031delG mutations hinder the C-terminal function of the AIRE protein, which is the putative interaction with transcriptional coactivators, leading to APECED of the siblings.

There was phenotypic variability between the 2 siblings. Case 1 had suffered from intractable thrush and ungual candidiasis since infancy and from hypoparathyroidism since childhood, whereas case 2 had mild ungual candidiasis alone. Such phenotypic variability is not unique to the siblings. To date, clinical features have been described in 21 of 63 patients, including the present siblings, with demonstrated AIRE mutations (Table 3Go) (6, 8). The review of the 21 patients including siblings from 6 families suggests that clinical features are variable not only among patients from different families but also among affected siblings. Furthermore, the phenotypic spectrum appears to be independent of the type of mutations and inexplicable by the age difference. The phenotypic diversity would primarily be due to the difference in genetic or environmental factors relevant to immune function (6, 8). Although the variations in the HLA genotype could be involved in the difference in immune function, the two siblings had an identical HLA genotype. Thus, it is likely that genetic and environmental factors other than HLA genotype, such as T cell receptor gene and viral infection (11), may be responsible for the phenotype variability in the siblings.


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  		Table 3. Clinical features of patients with AIRE gene mutations

 
Case 1 with severe candidiasis showed good responses to both in vivo delayed skin hypersensitivity test and in vitro lymphoblast stimulation test, and case 2 with mild candidiasis showed a good response to in vitro lymphoblast stimulation test. This suggests that cell-mediated immunity against Candida antigens was preserved in the siblings with candidiasis. This is noteworthy, because chronic mucocutaneous candidiasis is regarded as the clinical expression of a selective defect in cell-mediated immunity (12, 13, 14). Consistent with this, Childgren et al. (12) have reported that delayed skin sensitivity to Candida antigens is attenuated in patients with APECED, and preservation of cellular immunity has not been described in patients with APECED. At present, it remains unknown why cell-mediated immunity was preserved in the siblings with clinically discernible candidiasis, although it may be that mucocutaneous candidiasis becomes clinically discernible before the defective cellular immunity is demonstrated by immunological examinations.

It should be pointed out, however, that immunological examinations in cases 1 were carried out before the development of endocrinopathy. Thus, it is possible that cellular immunity in case 1 was more severely affected when she developed endocrinopathy. Consistent with this notion, patients with APECED usually show candidiasis as the first clinical signs and subsequently have endocrinopathy in later age (1, 2). However, it was impossible to reexamine the cellular immunity in case 1 after the development of hypoparathyroidism, and to our knowledge, there has been no report describing cellular immunity before and after the development of endocrinopathy. Thus, it remains to be clarified whether deterioration of cellular immunity may result in the development of endocrinopathy.

In summary, we found novel mutations of the AIRE gene in two siblings with variable manifestations of APECED. The results imply that loss of the C-terminus of AIRE protein plays a critical role in the development of APECED, and that the phenotypic spectrum is variable between siblings with the same mutations.


   Acknowledgments
 
We thank Dr. Georges Bordage (University of Illinois, Chicago, IL) for reviewing the manuscript.


   Footnotes
 
1 This work was supported in part by a grant for Pediatric Research from the Ministry of Health and Welfare, the Keio University Medical Science Fund, and the Pharmacia & Upjohn Fund for Growth and Development Research. Back

Received December 16, 1999.

Revised April 9, 2000.

Accepted April 13, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Ahonen P, Myllärniemi S, Sipilä I, Perheentupa J. 1990 Clinical variation of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med. 322:1829–1836.[Abstract]
  2. Betterle C, Greggio NA, Volpato M. 1998 Autoimmune polyglandular syndrome type 1. J Clin Endocrinol Metab. 83:1049–1055.[Free Full Text]
  3. Nagamine K, Peterson P, Scott HS, et al. 1997 Positional cloning of the APECED gene. Nat Genet. 17:393–398.[CrossRef][Medline]
  4. The Finnish-German APECED Consortium. 1997 An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet. 17:399–403.[CrossRef][Medline]
  5. Björses P, Pelto-Huikko M, Kaukonen J, Aaltonen J, Peltonen L, Ulmanen I. 1999 Localization of the APECED protein in distinct nuclear structures. Hum Mol Genet. 8:259–266.[Abstract/Free Full Text]
  6. Scott HS, Heino M, Peterson P, et al. 1998 Common mutations in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients of different origin. Mol Endocrinol. 12:1112–1119.[Abstract/Free Full Text]
  7. Pearce SHS, Cheetham T, Imrie H, et al. 1998 A common and recurrent 13-bp deletion in the autoimmune regulator gene in British kindreds with autoimnmune polyendocrinopathy type 1. Am J Hum Genet. 63:1675–1684.[CrossRef][Medline]
  8. Rosatelli MC, Scott HS, Meloni A, et al. 1998 A common mutation in Sardinian autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients. Hum Genet. 103:428–434.[CrossRef][Medline]
  9. Ward L, Paquette J, Seidman E, et al. 1999 Severe autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy in an adolescent girl with a novel AIRE mutation: response to immunosuppressive therapy. J Clin Endocrinol Metab. 84:833–852.
  10. Cooper DN, Krawczak M, Antonarakis SE. 1995 The nature and mechanisms of human gene mutation. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease, 7th Ed. New York: McGraw-Hill; 259–292.
  11. Kouskoff V, Korganow AS, Duchatelle V, Degott C, Benoist C, Mathis D. 1996 Organ-specific disease provoked by systemic autoimmunity. Cell. 87:811–822.[CrossRef][Medline]
  12. Childgren RA, Meuewissen HJ, Quie PG, Good RA, Hong R. 1969 The cellular immune defect in chronic mucocutaneous candidiasis. Lancet. 2:186–1288.[CrossRef]
  13. Arulanantham K, Dwyer JM, Genel M. 1979 Evidence for defective immunoregulation in the syndrome of familial candidiasis endocrinopathy. N Engl J Med. 300:164–168.[Abstract]
  14. Fidel PLJ, Sobel JD. 1994 The role of cell-mediated immunity in candidiasis. Trends Microbiol. 2:202–206.[CrossRef][Medline]



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