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Dipartimento di Endocrinologia e Metabolismo, Universita’ degli Studi di Pisa, 56124 Pisa, Italy
Address all correspondence and requests for reprints to: Filomena Cetani, M.D., Ph.D., Dipartimento di Endocrinologia e Metabolismo, Università di Pisa, Via Paradisa 2, 56124 Pisa, Italy. E-mail fcetani{at}yahoo.it
Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is a rare autosomal recessive disorder characterized by hypoparathyroidism, adrenal failure, chronic mucocutaneous candidiasis, and ectodermal dystrophies and other organ-specific autoimmune diseases. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is caused by mutations of the autoimmune regulator gene.
We identified an Italian family with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and a pattern of inheritance suggestive of a dominant mechanism. Serological and clinical studies showed a high prevalence of hypothyroid autoimmune thyroiditis in affected members with classical autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.
Direct sequencing of the entire coding region of the autoimmune regulator gene revealed the presence in the proband of a novel missense (G228W) mutation in exon 6 in a heterozygous state. The same heterozygous mutation was identified in all family members with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and/or hypothyroid autoimmune thyroiditis. None of the unaffected family members and 50 unrelated Italian controls carried the mutation.
In contrast with all other autoimmune regulator mutations reported in families, the novel G228W mutation acts in a dominant fashion in our family, as only one heterozygous mutation was found in the entire coding sequence of the autoimmune regulator gene in the proband. Moreover, analysis of the family tree showed direct transmission of the hypothyroid autoimmune thyroiditis/polyendocrinopathy-candidiasis-ectodermal dystrophy phenotype to the offspring in each generation in the absence of consanguinity, further supporting a dominant inheritance. The G228W closely cosegregated with hypothyroid autoimmune thyroiditis in our family, whereas a low penetrance of the full autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy phenotype was observed.
In conclusion, we report a novel mutation of the autoimmune regulator gene in a family with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, closely cosegregating with hypothyroid autoimmune thyroiditis. The G228W mutation acts in a dominant fashion and may shed light on the structure-function relationship of the autoimmune regulator protein.
AUTOIMMUNE POLYENDOCRINOPATHY-candidiasis-ectodermal dystrophy (APECED), also known as autoimmune polyendocrinopathy syndrome type I is, to date, the only organ-specific autoimmune disease described in humans that is inherited in a monogenic fashion. The typical disease components are hypoparathyroidism, primary adrenocortical failure and chronic mucocutaneous candidiasis (1, 2). APECED is clinically diagnosed in an individual with at least two of the three major features (2). However, failure of several other endocrine and nonendocrine organs is often found, such as autoimmune diabetes, primary hypogonadism, atrophic gastritis, vitiligo, alopecia, and autoimmune thyroiditis. Additional ectodermal dystrophies are represented by enamel dysplasia, nail dystrophies, and keratopathy. APECED is rare, but it is relatively more frequent in some ethnic groups, such as the Finns (incidence, 1:25,000), Sardinians (incidence, 1:14,000), and Iranian Jews (incidence, 1:9,000) (3, 4, 5). The disease is universally described as recessive autosomal, with high penetrance, with the first manifestations usually occurring during early childhood (2).
APECED is the first multiple autoimmune disease that has been shown to be caused by a defect in a single gene, named autoimmune regulator (AIRE) (6, 7, 8). The AIRE gene maps to chromosome 21q22.3 (4) and is expressed in immune-related organs, such as thymus, lymph nodes, and fetal liver (6, 9). The protein is predicted to function as a transcription coactivator because it includes a putative nuclear targeting signal, it is localized in the nucleus, and it contains four nuclear receptor-binding LXXLL motifs. However, the AIRE genomic targets and functions have not yet been described (4, 10, 11, 12, 13). A number of mutations have been described in the AIRE gene of patients with APECED, including single nucleotide substitutions, small insertions, and deletions. Reported pathogenic mutations are scattered through the coding sequence of AIRE (5, 6, 7, 8, 14, 15, 16), but at least four mutational hot spots have been suggested, in exons 2, 6, 8, and 10 (13).
In this paper we describe a novel mutation of the AIRE gene in an Italian family with APECED. This novel mutation appears to act in a dominant fashion, in contrast to all other mutations described in families to date. As an additional peculiarity, this mutation seems to carry an unusually high risk for hypothyroid autoimmune thyroiditis (hAT).
Subjects and Methods
Family description
The proband (III-1; Fig. 1
), a
woman who was 38 yr old at the time of diagnosis, was born at 40
wk gestation after an uncomplicated pregnancy and delivery. Her parents
were from central Italy and had no knowledge of Sardinian, Finnish, or
Jewish ancestry. She was diagnosed with idiopathic hypoparathyroidism
at age 5 yr and was subsequently treated with oral calcium and vitamin
D. Since adolescence she has had recurrent bouts of oral candidiasis.
At age 24 yr, hAT was detected, and
L-T4 treatment was started. She also
had enamel dysplasia at physical examination, but no other ectodermal
manifestations, such as nail dystrophies or vitiligo. Family history
was notable for the presence of a sister (III-3) with
hypoparathyroidism and of a mother (II-1) and maternal grandmother
(I-1) with hAT. A maternal aunt (II-3) had a history of childhood
hypocalcemia and died at age 40 yr of sudden death. No further relevant
information could be obtained. One of the proband’s two sons (IV-1)
also had hAT and a history of oral candidiasis since childhood. There
was no known consaguinity among any of family members. Therefore, only
the proband had typical APECED, as at least two of the three major
features of the syndrome are required to establish the diagnosis in
randomly selected cases (17). However, it is commonly
accepted that only one of these features is needed when ascertaining
siblings of proven APECED patients (5, 18, 19, 20). Indeed, a
good number of cases with only one manifestation and with homozygous
proven pathogenic mutations of the AIRE gene have been reported among
siblings of full APECED cases (21). Therefore, we also
classified individuals II-3 (hypoparathyroidism), III-3
(hypoparathyroidism), and IV-1 (chronic candidiasis) as APECED cases.
This prompted us to study the AIRE gene in this family. As all three
members of the family who had APECED also had hAT, for the purpose of
genetic studies we considered as affected members in this particular
family all subjects who had APECED, hAT, or both (Fig. 1). Our internal
review board approved the study, and all family members gave their
informed consent for both serological and genetic studies.
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Total serum calcium, free thyroid hormone levels, TSH, and cortisol were measured by commercial standard assays. Anti-Tg antibodies (TGAb) were measured by immunoradiometric assay (ICN Pharmaceuticals, Inc., High Wycomb, UK; normal, <50 U/ml). Thyroid peroxidase antibodies (TPOAb) were measured by immunoradiometric assay (AB-TPOK-3, DiaSorin, Inc., Saluggia, Italy) normal, <10 U/ml). Serum PTH was measured by immunoradiometric assay (Nichols Institute Diagnostics, San Capistrano, CA; reference range, 10–65 ng/liter). Serum anti-H+,K+-adenosine triphosphatase antibodies [antiparietal cell antibodies (APCA)] were measured by ELISA (Pharmacia & Upjohn, Inc., Freiburg, Germany; normal, <10 U/ml). Serum antiglutamic acid decarboxylase antibodies (anti-GAD) were measured by RIA (BioChem ImmunoSystems, Milan, Italy; normal, <1 U/ml). Serum anti-21hydroxylase antibodies (21-OH-Ab) were measured by RIA (CIS-Bio International, Gif-Sur-Yvette, France; normal, <1 U/ml).
Genetic analysis of the AIRE gene
Genomic DNA was isolated from peripheral blood samples taken
from the proband, her two siblings, her parents, and extended family
members (Fig. 1
). DNA was purified using a standard phenol-chloroform
extraction method. All 14 exons and their flanking exon-intron
boundaries of the AIRE gene (GenBank accession no. AB006684) were
amplified in the proband by PCR, using 11 pairs of oligonucleotide
primers, slightly modified from a previous study (8). The
region of interest detected in the proband (see Results) was
also amplified in all available family members. PCR amplification was
carried out in a 35-cycle PCR, in which the initial 1-min denaturation
of template DNA at 94 C was followed by 1 min at 50 or 54 C (Table 1) and 1 min at 72 C in a volume of 50
µl containing 1 µg DNA, 50 mmol/liter KCl, 10 mmol Tris-HCl (pH
8.3), 1 mmol (exons 1–4) or 1.5 mmol MgCl2
(exons 5–14), 0.01% gelatin, 0.2 mmol deoxynucleotide triphosphate, 5
U Taq polymerase (Cetus Corp., Berkeley, CA), and 100 mmol
of each primer. Dimethylsulfoxide (5%) was included for exons 1 and
2.
|
Linkage analysis
Classical two-point linkage analysis was performed using MLINK
software (22). Linkage with the detected AIRE mutation was
tested, assuming a dominant mode of inheritance and a 100% penetrance
as the only model. Gene frequencies for the mutation and for the
disease were estimated for analysis purposes at 1/100,000. LOD scores
were assessed over a wide range of 
values (0.001–0.500).
Results
Biochemical and immunological markers in family members
The relevant clinical and biochemical findings in all family
members are reported in Table 1. At the time of ascertainment, the
proband had an undetectable PTH level in the presence of mild
asymptomatic hypocalcemia (1.97 mmol/liter) while receiving calcium and
vitamin D treatment. Thyroid function tests where normal during
L-T4 treatment, with positive (299
U/ml) TPOAb and negative TGAb. 21-OH-Ab were positive (15 U/ml), with a
normal cortisol response to exogenous ACTH. APCA were also positive (37
U/ml). Anti-GAD were negative. A thyroid ultrasound showed a normal
sized gland with a typical diffuse hypoechoic pattern
(23). Liver and kidney function tests were normal.
Her elder son, a 17-yr-old boy (IV-1), had high titer TGAb and TPOAb,
with a slightly elevated TSH level while receiving
L-T4 treatment. He also had positive
APCA and anti-GAD tests. Serum PTH and calcium levels were normal. Her
younger son, a 14-yr-old boy (IV-2), had normal thyroid function tests
and negative organ-specific antibodies as well as normal serum PTH and
calcium levels. The proband’s mother (II-1), a 61-yr-old-woman, had
hAT and was mildly hypothyroid during
L-T4 treatment when first examined.
She had high titer TGAb and TPOAb levels, whereas all other
immunological markers studied were negative. The proband’s sister
(III-3) had undetectable serum PTH with a normal calcium level during
treatment with calcium and vitamin D. She also had medium TGAb titers,
high TPOAb titers with a mildly elevated TSH, and a typical hypoechoic
pattern at thyroid ultrasound and was therefore diagnosed as having
hAT. In addition, she had high titer anti-GAD, with normal fasting
glucose levels. She had no signs of active candidiasis, and all other
immunological markers studied were negative. The proband’s maternal
grandmother (I-1) had high titer TGAb and TPOAb, with a slightly
elevated TSH level during L-T4
treatment. She also had positive APCA and anti-GAD tests. Serum PTH and
calcium could not be tested in this patient, but she had no history,
symptoms, or signs of hypocalcemia. 21-OH-Ab were negative. No history
of candidiasis could be elicited. The maternal cousin of the proband
(III-5) was found to have previously undetected subclinical
hypothyroidism with high titer TGAb and TPOAb. All other
diagnosed family members had no history or findings suggestive
of candidiasis, hypocalcemia, or ectodermal dystrophy. They also had
negative immunological markers of endocrine autoimmunity, with the
exception of the maternal grandfather of the proband (I-2), who had
borderline positive APCA. Examination of the family tree (Fig. 1)
strongly suggested a dominant mode of inheritance of the complex
organ-specific autoimmunity trait.
Genetic analysis of the AIRE gene
The entire coding sequence of the AIRE gene was determined in the
proband. A novel heterozygous base substitution at position 809 of the
cDNA sequence (G to T) determining a change of glycine to tryptophan at
codon 228 (G228W) of exon 6 was detected. The remaining sequence was
entirely normal, with the exception of three common silent heterozygous
polymorphisms (588 C/T, S196S; 1197 T/C, A399A; and 1578 T/C D526D)
(8). None of these polymorphisms is predicted to alter the
amino acid sequence of the AIRE gene product. Direct sequencing of the
region of interest on exon 6 revealed the same heterozygous mutation in
all family members with autoimmunity, with the exception of the
proband’s maternal grandfather, who only had low titer APCA (Table 1).
Conversely, none of the unaffected family members inherited the
mutation from the maternal grandmother, nor was the mutation found in
the family members who married into the family (II-2 and III-2; Table 1). The mutation was not detected by direct sequencing the region of
interest in any of 50 unrelated normal controls of Italian origin.
Linkage analysis
Linkage analysis of the genetic data on the family yielded a
maximum LOD score of 2.1 at = 0.001, suggesting that the
detected mutation was indeed linked to the clinical phenotype.
Discussion
The typical components of APECED are hypoparathyroidism, primary adrenocortical failure, and chronic mucocutaneous candidiasis. APECED is clinically diagnosed in an individual with at least two of the three major features (1, 2). One major manifestation is sufficient to diagnose APECED in siblings of patients (5, 18, 19, 20). Autoimmune diabetes, primary hypogonadism, gastric autoimmunity, vitiligo, alopecia, and autoimmune thyroiditis are also often found, although with a much lower frequency (11). Additional qualifying findings are represented by enamel dysplasia, nail dystrophies, and keratopathy. The APECED phenotype is inherited in an autosomal recessive fashion (2). Original linkage studies have mapped the gene responsible for the syndrome to chromosome 21q22.3 (4). Subsequently, the gene for APECED, named AIRE, has been cloned and sequenced (6). Although the function of the AIRE gene product has not yet been clarified, the structure of the predicted protein suggests that it may act as a transcription coactivator (10, 11, 12, 13). After cloning of AIRE, mutations of the gene have been found in most families and individual patients with typical APECED.
In most reported cases, homozygous or compound heterozygous mutations of the AIRE gene have been documented (14, 15, 16, 24), in agreement with the evidence of a recessive mode of inheritance of APECED (2). In a minority of sporadic cases only one single heterozygous mutation has been found (13, 15). However, in the absence of family data, the possibility of mutations in the promoter region or in the intronic sequence affecting transcription and/or RNA splicing cannot be excluded in such cases. Thus, AIRE mutations reported to date seem to act through the classical loss of function mechanism, typical of recessive diseases.
We report a novel mutation of the AIRE gene in an APECED family with some peculiar features with respect to the mutations reported to date. The novel G228W mutation acts in a dominant fashion in our family, as shown by direct transmission of the disease to the offspring in each of four consecutive generations in the absence of consanguinity.
Moreover, only one heterozygous mutation was found in the entire coding sequence of the AIRE gene in the proband (III-1). The presence of a heterozygous known polymorphism (D526D) in exon 14, close to the 3'-end of the coding region, makes the presence of a large deletion of the second allele of the proband highly unlikely.
The glycine to tryptophan substitution at codon 228 and, consequently, the introduction of the aromatic side-chain of tryptophan, might alter the polarity of the protein. Moreover, a single mutation from the smallest and most flexible amino acid glycine to the bulkiest amino acid tryptophan will induce conformational modifications of the protein. These changes may then alter the binding/active site of AIRE, disrupting its function. Finally, recent data have shown that the AIRE gene product homodimerizes and that homodimerization is necessary for the transcriptional activity of the protein (12). Thus, it is possible that the G228W mutation acts through a dominant negative effect, i.e. by interfering with the homodimerization process and affecting the activity of the normal allele as well. This hypothesis is supported by the location of the G228W mutation, close to the 1–207 fragment of the protein that has been deemed necessary for homodimerization (12). Such a mechanism would explain the dominant mode of inheritance observed in our family.
The same mutation was found in two other family members with the full APECED phenotype (III-3 and IV-1). Interestingly, hAT was observed in all of these three members. All other members of the family with hAT (I-1, II-1, and III-5) also carried a heterozygous G228W mutation. All carriers of this mutation had, in adjunct to hAT, a variable admixture of serological markers of endocrine autoimmunity. In none of the members of the family without endocrine autoimmunity was the mutation observed. Thus, the G228W mutation appears to strongly cosegregate with the disease phenotype, as defined as hAT with or without APECED. This is further supported by the results of linkage analysis, showing suggestive evidence of linkage of the mutation to the phenotype. It seems, therefore, that the G228W mutation elicits an unusually high risk for hAT while showing lower penetrance for APECED. Recent data from association studies have excluded a different AIRE mutation (964del13) as a susceptibility factor for thyroid autoimmune disease (25). This observation is in keeping with the generally low prevalence of hAT in APECED, reported to be 6–10% (11). Although linkage analyses have yielded no evidence supporting 21q22.3 as a predisposing area for familial autoimmune thyroid disease and hAT (26), it will be interesting to determine whether this mutation may be important in a subset of families with thyroid autoimmunity.
In conclusion, we report a novel mutation of the AIRE gene in a family with APECED, closely cosegregating with hAT. In contrast with all other previously reported AIRE mutations, the G228W mutation acts in a dominant fashion and may shed light on the structure-function relationship of the AIRE protein.
Acknowledgments
We are grateful to all of the family members who graciously agreed to participate in the study.
Footnotes
This work was supported in part by University of Pisa (Fondi di Ateneo, to C.M. and G.B.), the Ministero dell’ Universita’ e della Ricerca Scientifica e Tecnologica (40%; Rome, Italy; to C.M.), and the Ministero della Sanita’ (Rome, Italy; to C.M.).
Abbreviations: AIRE, Autoimmune regulator; anti-GAD, antiglutamic acid decarboxylase antibodies; APCA, antiparietal cell antibodies; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; hAT, hypothyroid autoimmune thyroiditis; 21-OH-Ab, anti-21-hydroxylase antibodies; TGAb, anti-Tg antibodies; TPOAb, thyroid peroxidase antibodies.
Received March 20, 2001.
Accepted June 6, 2001.
References
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