Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED): a model disease to study molecular aspects of endocrine autoimmunity

Many of the organ-specific autoimmune diseases affect endocrine organs. This is due to the fact that the organ-specific autoantigens are often hormones or enzymes specifically engaged in the synthetic pathways resulting in hormone production. Autoimmune endocrine diseases include type 1 diabetes, hypo- and hyperthyroidism, Addison's disease, hypoparathyroidism, and gonadal failure. These diseases may occur as a solitary immunological abnormality but often coincide as polyendocrine autoimmune diseases, indicating a common aetiology in the pathogenesis. Autoimmune endocrine diseases have complex genetic predispositions and they are closely associated to certain HLA haplotypes. In addition to HLA, other genes are known to regulate the disposition to autoimmunity. Only a few autoimmune diseases with a monogenic background are known. An example is a syndrome regulated by a single gene, namely APECED, an acronym for autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, also known as autoimmune polyendocrinopathy syndrome type 1 (APS1). In this autosomal recessive disease, mutations in the AIRE (autoimmune regulator) gene cause organ-specific autoimmune destruction of several, mostly endocrine, tissues. This article reviews current knowledge of APECED and the AIRE gene behind this syndrome. APS1 (hereafter APECED) was first described in the 60s and 70s by Neufeld et al. [1] and Blizzard et al.[2] who described a rare syndrome affecting children in their early age. Later on, the group of Prof Perheentupa has significantly contributed to the clinical characterization of the syndrome [3,4]. Most of known autoimmune endocrine diseases may occur in patients with APECED, which they usually develop within their childhood and teenage years. Usually the first sign of the syndrome is chronic Candida infection, followed by autoimmune hypoparathyroidism and Addison's disease. At least two of these three major components need to be present for diagnosis. However, although the defect in APECED is inherited in an autosomal recessive manner, the clinical features are very heterogenous (Table 1). APECED is a rare disease but is more prevalent among some populations such as Finns (1/25 000) [5], Sardinians (1/14 400) [6] and Iranian Jews (1/9000) [7]. In Norway, the prevalence of APECED is reported as 1/80 000 [8]. The prevalence of the most common disease components of APECED, in Finnish patients. Adapted from [3] of postpubertal patients. The prevalence of the most common disease components of APECED, in Finnish patients. Adapted from [3] of postpubertal patients. Hypoparathyroidism, appearing within the first decade of life, is the most frequent, and sometimes the only, endocrine disease seen in APECED patients. For example, in the majority of Iranian Jews, and in approximately 20% of Finnish patients, hypoparathyroidism remained the only endocrinopathy [4]. Hypoparathyroidism is often followed by adrenocortical insufficiency, with an age at onset of about 4–12 years [4,9] but in several cases it may appear at 20 years of age [4]. Premature ovarian failure in females is far more common than primary testicular failure in males. Type 1 diabetes, hypophyseal failure, and autoimmune thyroid disease deserve to be mentioned as less common disease components of the syndrome. Interestingly, recent studies have indicated that autoimmune gastrointestinal disorders, which are relatively common among APECED patients, are, in fact, due to the immune reaction to the endocrine cells of the stomach and intestine (see below). The mucocutaneous candidiasis, appearing soon after birth, and the ectodermal dystrophy, affecting mainly nails and the tooth enamel, cannot be readily linked to autoimmunity. While the chronic candidiasis is most likely a part of the immunological dysregulation, the association of ectodermal dystrophy with autoimmunity is still open. Candida infection usually starts already within the first two years of life and appears in more chronic cases as oral thrush. Several cases of oral carcinoma have been reported, suggesting that oral candidiasis might be carcinogenic [10]. Of ectodermal skin diseases, alopecia, as patchy loss of hair, and vitiligo, as pigment-free skin areas, have been reported in approximately 40% and 25% of patients. In addition, urticaria-like erythema with fever was found in 9% of Finnish patients [4]. Splenic atrophy has been suspected to be relatively common among APECED patients. The exact reason for hypo- or asplenism is still open and it has been hypothesized to occur due to autoimmune-mediated destruction [11], or to depend on local AIRE gene dysfunction in the spleen [12]. The main immunological finding in the endocrine disorders of APECED is the existence of high levels of serum antibodies reacting specifically with components of the affected organs (Table 2). The nature of these autoantigens became clarified mainly in the early 90 s. Earlier work had demonstrated the presence of antibodies to 3–4 specific antigens present either in the cytosomal, mitochondrial, or ribosomal fraction in Addison's disease [13–15]. In 1992, the first adrenal autoantigen, steroid 17α-hydroxylase (P450c17), was recognized by expressional library screening [16]. Later on, two other enzymes linked to the steroidogenic synthetic pathway, namely steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc), were identified [17–19]. The three enzymes, P450c17, P450c21 and P450scc, belong to the cytochrome P450 superfamily and have significant similarities at the protein level. P450c21 is adrenal cortex specific; P450c17 and P450scc are also expressed in gonads, and the presence of autoantibodies to the latter two enzymes is also associated with hypogonadism in patients with APECED. The autoantibodies inhibit the steroidogenic enzyme activities in vitro[20], but a pathological role for the autoantibodies in vivo has not yet been proved [21]. Autoantigens in APECED. Only those autoimmune entities in APECED where the autoantigens are known are listed. Adapted from Heino et al. [56], Meriluoto et al. [90] and Immunobiology, 5th edition [91] This autoantigen in APECED has not been unequivocally proven. Autoantigens in APECED. Only those autoimmune entities in APECED where the autoantigens are known are listed. Adapted from Heino et al. [56], Meriluoto et al. [90] and Immunobiology, 5th edition [91] This autoantigen in APECED has not been unequivocally proven. Many other self-antigen targeting autoantibodies have been reported in APECED, often associated with a particular clinical manifestation (reviewed in [22,23]). In APECED, patient sera have specific autoantibodies to parathyroid glands as demonstrated by indirect immunofluorescence, and autoantibodies reacting with the calcium-sensing receptor, a protein specific to parathyroid glands, were initially reported [24], but this result was not supported by a more recent study by another group [25]. Autoantibodies to islet-cell specific autoantigens: GAD65, insulin, and IA2 are found in those with type 1 diabetes, and antithyroid peroxidase and thyroglobulin antibodies in APECED patients with thyroiditis (reviewed in [22]). Antibodies to another pancreatic islet cell expressed protein, aromatic l-amino acid decarboxylase, are described in APECED patients in association with chronic active hepatitis, vitiligo, or type 1 diabetes [26]. Interestingly, malabsorption appears to be a result of destruction of intestinal endocrine cells. Gastrointestinal dysfunction is associated with an autoimmune reaction to tryptophan hydroxylase in serotonin-producing enterochromaffin cells (EC) in the gastric antrum [27], histidine decarboxylase in histamine-producing enterochromaffin-like cells (ECL) in the gastric fundus [28], and to cholecystokinin-producing cells in the proximal portion of the small intestine [29]. It remains unknown why autoimmunity in APECED patients is predominantly targeted to proteins expressed in endocrine tissues and how the immune system specifically selects these antigens. Characteristically, many of the autoantibodies detected in the APECED syndrome are also found in corresponding sporadic clinical entities. The autoantibodies are often considered to arise as a consequence of tissue destruction, whereas the pathogenetic effect is mediated by T-cells. However, information about the cell-mediated immunity in APECED is still lacking. Despite this, the presence of autoantibodies can be used as a diagnostic marker as there generally is a good correlation between the autoantibodies and clinical disease and the appearance of autoantibodies often precedes the clinical manifestations. The AIRE gene, identified by positional cloning in 1997 by two independent groups, lies in chromosome 21q22.3 [30,31]. The gene, approximately 13 kb in length, contains 14 exons that encode a polypeptide of 545 amino acids [30,31]. Initial characterization of the AIRE protein, based on the amino acid sequence, revealed a conserved nuclear localization signal (NLS) in the N terminus; two plant homeodomain or PHD type zinc fingers and a proline rich region lying between these, in the C terminus; a SAND domain; and four LXXLL motifs, typical of nuclear receptor binding proteins [30–32]. It was soon discovered that the N terminus of AIRE also harbours an HSR domain, also found in Sp100 and Sp140, mediating Sp100-Sp100 homodimerization [33,34] (Fig. 1). Schematic of the AIRE protein showing the functional protein domains, and the distribution of the APECED-causing mutations for which functional and/or localization data is available (see also Table 3). HSR: homogenously staining region; SAND: Sp100, AIRE, NucP41/75 and DEAF-1; PHD: plant homeodomain zinc finger; PRR, proline rich region; L, LXXLL nuclear receptor interaction motif. The PHD zinc fingers are characteristically found in proteins involved in the regulation of transcription, typically at the chromatin level [35]. The structure is usually ascribed the function of mediating protein–protein interaction; at present more than 400 PHD finger-containing proteins are known [36]. The solution structures of the PHD fingers from KAP-1 and WSTF have been resolved, with results that support their role in mediating protein interactions [36,37]. The exact structure of the AIRE PHD zinc fingers has not been addressed, nor is there any data to corroborate the hypothesis that they mediate protein–protein interaction for AIRE. We and others have shown, however, that AIRE is a strong activator of transcription, and that the PHD fingers mediate this effect (see below and [38–40]). The SAND domain is found in a number of proteins with various functions (Sp100, AIRE, NucP41/75 and DEAF-1) [41]. It was originally suggested to function as a DNA binding domain, and recent data following the resolution of its structure confirmed the assumption [41,42]. A signature motif in the SAND domain, KDWK, seems to mediate the DNA binding of nuclear deaf-1-related (NUDR) [42]. Interestingly, the SAND domain seems to coexist almost invariably, in other nuclear proteins as well as in AIRE, with other functional protein domains, including chromatin–associated and protein interaction-mediating motifs [42]. The KDWK motif, however, is not found in the AIRE protein. Also, there are no unequivocal data to date to show that the SAND domain mediates DNA binding for AIRE, although Kumar et al. have reported in a recent paper [43] that AIRE is able to bind DNA oligonucleotides as homodimers or tetramers. The AIRE protein as a monomer was unable to bind DNA [43]. Our unpublished experiments showed that AIRE might actually not directly associate with DNA, but rather do so indirectly via some, at present unknown, intermediary protein (J. Pitkänen and P. Peterson, unpublished observation). The LXXLL motifs, four of which are found in the AIRE protein [30,31], mediate the binding of various proteins to nuclear receptors, mainly in a ligand-dependent manner [44]. These proteins then function as coactivators to nuclear receptors. Whether AIRE is somehow involved in mediating or regulating the effects of nuclear receptors remains to be seen, as no studies so far have addressed this interesting issue. Human AIRE expression is found in several tissues; the most prominent of these being the thymus. Other sites of AIRE expression include the lymph nodes, the spleen, and fetal liver [30,31,45]. AIRE expression in human peripheral blood monocytes and differentiated dendritic cells has also been demonstrated [46]. No significant expression is seen in the target organs of autoimmune destruction [45,47]. In the thymus, AIRE expression is seen in a subpopulation of medullary epithelial cells (MEC) [47]. The expression of AIRE in thymic epithelial cells has been further confirmed by isolation of thymocytes, epithelial, and dendritic cells from mouse thymi, which were then analysed by RT-PCR. AIRE expression was mainly seen in epithelial cells and, to a lesser degree, in dendritic cells, but not in thymocytes [48]. In mouse, a similar AIRE expression pattern emerges, with the notable exception that more tissues have been found positive for mouse AIRE than its human counterpart [46,49–52]. In addition to the sites of expression of human AIRE discussed above, mouse AIRE is also found in the bone marrow, the urinary tract, the genitals, the alimentary tract, the respiratory tract, the brain, and in endocrine organs including the adrenals and the thyroid gland [46,49–52]. To date, at least 49 APECED-causing patient mutations have been identified (Table 3) [6,8,30,31,40,53–67]. The mutations are distributed throughout the coding region of the gene (Fig. 1). The main types of mutations consist of either nonsense or frame shift mutations resulting in a truncated polypeptide, or single amino acid-changing missense Most of the mutations occur in the functional protein of AIRE described of these affect the HSR domain in the N a single exception are missense mutations are found in the PHD four in the SAND domain, in the domain and three affect the LXXLL The effect of APECED-causing mutations on the function and localization of the AIRE protein. In addition, the effect of mutations to the structure of the PHD zinc fingers are and the first and or and of the conserved PHD fingers to Adapted from et al. Pitkänen et al. and of not patient in results between no data The effect of APECED-causing mutations on the function and localization of the AIRE protein. In addition, the effect of mutations to the structure of the PHD zinc fingers are and the first and or and of the conserved PHD fingers to Adapted from et al. 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