AIRE Variations in Addison's Disease and Autoimmune Polyendocrine Syndromes

Genes and Immunity (2008) 9, 130–136 & 2008 Nature Publishing Group All rights reserved 1466-4879/08 $30.00 www.nature.com/gene ORIGINAL ARTICLE AIRE variations in Addison’s disease and autoimmune polyendocrine syndromes (APS): partial gene deletions contribute to APS I AS Bøe Wolff1,2,3,7, B Oftedal1,2,7, S Johansson3,4, O Bruland3, K Løva˚s1,2, A Meager5, C Pedersen6, ES Husebye1,2 and PM Knappskog3,4 1Institute of Medicine, University of Bergen, Bergen, Norway; 2Department of Medicine, Haukeland University Hospital, Bergen, Norway; 3Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway; 4Department of Clinical Medicine, University of Bergen, Bergen, Norway; 5Biotherapeutics Group, The National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Herts, UK and 6Department of Pediatrics, Kolding Hospital, Kolding, Denmark Autoimmune Addison’s disease (AAD) is often associated with other components in autoimmune polyendocrine syndromes (APS). Whereas APS I is caused by mutations in the AIRE gene, the susceptibility genes for AAD and APS II are unclear. In the present study, we investigated whether polymorphisms or copy number variations in the AIRE gene were associated with AAD and APS II. First, nine SNPs in the AIRE gene were analyzed in 311 patients with AAD and APS II and 521 healthy controls, identifying no associated risk. Second, in a subgroup of 25 of these patients, AIRE sequencing revealed three novel polymorphisms. Finally, the AIRE copy number was determined by duplex quantitative PCR in 14 patients with APS I, 161 patients with AAD and APS II and in 39 healthy subjects. In two Scandinavian APS I patients previously reported to be homozygous for common AIRE mutations, we identified large deletions of the AIRE gene covering at least exon 2 to exon 8. We conclude that polymorphisms in the AIRE gene are not associated with AAD and APS II. We further suggest that DNA analysis of the parents of patients found to be homozygous for mutations in AIRE, always should be performed. Genes and Immunity (2008) 9, 130–136; doi:10.1038/sj.gene.6364457; published online 17 January 2008 Keywords: AIRE; autoimmunity; Addison’s disease; APS I; copy number; SNP Introduction association to the MHC class 2 haplotypes DR3-DQ2, DR4-DQ85–8 and to MHC class I chain-related A Autoimmunity is the most common cause of primary (MICA)6,9 have been noted, but other genes are probably adrenal failure (autoimmune Addison’s disease (AAD)) also involved. However, owing to the rarity of AAD, in Western countries. It affects women more often than many studies have been underpowered and therefore men, but can commence at virtually any age. A hallmark clear associations have been difficult to identify. of AAD is the preponderance of multiorgan autoimmu- Autoimmune polyendocrine syndrome type I (APS I) nity, most commonly as part of an autoimmune is an even rarer disease,10,11 typically presenting with the polyendocrine syndrome type II (APS II), that is, the major clinical manifestations chronic mucocutanous combination of AAD with autoimmune thyroid disease candidiasis, primary hypoparathyroidism and AAD.12 and/or type 1 diabetes. It is assumed that both genetic APS I is caused by mutations in the autoimmune and environmental factors contribute to the risk of regulator (AIRE) gene, encoding the transcription factor developing AAD, similar to the situation in type 1 AIRE that regulates expression of organ-specific trans- diabetes and many other autoimmune diseases. Several cripts in thymic medullary epithelial cells. Thereby, genes and genetic regions have been associated with AIRE regulates negative selection of autoreactive AAD, notably the major histocompatibility complex thymocytes.13 Thus polymorphisms or other variations (MCH), and the genes encoding the cytotoxic T lympho- in AIRE could potentially contribute, together with other cyte antigen 4 (CTLA-4)1–3 and protein tyrosine genes, to autoimmunity in other and more common phosphatase non-receptor 22 (PTNP22).4 Particularly strong autoimmune diseases.14,15 However, so far no strong evidence has been presented to substantiate this in humans, neither studies searching for mutations in the Correspondence: Dr AS Bøe Wolff, Institute of Medicine, University AIRE gene, or in single nucleotide polymorphism (SNP) of Bergen, Haukeland University Hospital, Bergen N-5021, Norway. analyses of the AIRE gene region.16,17 Since AAD is one of E-mail: [email protected] 7These authors contributed equally to this work. the main manifestations of APS I, we asked whether Received 2 November 2007; revised 19 November 2007; accepted 26 common and/or rare genetic variants in AIRE are November 2007; published online 17 January 2008 associated with AAD. Analysis of AIRE in Addison’s disease AS Bøe Wolff et al 131 Subjects and methods probes are commercially available (Applied Biosystems). The Taqman allelic discrimination assay was performed Subjects essentially as described by the manufacturer (Applied Patients were recruited to the Norwegian Addison Biosystems), although we used less volume of the Registry by contacting departments of internal medicine reagents than recommended. Briefly, 2 ml of patient or and endocrinology at all hospitals in Norway. A search of control DNA (concentration 5–50 ng mlÀ1) were applied the records of each hospital was performed using into wells of a 384-well optical readable plate. The DNA ICD-8-10 codes. Altogether 311 patients with confirmed was allowed to dry at room temperature over night. Addison’s disease were recruited to the study. The A THEONYX robot (Aviso, Greiz-Gommla, Germany) diagnosis of AAD was based on typical biochemical was used to aliquot 2 ml of Taqman mastermix including findings as low morning serum cortisol concentration À1 primers and probes into each well of the plate. Negative (o150 nmol l ) and simultaneously high level of ACTH controls (water) and positive controls (known genotype, 4 À1 18 ( 20 pmol l ). Among those recruited, 25 patients run on several plates) were included. The samples were with Addison’s disease and APS I-like features were analyzed on the ABI 7900HT Genetic Analyzer and SDS selected for sequencing of the AIRE gene on the basis of 2.1/2.2 software (Applied Biosystems). one of the following criteria; Addison’s disease diag- nosed at o20 years of age, autoantibodies against tryptophan hydroxylase (TPH-1), tyrosin hydroxylase AIRE copy number (copy number variation (CNV assay)) The analysis was performed by running a duplex (TH), aromatic L-amino acid decarboxylase (AADC), side-chain cleavage enzyme (SCC) or 17a-hydroxylase Taqman real-time PCR assay. For every sample, the (17OH) and the presence of ectodermal manifestations fluorescent signals from the target gene, AIRE and from the reference gene, Rb-1 (Retinoblastoma-1, retino- associated with APS I. Previously, these patients had 23 been screened for the most common Norwegian AIRE blastoma susceptibility protein), were detected using mutations, which identified one patient with mutations different probes. The reference gene is known to be in the AIRE gene.19 In this study, all the exons and available in two copies in the genome. Using a standard flanking introns of the AIRE gene were sequenced. Blood curve for each gene, the number of AIRE-copies can be from 521 healthy individuals were collected as normal calculated as the relative relationship between the signal controls. DNA from 161 AAD patients, 14 patients with from AIRE and the signal from the Rb-1-gene. The assay m m APS I and 39 healthy blood donors were examined for mixture consisted of (for a 10 l reaction) 5 l(2Â ) m AIRE copy numbers. Thirteen of these Norwegian Taqman universal PCR mastermix, 0.1 l of Amplitaq m m patients had previously been characterized as homo- Gold, 2.5 lofdH2O, 0.7 l of Rb-1 primer/probe mM mM m zygous for a mutation in the AIRE gene while no mastermix (22.5 primers and 5 probe), 0.7 lof mM mutations had been found in one patient.20 One Danish AIRE-1 primer/probe mastermix (5.63 primers and mM female with APS I (AAD, hypoparathyroidism, ovarial 1.25 probe, primers/probe No 1, Table 1)) and 15 ng m insufficiency, keratitis, bilateral ptosis, nail pitting and DNA (1 l). Most samples were run in duplicates. alopecia), which previously had been genotyped homo- Primers and probes were designed by Beacon Designer zygous for the Finnish major mutation (c.769C4T) in v. 2.1 (Premier Biosoft International) (Table 1). All AIRE, was also included in the AIRE copy number reagents were purchased from Applied Biosystems. analysis. Aberrant copy number findings were verified by a Taqman real-time PCR duplex assay using primers and probes at different locations in AIRE (primers/probe No Assay of antibodies 2–6, see Table 1), and by using another reference gene Antibodies against SCC, 17OH, AADC, TPH-1 and TH (peripheral myelin protein 22, PMP22). In one of the were assayed by a method based on the in vitro cases (N1), samples from the parents and four siblings transcribed and translated protein as described by were obtained and analyzed to confirm our findings in Ekwall et al.21 Interferon antibodies were determined in the offspring. the 25 APS I-like patients which were AIRE sequenced as described by Meager et al.22 by enzyme-linked immuno- sorbent assay (ELISA) and human cell line-based Genetic analysis and statistics bioassay for neutralization of type I IFN activity as Haplotype mapping (Hap Map) data was used to tag all described previously. known variation with a 5% minor allele frequency, with the pairwise tagging method and cutoff and r2X0.8 using the Haploview software.24 Single point analysis and Mutational analysis of the AIRE gene haplotype estimations were performed using Haploview DNA was extracted from 3–10 ml of patient EDTA blood and Unphased software.25 P-values presented are not samples according to standard protocols.

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