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Department of Medicine, University of Birmingham and Birmingham Heartlands and Queen Elizabeth Hospitals, Birmingham B9 5SS, United Kingdom
Address correspondence to: Dr. S. C. L. Gough, Department of Medicine, University of Birmingham, Birmingham Heartlands Hospital, Bordesely Green East, Birmingham B9 5SS, United Kingdom. E-mail: s.c.gough{at}bham.ac.uk
Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare monogenic autoimmune disease with endocrine components including type 1 diabetes, adrenal failure, and thyroid dysfunction, with major autoantibodies directed against adrenal, pancreas, and thyroid tissue. A 13-bp deletion in exon 8 of the autoimmune regulator (AIRE1) gene on chromosome 21q22.3 accounts for more than 70% of mutant alleles in United Kingdom subjects with APECED. To determine whether this polymorphism contributes to disease susceptibility in subjects with autoimmune disease in general, we screened 302 patients with Graves’ disease, 154 patients with autoimmune hypothyroidism, 235 patients with type 1 diabetes, and 318 control subjects for the 13-bp deletion of the AIRE1 gene. The mutation was present in only 1 (0.33%) patient with Graves’ disease, 1 patient with autoimmune hypothyroidism (0.6%), and 1 (0.315) of the control subjects. No patients with type 1 diabetes were found to carry the mutation. We conclude, therefore, that the 13-bp deletion of the AIRE1 gene is not a susceptibility locus for the more common autoimmune endocrinopathies in the United Kingdom.
AUTOIMMUNE diseases are common complex disorders in which a number of genetic and environmental factors are believed to contribute to the etiology. Family studies have shown that autoimmune diseases tend to cluster within families, implying that some susceptibility genes may be common to different autoimmune diseases (1). Association between the major histocompatibility complex (MHC) class II molecules and a number of autoimmune diseases has long been documented (1). Further support for the sharing of autoimmune loci between related disorders comes from association of polymorphism of the cytotoxic T lymphocyte associated-4 (CTLA4) gene with type 1 diabetes (2, 3, 4), Graves’ disease (3, 4, 5, 6, 7), autoimmune hypothyroidism (6, 8), and Addison’s disease (8, 9).
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare monogenic autoimmune disease, with no known MHC-human leukocyte antigen (HLA) association (10). Typically, the disease includes the triad of hypoparathyroidism, primary adrenocortical failure, and chronic mucocutaneous candidiasis. However, the clinical picture can be highly variable with a phenotype comprised of a number of nonendocrine and endocrine components including type 1 diabetes, adrenal failure (Addison’s disease), and thyroid dysfunction (11), with major autoantibodies directed against adrenal (12, 13), pancreas (14, 15), and thyroid tissue (16).
The gene responsible for APECED has been mapped to chromosome 21q22.3 and designated the autoimmune regulator gene (AIRE1) (17, 18). The AIRE1 gene is widely expressed in lymphoid tissues including thymus, lymph node, and spleen, as well as in the adrenal cortex and thyroid, making it a good candidate gene for other autoimmune disorders (17, 18). Although a number of polymorphic sites have been identified within the AIRE gene, a 13-bp deletion at nucleotide 964 in exon 8 (964del13) accounts for more than 70% of APECED alleles in United Kingdom subjects (19). The aim of this study was to determine whether this frequent polymorphism has a role in the pathogenesis of more common endocrinopathies including type 1 diabetes, Graves’ disease, and autoimmune hypothyroidism.
Patients and Methods
White patients of British origin with Graves’ disease and autoimmune hypothyroidism were recruited from thyroid clinics as described previously (20). Briefly, patients with Graves’ disease were defined by the presence of biochemical hyperthyroidism together with two of the following criteria: diffuse goiter, a significant titre of thyroid peroxidase, thyroglobulin or thyrotropin-receptor (TSH-R) autoantibodies, or the presence of dysthyroid eye disease. The diagnosis of autoimmune hypothyroidism was based on the presence of positive thyroid autoantibodies (thyroid peroxidase and/or thyroglobulin) and biochemical evidence of hypothyroidism. DNA samples from the type 1 diabetes British Diabetic Association Warren Repository (21) were used to study patients with type 1 diabetes. Ethnically matched control subjects, with no history of autoimmune disease, were bled at various sites including the Blood Transfusion Service, Birmingham Heartlands Hospital and the Queen Elizabeth Hospital, Birmingham. In total, DNA was obtained from 302 patients with Graves’ disease, 154 patients with autoimmune hypothyroidism, 235 patients with type 1 diabetes, and 318 control subjects. The study was approved by the local ethics committees, and all subjects gave informed, written consent.
DNA was extracted from the whole blood using the Nucleon Bacc II kit
(Nucleon Biosciences, UK). Amplification of the target DNA in
exon 8 of the AIRE1 gene was carried out by PCR using
primers 5' CAC CCC AGC CCA GTC TGC ATG 3' and 5' CTT CAG GGT CAG TGG
GTG GAG 3'. The reaction was carried out in a final volume of 50 µL
containing 200 
g genomic DNA, 50 
mol of each primer,
200 µM/L dNTP’s, 1 mM magnesium
chloride, 50 mmol potassium chloride, 10 mmol HCl (Tris) to give pH of
8.3, 1U Taq DNA polymerase, and 4% dimethyl sulphoxide.
Amplification was performed in an MJ Research, Inc. Tetrad
thermal cycler. After an initial denaturation at 94 C for 5 min,
PCR amplification was performed using 35 cycles. Each cycle consisted
of 30 sec at 93 C, 30 sec at 63.5 C, and 30 sec at 72 C. This produced
a 229-bp amplicon, which was visualized on a 2% agarose gel stained
with ethidium bromide. Restriction fragment length polymorphism (RFLP)
analysis was performed on 8 µL of PCR product using 10 IU of BsrB1
(New England Biolabs, Inc., Beverly, MA) enzyme at 37 C
over 2 h. This yielded two fragments of 140 bp and 89 bp in the
presence of wild-type sequence and a single fragment of 216 bp in the
presence of mutation with 13 bp deletion. These products were resolved
by electrophoresis on a 4% Nusieve agarose (3:1) gel stained with
ethidium bromide.
Statistical analysis
Comparison of frequencies of the 13-bp deletion of the AIRE1 gene between patients and controls were performed using Fisher’s exact test.
Results
A heterozygous 13-bp deletion was found in 1 out of 302 patients (0.33%) with Graves’ disease and in 1 out of 318 of the normal controls (0.31%) (P = 0.5). The normal control with the deletion had normal thyroid function and serum biochemistry and was negative for thyroid peroxidase and thyroglobulin autoantibodies. The subject with the 13-bp deletion in the Graves’ disease population was a woman who had thyrotoxicosis diagnosed at the age of 25 yr. At onset she had dysthyroid eye disease and had no family history of autoimmunity including thyroid disease. The 13-bp deletion was found in 1 out of 154 patients (0.6%) with autoimmune hypothyroidism (P = 0.5, compared with controls) and in no patient with type 1 diabetes. The patients with thyroid disease and the 13-bp deletion had no clinical evidence of other endocrinopathy. The HLA status of all subjects has previously been ascertained. The control subject and the patient with Graves’ disease were DR3 positive, and the patient with autoimmune hypothyroidism was DR3 negative. The presence of the heterozygous deletion in all three subjects was confirmed by sequence analysis (using ABI 377 PRISM DNA sequencer).
Discussion
Both type 1 diabetes and autoimmune thyroid disease are complex polygenic disorders in which a number of susceptibility loci are likely to contribute to disease (1). Candidate gene studies have identified a number of susceptibility loci, including the MHC-HLA region on chromosome 6p, the insulin gene region on chromosome 11q, and the CTLA4 gene region on chromosome 2q33, as susceptibility loci for type 1 diabetes (3, 22) and the MHC-HLA region (20) and CTLA4 gene region in Graves’ disease (3, 4, 5, 6, 7). Genome-wide searches in both diseases have revealed further susceptibility loci for type 1 diabetes (23) and Graves’ disease (24, 25, 26). Both type 1 diabetes and thyroid dysfunction are features of APECED, and antibodies to pancreas (14, 15) and thyroid tissue (16) have been detected in these patients. A mutation involved in the development of APECED is, therefore, a candidate for these other more common autoimmune diseases.
In this study we screened patients with, Graves’ disease and autoimmune hypothyroidism and type 1 diabetes for a 13-bp deletion in exon 1 of the AIRE1 gene, which accounts for more than 70% of APECED alleles in British subjects (19). The mutation was found in one subject with Graves’ disease and in one member of the control group. With respect to the patient with Graves’ disease, clinical evaluation found no evidence of other autoimmune phenomena or indeed any obvious distinguishing characteristics. As a result of the large size of the data sets used, we can be confident that the 13-bp deletion of the AIRE1 gene is not a susceptibility locus for the more common autoimmune endocrinopathies including type 1 diabetes, autoimmune hypothyroidism, or Graves’ disease in the United Kingdom.
Acknowledgments
We acknowledge the help of J. Daykin, J. Carr-Smith, A. Daly (University of Birmingham), M. Armitage (Royal Bournemouth Hospital), A. Hattersley (Royal Devon & Exeter Hospital), P. Dodson (Birmingham Heartlands Hospital) in recruiting patients, and S.H.S. Pearce (University of Newcastle upon Tyne) for PCR details and for the supply of primers.
Footnotes
1 This work was supported by an award of a project grant from the
Wellcome Trust (Grant #M/95/3717), the Trustees of the former United
Hospitals and Eli Lilly & Co. (UK). R. Nithiyananthan is a
Wellcome Trust Research Fellow. 
Received July 20, 1999.
References
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