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Polymorphisms in the Cytotoxic T Lymphocyte Antigen-4 Gene Region Confer Susceptibility to Addison’s Disease

Institute of Medical Genetics (A.B., H.E.A., D.E.U.), Ullevål University Hospital, University of Oslo, 0315 Oslo, Norway; Institute of Immunology (B.A.L.), Rikshospitalet University Hospital, 0027 Oslo, Norway; Departments of Paediatrics and Clinical Molecular Biology (A.G.M.), Akershus University Hospital, 1474 Nordbyhagen, Norway; Division of Clinical Sciences (North) (E.H.K., A.P.W.), University of Sheffield, Sheffield S5 7AU, United Kingdom; and Division of Endocrinology, Institute of Medicine (E.S.H.), Haukland University Hospital, N-5021 Bergen, Norway

Address all correspondence and requests for reprints to: Dr. Anne Blomhoff, Institute of Medical Genetics, University of Oslo, P.O. Box 1036, Blindern, NO-0315 Oslo, Norway. E-mail: anne.blomhoff{at}ioks.uio.no.

	Abstract

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The cytotoxic T lymphocyte antigen-4 (CTLA4) gene on chromosome 2q33 encodes a key regulator in the adaptive immune system. The CTLA4 surface molecule is expressed on activated T lymphocytes and involved in down-regulation of the immune response. Previous studies on a possible association between autoimmune Addison’s disease and CTLA4 polymorphisms have shown conflicting results. A recent study identified new candidate polymorphisms in the CTLA4 region, influencing gene splicing and thereby the relative abundance of soluble CTLA4. We genotyped 134 patients with Addison’s disease and 413 healthy controls from Norway and United Kingdom for these newly identified polymorphisms. Our data demonstrate that the same polymorphisms that have recently been demonstrated to confer susceptibility to autoimmune thyroid disease and type 1 diabetes also confer susceptibility to Addison’s disease. This finding suggests that polymorphisms in CTLA4 confer general risk to develop autoimmunity and identifies a potential therapeutic target in the prevention of autoimmune endocrine disorders.

	Introduction

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ADDISON'S DISEASE, ALSO known as primary adrenal insufficiency, is a rare disease with a prevalence of 4–14 per 100,000 in Caucasians (1). The most common cause is the autoimmune destruction of the adrenocortical tissue. The disease may occur isolated or as a part of an autoimmune polyendocrine syndrome (APS I or APS II) (2). Whereas APS I is a rare autosomal recessive disease (3), both isolated Addison’s disease and APS II are genetically complex diseases. APS II comprises Addison’s disease with type 1 diabetes and/or autoimmune thyroid disease (2). Addison’s disease, like most other autoimmune diseases, is associated with the histocompatibility leukocyte antigen (HLA) complex on chromosome 6p21 (4).

The cytotoxic T lymphocyte antigen-4 (CTLA4) gene, located on chromosome 2q33, has been reported to be associated with several autoimmune diseases, e.g. Graves’ disease, type 1 diabetes, and Hashimoto’s thyroiditis (5). The gene encodes the CTLA4 molecule, which is a member of the immunoglobulin superfamily (6) and a surface molecule on activated T lymphocytes. The molecule is homologous to CD28, but with opposing effects on T-cell activation because it down-regulates the immune response upon B7 engagement (7). It has proven to be an important candidate gene for T cell-mediated autoimmune diseases. The CTLA4 polymorphisms tested in Addison’s disease, as well as in most studies of other autoimmune diseases, have been the +49A/G (Thr¹⁷Ala) polymorphism in exon 1 and the (AT)_n dinucleotide repeat polymorphism in the 3'-untranslated region. One study found significant association with the +49A/G polymorphism (8). In another study, no association was detectable for the +49A/G polymorphism in the total material, whereas a weak association was observed after stratification for a particular HLA DQA1 allele (9). In a third study (10), a weak association between the 3'-untranslated region (AT)_n polymorphism was observed in patients from United Kingdom, whereas no association was observed in Norwegian patients.

In a recent publication, Ueda et al. (11) performed extensive linkage disequilibrium mapping of this gene region in type 1 diabetes and autoimmune thyroid disorders (Graves’ disease and autoimmune hypothyroidism). This study strongly suggested that polymorphisms within the CTLA4 region, and not the neighboring genes CD28 and ICOS, were primarily involved in susceptibility. Furthermore, the +49A/G and the 3'-(AT)_n polymorphisms were excluded as etiological polymorphisms. Disease susceptibility was mapped to a 6.1-kb region 3' of the CTLA4 gene, and the polymorphisms were suggested to affect the relative amount of soluble and full-length (membrane bound) CTLA4 produced. The candidate polymorphisms most likely to cause this effect and to be involved in disease susceptibility have not been analyzed previously in Addison’s disease. We wanted to test whether these newly identified polymorphisms are likely to be of importance in Addison’s disease. We therefore typed patients and controls for five of these novel single nucleotide polymorphisms (SNPs) as well as the +49A/G SNP in exon 1 in patients and healthy controls from Norway and the United Kingdom.

	Subjects and Methods

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A total of 134 patients with autoimmune Addison’s disease (94 from Norway and 40 from the United Kingdom) and 413 healthy controls (273 from Norway and 140 from the United Kingdom) were included in the study. The patients had either isolated Addison’s disease or APS II (Norwegian dataset, n = 54 + 40; United Kingdom dataset, n = 18 + 22, respectively). Patients with APS I were not included in the study. The characteristics of the majority of these patients are described elsewhere (10, 12). We analyzed the five most strongly associated SNPs reported by Ueda et al. (11) and the +49A/G SNP. The six SNPs were MH30 upstream of the CTLA4 gene, +49A/G in exon 1, and CT60, JO31, JO30, and JO27_1 downstream of CTLA4 (11). The SNPs were analyzed with TaqMan Minor Groove Binder chemistry assay (Applied Biosystems, Cheshire, UK), using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Sequences on probes and primers are available on request. CT60 was additionally typed using a PCR-restriction fragment length polymorphism assay.

The allele frequencies among patients and controls were compared using Fisher’s exact test. Because previous studies have shown a tendency for the same alleles to be involved in Addison’s disease, type 1 diabetes, and autoimmune thyroid disease (8, 9, 10, 11, 12), we performed a one-sided Fisher’s exact test. For all statistical tests, P < 0.05 was considered significant. Haplotypes were constructed using the expectation maximization algorithm as implemented in the program Unphased (window size 6) in the GLUE interface (http://www.hgmp.mrc.ac.uk). The study was approved by appropriate ethical committees and based on informed consent.

	Results

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Table 1 shows the distribution of CTLA4 polymorphisms in Addison’s disease patients and controls. The alleles that are increased among patients compared with the controls are shown, and markers MH30, CT60, JO30, and JO27_1 were found to be positively associated with disease in the Norwegian dataset. In the smaller United Kingdom dataset, a significant difference was observed only for the JO30 polymorphism. There were no differences in allele frequencies in either patients or controls, respectively, when comparing Norwegian and United Kingdom data (data not shown). Hence, we analyzed the combined data and found significant association for all markers except +49A/G. For the associated markers, there were no significant difference in APS II and isolated Addison (data not shown). The success rate of genotyping was >97% for all markers, and only the allele frequencies are presented (the actual number of alleles are available on request). There is strong linkage disequilibrium between the markers analyzed, and analysis of constructed haplotypes revealed that there were three predominating haplotypes. These three haplotypes had the following alleles for markers MH30-+49AG-CT60-JO31-JO30-JO27: GAGGGT, GGGGGT, and CAATAG. All other haplotypes were rare with a frequency of <5%. None of the haplotypes were significantly associated with Addison’s disease (haplotypes with <5% frequency were excluded). A comparison of these did not allow any conclusion as to which of the SNPs (MH30, CT60, JO31, JO30, or JO27_1) are most likely to be of primary importance. There were no significant departures from Hardy-Weinberg equilibrium in any of the populations analyzed. The +49A/G in United Kingdom controls and JO27_1 polymorphism in United Kingdom cases showed a tendency of departure from Hardy-Weinberg (P_uncorr = 0.04 and 0.03, respectively). However, after a semiconservative Bonferroni correction (correcting for the number of markers, but not the number of populations analyzed), this was not significant (P_corr = 0.2 and 0.2, respectively).

	Discussion

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We found that CTLA4 polymorphisms conferred risk irrespective of HLA genotypes contrary to a previous report (data not shown) (9).

Taken together, our results strongly suggest that, in Addison’s disease, particular alleles at polymorphisms in the CTLA4 region are conferring increased risk. Furthermore, the alleles involved are identical with the ones recently identified to be involved in other autoimmune diseases, namely type 1 diabetes and autoimmune thyroid disorders. This suggests that these CTLA4 SNPs may confer susceptibility to endocrine autoimmunity in general and not be limited to certain diseases. Moreover, our data add weight to the idea that the association is due to an effect on differential CTLA4 splicing. How differential splicing and relative amount of soluble vs. membrane-bound CTLA4 may affect pathogenesis is presently unknown, but in light of the key role of CTLA4 in adaptive immunity, numerous possibilities can be envisaged (5, 11). The identification of candidate CTLA4 polymorphisms in Addison’s disease identifies a potential target for future intervention trials attempting to prevent disease development.

	Acknowledgments

 
We thank Beate Skinningsrud for excellent technical assistance.

	Footnotes

 
This work was supported by University of Oslo, Norwegian Diabetes Association, Novo Nordisk Research Foundation, Norwegian Research Council, Haukeland University Hospital, and Innovest.

Abbreviations: APS, Autoimmune polyendocrine syndrome; CTLA4, cytotoxic T lymphocyte antigen-4; HLA, histocompatibility leukocyte antigen; SNP, single nucleotide polymorphism.

Received October 23, 2003.

Accepted March 18, 2004.

	References

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