Evidence from autoimmune thyroiditis of skewed X-chromosome inactivation in female predisposition to autoimmunity

European Journal of Human Genetics (2006) 14, 791–797 & 2006 Nature Publishing Group All rights reserved 1018-4813/06 $30.00 www.nature.com/ejhg ARTICLE Evidence from autoimmune thyroiditis of skewed X-chromosome inactivation in female predisposition to autoimmunity Tayfun Ozcelik*,1, Elif Uz1, Cemaliye B Akyerli1, Sevgi Bagislar1, Chigdem A Mustafa1, Alptekin Gursoy2, Nurten Akarsu3, Gokce Toruner4, Nuri Kamel2 and Sevim Gullu2 1 Department of Molecular Biology and Genetics, Bilkent University, Faculty of Science, Ankara, Turkey; 2Department of Endocrinology and Metabolic Diseases, Ankara University, School of Medicine, Sihhiye, Ankara, Turkey; 3Gene Mapping Laboratory, Pediatric Hematology Unit, Department of Pediatrics, Hacettepe University, Medical Faculty, Sihhiye, Ankara, Turkey; 4Center for Human and Molecular Genetics, UMDNJ – New Jersey Medical School, Newark, NJ, USA The etiologic factors in the development of autoimmune thyroid diseases (AITDs) are not fully understood. We investigated the role of skewed X-chromosome inactivation (XCI) mosaicism in female predisposition to AITDs. One hundred and ten female AITDs patients (81 Hashimoto’s thyroiditis (HT), 29 Graves’ disease (GD)), and 160 female controls were analyzed for the androgen receptor locus by the HpaII/polymerase chain reaction assay to assess XCI patterns in DNA extracted from peripheral blood cells. In addition, thyroid biopsy, buccal mucosa, and hair follicle specimens were obtained from five patients whose blood revealed an extremely skewed pattern of XCI, and the analysis was repeated. Skewed XCI was observed in DNA from peripheral blood cells in 28 of 83 informative patients (34%) as compared with 10 of 124 informative controls (8%, Po0.0001). Extreme skewing was present in 16 patients (19%), but only in three controls (2.4%, Po0.0001). The buccal mucosa, and although less marked, the thyroid specimens also showed skewing. Analysis of two familial cases showed that only the affected individuals demonstrate skewed XCI patterns. Based on these results, skewed XCI mosaicism may play a significant role in the pathogenesis of AITDs. European Journal of Human Genetics (2006) 14, 791–797. doi:10.1038/sj.ejhg.5201614; published online 5 April 2006 Keywords: X chromosome inactivation; autoimmune thyroid disease; female predisposition to autoimmunity Introduction Hashimoto’s thyroiditis (HD) and Graves’ disease (GD) are autoimmune thyroid diseases associated with multiple genetic factors. Although the pathogenesis is poorly understood, a widely accepted model suggests an inherited *Correspondence: Professor T Ozcelik, Department of Molecular Biology and Genetics, Faculty of Science, B-242, Bilkent University, Bilkent, Ankara 06800, Turkey. Tel: þ 90 312 2902139; Fax: þ 90 312 2665097; E-mail: Received 11 November 2005; revised 19 January 2006; accepted 10 February 2006; published online 5 April 2006 background, which predisposes the subjects to autoimmunity. Additional intrinsic and extrinsic factors such as hormones and the environment may ultimately trigger or contribute to the development of the disease phenotype.1 Extensive linkage genome screens during the past decade have resulted in the identification of several thyroidspecific susceptibility genes and/or loci, but confirmation through multiple population studies is still awaited for the majority of these loci.1,2 A common feature of autoimmune diseases, including autoimmune thyroid diseases (AITDs), is an increased prevalence in women when compared with men. The most striking sex differences are Skewed XCI in autoimmune thyroid disease T Ozcelik et al 792 observed in AITDs, scleroderma, Sjögren’s syndrome, and systemic lupus erythematosus, which are diseases where over 80% of the patients are females.3 It has been demonstrated that risk of autoimmunity could be increased by a lack of exposure to self-antigens in the thymus and the presence of autoreactive T cells.4 – 6 Disturbances in the X-chromosome inactivation (XCI) process provide a potential mechanism whereby the lack of exposure to self-antigens could occur,7,8 including AITDs.9,10 X-chromosome inactivation is a physiologic process that takes place in early female development and results in the transcriptional silencing of one of the pair of X chromosomes.11 As a result of this epigenetic regulation, a random inactivation of the X chromosome inherited from either parent occurs and normal female subjects are thus a mosaic of two cell populations. It is therefore an attractive hypothesis that skewed XCI could lead to the escape of X-linked self-antigens from presentation in the thymus or in other peripheral sites that are involved in tolerance induction, inadequate thymic deletion, and finally loss of T-cell tolerance. Indeed, we recently observed skewed XCI in blood cells of women with scleroderma.12 Based on our observation that an association exists between skewed XCI and female predisposition to autoimmunity, we hypothesized that skewed XCI may be involved in the pathogenesis of AITDs, particularly in the hematopoietic compartment. We observed extremely skewed XCI in the blood samples of a significant proportion of female patients with AITDs. Methods Patients and pedigree analysis Caucasian women diagnosed with AITDs (n ¼ 110), and healthy female controls with no history of autoimmune disease and cancer (n ¼ 160) were included in the study. Among the patients, 81 were diagnosed with HT and 29 with GD. The mean ages were 44.8714.1 (mean7SD) years for AITDs (46714.2 years in the Hashimoto patients, and 40.6713.2 years in the Graves’ patients), and 46710 for controls. The duration of the symptoms was 5.777.4 years among the AITDs patients (5.777 years in the Hashimoto patients and 678.5 years in the Graves’ patients). The mean age of diagnosis was 39712 years. All of the patients had attended the outpatient clinics of the Endocrinology and Metabolic Diseases Department of Ankara University School of Medicine for at least 1 year since the onset of disease. Patients were randomly chosen for the study. All clinical investigations described in this manuscript were conducted in accordance with the guidelines in the Declaration of Helsinki (http://www.wma.net). The ethics review board of the participating institutions approved the study protocol. Informed consent was obtained from all subjects. European Journal of Human Genetics The diagnosis of HD was made by the existence of a firm goitre in combination with elevated thyroid auto-antibodies (thyroglobulin and/or thyroid peroxidase), a low ultrasonographic echogenity of the gland, and demonstration of lymphocytic infiltration by fine-needle aspiration biopsy and/or biochemical hypothyroidism. The diagnosis of GD was based on biochemical hyperthyroidism, and a diffuse symmetrical goitre in combination with positive thyroid antibodies (thyroglobulin, thyroid peroxidase or TSH receptor). In addition, thyroid ophthalmopathy and/ or d (...truncated)