Title: SNPs at the PRR3, ABCF1 and GNL1 genes in the HLA class I region are associated with Graves ophthalmopathy in a gender-dependent manner
Authors: Yu-Huei Liu, PhD,1, 2 Yi-Ju Chen, MS, 1 Hsin-Hung Wu, PhD, 3 Tzu-Yuan Wang, MD, 4,5 Fuu-Jen Tsai, MD, PhD1, 6,7, 8, 9,10
1Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan; 2Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan; 3 Department of Business Administration, National Changhua University of Education, Changhua, Taiwan. 4Division of
Endocrinology and Metabolism, Department of Medicine, China Medical University Hospital, Taichung, Taiwan; 5 Department of Endocrinology and Metabolism, College of Chinese Medicine, China Medical University, Taichung, Taiwan; 6Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan; 7School of Chinese Medicine, China Medical University, Taichung, Taiwan; 8School of Post-
Baccalaureate Chinese Medicine, China Medical University, Taichung, Taiwan;
9Department of Biotechnology, Asia University, Taichung, Taiwan, 10Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan.
Meeting Presentation: None
Financial Support: This study was supported by China Medical University (grant number: CMU-101-N1-08), Taichung, Taiwan. The sponsor/funding organization had no role in the design or conduct of this research.
Conflict of Interest: No conflicting relationship exists for any author.
Running title: SNPs at the PRR3, ABCF1 and GNL1 genes in the HLA class I region and GO
Address for reprints:
Fuu-Jen Tsai, MD, PhD.
Department of Medical Genetics and Medical Research, China Medical University Hospital (No.2 Yuh-Der Road, 404 Taichung, Taiwan)
Telephone: 886-4-22062121 Ext 2041; Fax: 886-4-22033295 E-mail: [email protected]
Abstract
Objective:To investigate whether a conserved HLA class I region influenced the development of Graves ophthalmopathy (GO) in patients with Graves disease (GD) in a Taiwan-Chinese population.
Design: Case-control study.
Participants: Four hundred and sixty-eight Taiwan-Chinese patients with GD; 200 of these patients had GO while 268 did not.
Methods: Five single nucleotide polymorphisms (SNPs) between the HLA-A and HLA-C loci were genotyped.
Main Outcome Measures: Mann-Whitney test and chi-square test with Bonferroni correction were used. The odds ratios (ORs) were estimated by applying
unconditional logistic regression with a 95% confidence interval (CI).
Results: Strong gender effects on the distribution of the SNPs were apparent: male GD patients carrying an A allele at rs2074503 in the PRR3 gene tended to avoid acquiring GO (p = 0.008, OR = 0.450, 95% CI = 0.248–0.819), whereas female patients tended to develop GO (p = 0.01, OR = 1.486, 95% CI = 1.098–2.012). In addition, only the female GD patients with a T allele at rs1264439 in the ABCF-1 gene tended to develop GO (p = 0.005, OR = 1.539, 95% CI = 1.139–2.081). Analysis of the haplotype blocks of the SNPs rs2074505 (GNL1) and rs2074503 (PRR3) showed that haplotype HA1 was underrepresented in male GO patients (p = 0.004, OR = 0.418, 95% CI = 0.228–0.767), whereas HA-4 was under-represented in female GO patients (p = 0.007, OR = 0.660, 95% CI = 0.490–0.895).
Conclusions: The results suggested that SNPs at PRR3 and ABCF1 genes and the haplotype composed by SNPs at GNL1 and PRR3 between the HLA-A and HLA-C genes tended to predict Graves ophthalmopathy in a gender-dependent manner in patients with Graves disease in Taiwan.
Introduction
Graves disease (GD; Online Mendelian Inheritance in Man [OMIM] 275000 in Entrez databases from The National Center for Biotechnology Information [NCBI]) is the most common autoimmune thyroid disease (OMIM 608173). GD is a complex disorder that develops through interactions between susceptibility genes and
environmental agents such as infection and stress.1-3 GD affects women 5- to 10-fold more frequently than men and is characterized by hyperthyroidism, diffuse goiter, the presence of autoantibodies targeting the thyroid-stimulating hormone receptor, and hyperthyroid features such as weight loss, fatigue, weakness, rapid heartbeat, and hand tremors. Some GD patients also experience extrathyroidal manifestations such as Graves ophthalmopathy (GO) and thyroid dermopathy. Of these disorders, GO is the most common organ-specific endocrine disease and affects 25–50% of patients with GD.4
The etiological factors of GO remain unknown; however, GO is generally believed to be associated with genetic predispositions and environmental factors, including exposure to cigarette smoke, high dietary iodine intake, and stressful life events.5 Many genetic loci influencing GO risk have been identified using genome- wide linkage and candidate gene-association studies.6, 7 The human leukocyte antigen (HLA) region was the first GO susceptibility gene region to be identified in different ethnic groups, however, this region does not account for the all genetic information associated with GO.7, 8 Additional studies have identified several other genetic loci associated with GD and GO, primarily in genes involved in immune regulation including cytotoxic T-lymphocyte-associated protein 4 (CTLA4),7 intracellular adhesion molecule 1 (ICAM1),9 integrin alpha E (ITGAE),10 interferon gamma
(IFNγ),11, 12 tumor necrosis factor (TNF),11, 13 interleukin 1 beta (IL1β)14-16 and others.17-
19
Using whole-genome screening technology, the HLA region on chromosome 6 was identified as containing susceptibility loci for GD and the related disorder GO.
These data indicated that other genes within and surrounding the HLA region could be additional factors for GD.20, 21 The evolution of the HLA class I region, especially its hitchhiking region, has been suggested to play a potential role in the generation of new disease alleles in humans.22 However, it has not been fully elucidated whether polymorphisms in the conserved region of HLA class I are involved in GO
pathogenesis. The aim of the present study was to assess whether genes in the conserved region between HLA-A and HLA-C22 played a role in the development of GO in a Taiwan-Chinese population with GD.
Methods
Patients and DNA isolation
This study was performed according to the principles of the Declaration of Helsinki, and the participants provided written informed consent. The study was approved by the Medical Ethics Committee of China Medical University Hospital.
The study enrolled 468 GD patients from the China Medical University Hospital in Taiwan; of these patients, 200 had GO, while 268 did not. Detailed descriptions of the inclusion/exclusion criteria have been published elsewhere.10, 14, 17-19, 23, 24 The inclusion criteria were as follows: (i) patients were self-reported non-aboriginal Taiwanese, and none of the parents or grandparents had an aboriginal background; (ii) patients had to be able to understand the risks and benefits of the protocol and be able to give
informed consent; (iii) patients had typical clinical features including
hyperthyroidism, diffuse enlargement of the thyroid gland, increased free thyroxine or triiodothyronine levels, suppressed thyroid stimulating hormone levels, positive thyrotrophin-receptor autoantibodies, and with or without antimicrosomal or
antithyroglobulin antibodies; (iv) patients had to satisfy the diagnostic criteria of GD at the time of examination; and (v) the stored genomic DNA had to be sufficient for analysis in the current study. The exclusion criteria were as follows: (i) patients were not able to understand or give informed consent; (ii) patients who were pregnant or had delivered one or more babies within one year; or (iii) the stored genomic DNA was not sufficient for analysis in the current study. The methods for drawing blood, handling and storing DNA samples and quality assurance have been described elsewhere.10, 14, 17-19, 23, 24
Single nucleotide polymorphism (SNP) genotyping
Five SNPs between the HLA-A and HLA-C genes that had been identified and mapped by other researchers,22 were analyzed: rs2105960 in the RAS oncogene family pseudogene 1 (RANP1) gene; rs1264457 in the coding region of the major histocompatibility complex class I, E (HLA-E) gene; rs2074505 in coding region of the guanine nucleotide binding protein-like 1 (GNL1) gene; rs2074503 in the 3′- untranslated region (UTR) of the proline-rich 3 (PRR3) gene; and rs1264439 near the 3′-endof the ATP-binding cassette, subfamily F, member 1 (ABCF-1) gene. The
genotyping was performed using the Assay-on-Demand system for allelic
discrimination analysis and detection, according to the manufacturer’s instructions (Applied Biosystems). The polymerase chain reaction (PCR) mixture contained 10 ng of genomic DNA, 10 μL TaqMan master mix, and 0.125 μL of the 40× assay mix.
The reactions were performed in 96-well plates on a ViiA 7 Real-Time PCR System (Applied Biosystems).
Statistical analyses
Statistical analyses were performed using PASW Statistics 18.0 software. The associations between gender and age were estimated using the Mann-Whitney test.
The associations between gender and all other demographic characteristics and the associations between each polymorphism and GO were examined using the chi-square (2) test. A two-tailed p-value less than 0.05 with Bonferroni correction (threshold p- value was 0.05/n, where n is the number of all independent tests performed together) was considered statistically significant.25 The polymorphism frequencies were
compared between GD patients with and without GO, and the odds ratios (ORs) were estimated by applying unconditional logistic regression with a 95% confidence interval (CI). The screening for linkage disequilibrium was performed using Haploview ver. 4.1.26
Results
Patient characteristics and their correlations with GO in patients with GD
The study enrolled 468 GD patients with a female-to-male ratio of 3.85. The frequency distributions were compared between male and female GD patients with or without GO for clinical characteristics such as goiter, nodular hyperplasia, myxedema, vitiligo, age, and smoking history, and for whether they had undergone thyroid gland surgery. As demonstrated in Table 1, none of the clinical features (GO, goiter, nodular hyperplasia, myxedema, and vitiligo) or environmental factors (smoking and treatment strategies) were significantly associated with GO in patients with GD.
Alleles and genotypes of the PRR3 and ABCF-1 genes contributed to GO development in a gender-dependent manner
To determine whether gene polymorphisms in the HLA class I region influenced the development of GO in patients with GD, five SNPs between the HLA-A and HLA- C genes were genotyped for all patients. No significant associations were found between the polymorphisms tested and the clinical features (GO, goiter, nodular hyperplasia, myxedema, and vitiligo) or environmental factors (smoking and treatment strategies; data not shown).
When gender-stratification analyses were performed, we found that two of the SNPs showed opposite effects in male and female GD patients (Table 2). Male patients tended to avoid developing GO if they carried the A allele at rs2074503 (p = 0.008, OR = 0.450, 95% CI = 0.248–0.819). In contrast, female patients carrying the same alleles tended to develop GO (rs2074503: p = 0.010, OR = 1.486, 95% CI = 1.098–2.012; rs1264439: p = 0.005, OR = 1.539, 95% CI = 1.139–2.081). The observations remained significant after applying Bonferroni corrections. Genotypic analyses further showed that the SNP rs2074503 met the criteria for significance after Bonferroni correction in men (Table 3). These results suggested that the SNPs
rs2074503 in the PRR3 gene and rs1264439 in the ABCF-1 gene were associated with the development of GO in a gender-dependent manner.
Haplotypes of loci in the GNLI and PRR3 genes were associated with GO in a gender-dependent manner
Linkage disequilibrium (LD) r2 values were calculated for the five SNPs between the HLA-A and HLA-C genes (Figure 1). Two of them, rs2074505 (GNL1) and
rs2074503 (PRR3), were in strong LD (r2>0.85) in male and female patients without GO, but not in those with GO. This difference led us to further estimate the
associations of haplotypes composed of the 2 SNPs with GO (Table 4, available at http://aaojournal.org.). Overall, the distributions of haplotypes in male and female patients were significantly different from one another (p = 0.005 and p = 0.013, respectively). Haplotype-specific analyses showed that haplotype HA1 was associated with a tendency to not have GO in the male patients (p = 0.004; OR = 0.418, 95% CI
= 0.228–0.767), whereas haplotype HA4 was associated with a tendency to not have GO in the female patients (p = 0.007; OR = 0.660, 95% CI = 0.490–0.895). Statistical significance values met the criteria after Bonferroni corrections. These results showed that haplotypes HA1 and HA4 tended to be associated with the presence of GO in GD patients in a gender-dependent manner.
Discussion
Genetic predisposition and environmental factors are thought to be the major causes of GO, 5 and genome-wide linkage and candidate gene-association studies have identified many genetic loci that influence the susceptibility of patients to GO development, primarily for genes involved in immune regulation. The susceptibility
loci for familial GD and GO have been identified in the HLA region of chromosome 6.20, 21 In addition, the same region has been also reported as containing a genetic polymorphism indicating susceptibility to ankylosing spondylitis, another
autoimmune disease.27 Moreover, the rapid evolution of the HLA class I region may contribute to the emergence of new diseases including autoimmune diseases,22, 28 thereby increasing researchers’ interest in investigating whether additional genes within the HLA class I region influenced GO susceptibility. In this study, we identified new associations between GO and PRR3, ABCF1and GNL1-PRR3
haplotypes in patients with GD. To the best of our knowledge, this is the first study to demonstrate that new polymorphisms in the conserved HLA class region may be associated with GO in GD. Our results supported the results of other whole-genome screening studies in that loci close to, but distinct from, the HLA region were found to be susceptibility loci for familial GD.20, 21, 29
Histological examination of tissues from patients with GO demonstrated an increase in orbital volume which in part due to the deposition of collagen,
glycosaminoglycans and fat. Nevertheless, genes involved in cell growth regulation, DNA transcription, and protein synthesis should also be considered.30 In this study, rs2074503, located within the 3′-UTR of PRR3, showed a strong association with GO and may be a target of microRNAs. PRR3 is located on chromosome 6p21.33 and encodes a protein with a putative CCCH zinc finger-superfamily proline-rich domain which is frequently observed in DNA-binding proteins; therefore, the polymorphism may contribute to autoimmune diseases such as GO through initiation of the
transcription of immunologically relevant genes.31 Moreover, GNL1, where SNP rs2074505 located on (chromosome 6p21.3), encodes a member of the large nucleolar GTPases and acts as a pivotal coordinator of signaling cascades that regulate cell growth and proliferation.32 In addition, ABCF1, where SNP rs1264439 located on (chromosome 6p21.33), plays a role in binding to ribosomes, promoting translational initiation and mediating the inflammatory response.33, 34 Elucidation of the
mechanisms through which cell growth-related processes influence the development of GO will require additional studies, and the results of our study should be confirmed in a greater number of patients.
We identified one allele at a locus that predicted a tendency to develop GO in women but to avoid develop GO in men, suggesting that distinct gender-associated factors mediated the distribution of these SNPs. Indeed, others and our previous studies have shown that female gender is one of the nongenetic factors associated with a tendency to develop GO.10, 35-38 Although estrogens –dependent exacerbation of disease can be inhibited by androgens in a number of experimental animal models,39, 40 and an experimental animal model for GO has been successfully established,41 the
influence of sex hormones on disease susceptibility to GO requires further
investigations. Clinical observations have shown that gender differences in GD and other autoimmune disorders extend beyond hormonal differences,38, 42 and both epigenetic X-chromosome inactivation43-46 and X-linked inheritance47, 48 have been suggested to be indicative of a tendency toward disease susceptibility to GD, however, a similar mechanism for disease susceptibility to GO has not yet been identified. Because epigenetic, genetic and transcriptional alternations may contribute to the development of GO, it is reasonable to further investigate how the genetic effects due to gender differences may affect the development of GO.
In conclusion, the present study showed that two new SNPs at PRR3 and ABCF1 genes and the haplotype composed by SNPs at GNLI and PRR3 in the HLA class I region were associated with GO in a gender-dependent manner, which may indicate trends in Taiwan-Chinese patients with GD. Further genetic and biological studies on these specific SNPs and haplotypes may provide valuable insights into the genetic components influencing the development of GO in patients with GD. In addition, identifying new disease susceptibility variants for the same disease through genetic association studies will improve our understanding of the gene-gene interactions in this complex disease.
References
1. Ginsberg J. Diagnosis and management of Graves' disease. CMAJ 2003;168:575-85.
2. Villano MJ, Huber AK, Greenberg DA, et al. Autoimmune thyroiditis and diabetes: dissecting the joint genetic susceptibility in a large cohort of multiplex families. J Clin Endocrinol Metab 2009;94:1458-66.
3. Huber A, Menconi F, Corathers S, et al. Joint genetic susceptibility to type 1 diabetes and autoimmune thyroiditis: from epidemiology to mechanisms. Endocr Rev 2008;29:697-725.
4. McIver B, Morris JC. The pathogenesis of Graves' disease. Endocrinol Metab Clin North Am 1998;27:73-89.
5. Stan MN, Bahn RS. Risk factors for development or deterioration of Graves' ophthalmopathy. Thyroid 2010;20:777-83.
6. Gianoukakis AG, Smith TJ. Recent insights into the pathogenesis and
management of thyroid-associated ophthalmopathy. Curr Opin Endocrinol Diabetes Obes 2008;15:446-52.
7. Bednarczuk T, Gopinath B, Ploski R, Wall JR. Susceptibility genes in Graves' ophthalmopathy: searching for a needle in a haystack? Clin Endocrinol (Oxf)
2007;67:3-19.
8. Weetman AP, Zhang L, Webb S, Shine B. Analysis of HLA-DQB and HLA- DPB alleles in Graves' disease by oligonucleotide probing of enzymatically amplified DNA. Clin Endocrinol (Oxf) 1990;33:65-71.
9. Wang S, Sun H, Chen HY, et al. Intercellular adhesion molecule 1 gene polymorphisms do not contribute to Graves' disease in Chinese patients. Endocrine 2007;31:114-8.
10. Liu YH, Chen RH, Chen WC, et al. Disease association of the CD103
polymorphisms in Taiwan Chinese Graves' ophthalmopathy patients. Ophthalmology 2010;117:1645-51.
11. Anvari M, Khalilzadeh O, Esteghamati A, et al. Genetic susceptibility to Graves' ophthalmopathy: the role of polymorphisms in proinflammatory cytokine genes. Eye (Lond) 2010;24:1058-63.
12. Siegmund T, Usadel KH, Donner H, et al. Interferon-gamma gene microsatellite polymorphisms in patients with Graves' disease. Thyroid 1998;8:1013-7.
13. Bednarczuk T, Hiromatsu Y, Seki N, et al. Association of tumor necrosis factor and human leukocyte antigen DRB1 alleles with Graves' ophthalmopathy. Hum Immunol 2004;65:632-9.
14. Liu YH, Chen RH, Wu HH, et al. Association of interleukin-1beta (IL1B) polymorphisms with Graves' ophthalmopathy in Taiwan Chinese patients. Invest Ophthalmol Vis Sci 2010;51:6238-46.
15. Khalilzadeh O, Anvari M, Esteghamati A, et al. Graves' ophthalmopathy and gene polymorphisms in interleukin-1alpha, interleukin-1beta, interleukin-1 receptor and interleukin-1 receptor antagonist. Clin Experiment Ophthalmol 2009;37:614-9.
16. Liu N, Li X, Liu C, et al. The association of interleukin-1alpha and interleukin- 1beta polymorphisms with the risk of Graves' disease in a case-control study and meta-analysis. Hum Immunol 2010;71:397-401.
17. Liu YH, Wan L, Chang CT, et al. Association between copy number variation of complement component C4 and Graves' disease. J Biomed Sci 2011;18:71.
18. Liu YH, Chen CC, Liao LL, et al. Association of IL12B polymorphisms with susceptibility to Graves ophthalmopathy in a Taiwan Chinese population. J Biomed Sci 2012;19:97.
19. Liao WL, Chen RH, Lin HJ, et al. Toll-like receptor gene polymorphisms are associated with susceptibility to Graves' ophthalmopathy in Taiwan males. BMC Med Genet 2010;11:154.
20. Tomer Y, Barbesino G, Greenberg DA, et al. Mapping the major susceptibility loci for familial Graves' and Hashimoto's diseases: evidence for genetic heterogeneity and gene interactions. J Clin Endocrinol Metab 1999;84:4656-64.
21. Tomer Y, Ban Y, Concepcion E, et al. Common and unique susceptibility loci in Graves and Hashimoto diseases: results of whole-genome screening in a data set of 102 multiplex families. Am J Hum Genet 2003;73:736-47.
22. Shiina T, Ota M, Shimizu S, et al. Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity. Genetics 2006;173:1555-70.
23. Liao WL, Chen RH, Lin HJ, et al. The association between polymorphisms of B7 molecules (CD80 and CD86) and Graves' ophthalmopathy in a Taiwanese population. Ophthalmology 2011;118:553-7.
24. Tsai KH, Tsai FJ, Lin HJ, et al. Thymic stromal lymphopoietin gene promoter polymorphisms and expression levels in Graves' disease and Graves' ophthalmopathy.
BMC Med Genet 2012;13:116.
25. Bland JM, Altman DG. Multiple significance tests: the Bonferroni method. BMJ 1995;310:170.
26. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263-5.
27. Paladini F, Belfiore F, Cocco E, et al. HLA-E gene polymorphism associates with ankylosing spondylitis in Sardinia. Arthritis Res Ther 2009;11:R171.
28. Eriksson N, Tung JY, Kiefer AK, et al. Novel associations for hypothyroidism include known autoimmune risk loci. PLoS One 2012;7:e34442.
29. Burton PR, Clayton DG, Cardon LR, et al. Association scan of 14,500
nonsynonymous SNPs in four diseases identifies autoimmunity variants. Nat Genet 2007;39:1329-37.
30. Bahn RS, Heufelder AE. Pathogenesis of Graves' ophthalmopathy. N Engl J Med 1993;329:1468-75.
31. Blach-Olszewska Z, Leszek J. Mechanisms of over-activated innate immune system regulation in autoimmune and neurodegenerative disorders. Neuropsychiatr Dis Treat 2007;3:365-72.
32. Boddapati N, Anbarasu K, Suryaraja R, et al. Subcellular distribution of the human putative nucleolar GTPase GNL1 is regulated by a novel arginine/lysine-rich domain and a GTP binding domain in a cell cycle-dependent manner. J Mol Biol 2012;416:346-66.
33. Richard M, Drouin R, Beaulieu AD. ABC50, a novel human ATP-binding cassette protein found in tumor necrosis factor-alpha-stimulated synoviocytes.
Genomics 1998;53:137-45.
34. Tyzack JK, Wang X, Belsham GJ, Proud CG. ABC50 interacts with eukaryotic initiation factor 2 and associates with the ribosome in an ATP-dependent manner. J Biol Chem 2000;275:34131-9.
35. Manji N, Carr-Smith JD, Boelaert K, et al. Influences of age, gender, smoking, and family history on autoimmune thyroid disease phenotype. J Clin Endocrinol Metab 2006;91:4873-80.
36. Acharya SH, Avenell A, Philip S, et al. Radioiodine therapy (RAI) for Graves' disease (GD) and the effect on ophthalmopathy: a systematic review. Clin Endocrinol (Oxf) 2008;69:943-50.
37. Rapoport B, Alsabeh R, Aftergood D, McLachlan SM. Elephantiasic pretibial myxedema: insight into and a hypothesis regarding the pathogenesis of the
extrathyroidal manifestations of Graves' disease. Thyroid 2000;10:685-92.
38. Fairweather D, Frisancho-Kiss S, Rose NR. Sex differences in autoimmune disease from a pathological perspective. Am J Pathol 2008;173:600-9.
39. Estienne V, Duthoit C, Reichert M, et al. Androgen-dependent expression of FcgammaRIIB2 by thyrocytes from patients with autoimmune Graves' disease: a possible molecular clue for sex dependence of autoimmune disease. FASEB J 2002;16:1087-92.
40. Cutolo M, Sulli A, Capellino S, et al. Sex hormones influence on the immune system: basic and clinical aspects in autoimmunity. Lupus 2004;13:635-8.
41. Moshkelgosha S, So PW, Deasy N, et al. Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves' orbitopathy induced by thyrotropin receptor plasmid-in vivo electroporation.
Endocrinology;154:3008-15.
42. Whitacre CC. Sex differences in autoimmune disease. Nat Immunol 2001;2:777- 80.
43. Brix TH, Knudsen GP, Kristiansen M, et al. High frequency of skewed X- chromosome inactivation in females with autoimmune thyroid disease: a possible explanation for the female predisposition to thyroid autoimmunity. J Clin Endocrinol Metab 2005;90:5949-53.
44. Ozcelik T, Uz E, Akyerli CB, et al. Evidence from autoimmune thyroiditis of skewed X-chromosome inactivation in female predisposition to autoimmunity. Eur J Hum Genet 2006;14:791-7.
45. Yin X, Latif R, Tomer Y, Davies TF. Thyroid epigenetics: X chromosome inactivation in patients with autoimmune thyroid disease. Ann N Y Acad Sci 2007;1110:193-200.
46. Simmonds MJ, Kavvoura FK, Brand OJ, et al. Skewed X chromosome
inactivation and female preponderance in autoimmune thyroid disease: an association study and meta-analysis. J Clin Endocrinol Metab;99:E127-31.
47. Szymanski K, Miskiewicz P, Pirko K, et al. rs3827440, a nonsynonymous single nucleotide polymorphism within GPR174 gene in X chromosome, is associated with
Graves' disease in Polish Caucasian population. Tissue Antigens;83:41-4.
48. Chu X, Shen M, Xie F, et al. An X chromosome-wide association analysis identifies variants in GPR174 as a risk factor for Graves' disease. J Med
Genet;50:479-85.
