Clinical Endocrinology (2014)

doi: 10.1111/cen.12640

ORIGINAL ARTICLE

The association of thyroid peroxidase antibody risk loci with susceptibility to and phenotype of Graves’ disease  ski†, Robin P. Peeters‡, Piotr Miskiewicz*, Eleonora Porcu§,¶, Giorgio Pistis§,¶, Aleksander Kus*, Konrad Szyman Serena Sanna§, Silvia Naitza§, Rafał Płoski†, Marco Medici‡ and Tomasz Bednarczuk* *Department of Internal Medicine and Endocrinology, Medical University of Warsaw, Warsaw, †Department of Medical Genetics, Centre for Biostructure, Medical University of Warsaw, Warsaw, Poland, ‡Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands, §Istituto di Ricerca Genetica e Biomedica (IRGB), Consiglio Nazionale delle Ricerche, c/o Cittadella Universitaria di Monserrato, Monserrato, Cagliari, and ¶Dipartimento di Scienze Biomediche, Universita di Sassari, Sassari, Italy

Summary Background Despite great progress, the genetic basis of Graves’ disease (GD) remains poorly understood. Recently, a population-based genomewide association study (GWAS) identified five novel loci (ATXN2/SH2B3, MAGI3, BACH2, TPO and KALRN) as significantly associated with the presence of thyroid peroxidase autoantibodies (TPOAbs), whereas several other loci showed suggestive association. Methods In this study, we investigated 16 single nucleotide polymorphisms (SNPs) associated with TPOAbs for the association with susceptibility to and phenotype of GD in a cohort of 647 patients with GD and 769 controls from a Polish Caucasian population. Results SNPs within/near HCP5 (rs3094228, P = 1
6 9 10 12, OR = 1
88), MAGI3 (rs1230666, P = 1
9 9 10 5, OR = 1
51) and ATXN2/SH2B3 (rs653178, P = 0
0015, OR = 1
28) loci were significantly associated with susceptibility to GD. Allele frequencies differed significantly in subgroups of patients with GD stratified by age of GD onset for HCP5 (P = 0
0014, OR = 1
50) and showed a suggestive difference for MAGI3 (P = 0
0035, OR = 1
50) SNPs. Although rs11675434 located near TPO showed no association with GD susceptibility, it was significantly associated with the presence of clinically evident Graves’ ophthalmopathy (GO, P = 5
2 9 10 5, OR = 1
64), and this effect was independent from smoking status, age of GD onset and gender. Conclusions This is the first study showing an association of the ATXN2/SH2B3 locus with susceptibility to GD. Furthermore, we observed a novel significant association within the HLA region at a SNP located near HCP5 and confirmed the association of the MAGI3 locus with GD susceptibility. HCP5 and

Correspondence: Tomasz Bednarczuk, Department of Internal Medicine and Endocrinology, Medical University of Warsaw, ul. Banacha 1a, 02-097 Warsaw, Poland. Tel.:+48 22 599 29 75; Fax: +48 22 599 19 75; E-mail: [email protected] © 2014 John Wiley & Sons Ltd

MAGI3 SNPs were further correlated with age of GD onset. Finally, we identified TPO as a new susceptibility locus for GO. (Received 1 August 2014; returned for revision 19 September 2014; finally revised 13 October 2014; accepted 17 October 2014)

Introduction Graves’ disease (GD), which is the most common cause of hyperthyroidism in countries with sufficient iodine intake, exhibits a complex pathogenesis affected by genetic, environmental and existential factors. It is estimated that the genetic impact is responsible for up to 79% of the susceptibility.1 The genetic predisposition to GD is likely to be polygenic, that is due to numerous polymorphisms with small individual effects. The most consistent associations with susceptibility to GD have been documented for polymorphisms within the HLA region as well as near or within CTLA-4, PTPN22, FCRL3, TSHR, TG, IL2RA, FOXP3 and CD40 genes.2,3 Most of these loci are associated with shared susceptibility to other autoimmune diseases, and TSHR is the only one specifically associated with GD.4 As these loci only explain approximately 10% of the genetic susceptibility to GD,5 various efforts have been performed to increase the number of risk loci, with only limited success. Whilst the autoimmune thyroid disease (AITD), including GD and Hashimoto’s thyroiditis (HT), is the most common autoimmune disorder, there have been surprisingly few genomewide association studies (GWAS) performed in GD so far, although they identified several novel risk loci.5–8 Another way to improve the understanding of the genetic basis of both Mendelian and complex diseases is to perform GWAS on related quantitative clinical parameters.9,10 In a recent multicentre population-based GWAS conducted by Medici et al. 11 for serum levels of thyroid peroxidase antibodies (TPOAbs), the authors reported five novel TPOAb risk loci, including ATXN2/SH2B3, MAGI3, BACH2, TPO and KALRN, as well as several additional SNPs that showed borderline significant associations. The appearance of TPOAbs 1

2 A. Kus et al. usually precedes the development of overt thyroid disease, including both HT and GD. As the majority of newly identified SNPs by Medici et al. have not been investigated in GD so far, the aim of the current study was to assess whether these variants are associated with susceptibility to and phenotype of GD.

Materials and methods Subjects A total of 647 unrelated patients with GD (517 females, 130 males) from the Polish Caucasian population were consecutively recruited from the Department of Internal Medicine and Endocrinology, Medical University of Warsaw. The analysed group comprises an extension of the cohort described in previous studies.12–15 The diagnosis of GD was established on the basis of clinical and biochemical symptoms of hyperthyroidism accompanied by diffuse goitre, detectable TSH receptor autoantibodies and/or increased radioiodine uptake, as previously described.12–15 The severity of the Graves’ ophthalmopathy was assessed using NOSPECS classification and class III or higher was considered clinically evident. The clinical characteristics of the study group are shown in Table 1. The control group consisted of 769 Polish unrelated adults (559 females, 210 males). They were randomly recruited among subjects requesting paternity testing who gave their consent for anonymous use of their genetic material for scientific research. This cohort has been used in previous studies.13 Written informed consents were collected from all of the patients, and the local ethical committee of Medical University of Warsaw approved the study. SNPs selection All 16 SNPs analysed in this study were selected from the study by Medici et al.11 rs73191621 was analysed instead of rs2010099 Table 1. Clinical characteristics of the study group Characteristics

N*

n (%)

Male Age of onset in years (mean  SD) Disease duration in years (mean  SD) Ophthalmopathy (NOSPECS class) Class 0–II Class III Class IV Class V Class VI Cigarette smokers Family history of AITD Therapy for hyperthyroidism Antithyroid drugs Radioactive iodine Surgery

647 609 609 603

130 (20
1) 39
98  14
78 4
78  6
09

*N, number available for analysis.

587 562 600

388 105 89 8 13 263 169

(64
3) (17
4) (14
8) (1
3) (2
2) (44
8) (30
1)

196 (32
7) 319 (53
2) 86 (14
3)

(D’ = 1
0, r2 = 1
0 in 1000 Genomes Phase I CEU population). The characteristics of the selected SNPs are shown in Table 2. Genotyping The SNPs were genotyped using TaqManâ SNP Genotyping Assays in OpenArray format (TaqManâ OpenArray Genotyping Plate Custom Format 16 kit) provided by Applied Biosystems, Woolston, Warrington, UK. The genotyping procedure was performed according to the manufacturer’s protocol on the QuantStudioTM 12K Flex instrument (Life Technologies Corporation, Carlsbad, California, USA). The overall genotyping call rate was 96
5%, whereas the genotyping call rate for each SNP is shown in Table 2. One SNP (rs301799, RERE) failed to reach a 95% call rate and was excluded from further analysis. Statistical analyses Genotype distributions of all SNPs among patients and controls were tested for Hardy–Weinberg equilibrium, showing no evidence of deviation. The allele and genotype frequencies in patients and controls were compared and analysed for association with susceptibility to GD using a chi-sqaure test. To prevent false-positive findings due to multiple testing, a 0
05/15 = 3
33 9 10 3 P-value cut-off level was used to declare statistical significance. All SNPs were subsequently analysed for genotype–phenotype correlations. The risk allele frequencies in subgroups of patients with GD stratified by the presence of clinically evident Graves’ ophthalmopathy (GO, assessed according to the NOSPECS classification (NOSPECS class 0–II vs NOSPECS class III–VI)) and age of GD onset (age of GD onset