GPR174 and ITM2A Gene Polymorphisms rs3827440 and rs5912838 on the X chromosome in Korean Children with Autoimmune Thyroid Disease

(1) Background: Autoimmune thyroid diseases (AITDs) are female predominant and much attention has been focused on G protein-coupled receptor 174 (GPR174) and integral membrane protein 2A (ITM2A) on the X chromosome as Grave’s disease (GD) susceptible locus. (2) Methods: We genotyped four single nucleotide polymorphisms (SNPs), rs3810712, rs3810711, rs3827440, and rs5912838, of GPR174 and ITM2A in 115 Korean children with AITD (M = 25 and F = 90; GD = 74 (14.7 ± 3.6 years), HD = 41 (13.4 ± 3.2 years); GD-thyroid-associated ophthalmopathy (TAO) = 40, GD-non-TAO=34) and 204 healthy Korean individuals (M = 104 and F = 100). The data were analyzed by sex-stratified or combined. (3) Results: Three SNPs, rs3810712, rs3810711 and rs3827440, were found to be in perfect linkage disequilibrium (D’ = 1, r2 = 1). In AITD, HD, GD, GD-TAO, and GD-non-TAO patients, rs3827440 TT/T and rs5912838 AA/A were susceptible and rs3827440 CC/C and rs5912838 CC/C were protective genotypes. When analyzed by sex, rs3827440 TT and rs5912838 AA were susceptible and rs3827440 CC and rs5912838 CC were protective genotypes in female AITD, GD, GD-TAO, and GD-non-TAO subjects. In male AITD patients, rs3827440 T and rs5912838 A were susceptible and rs3827440 C and rs5912838 C were protective genotypes. (4) Conclusions: Polymorphisms in GPR174 and ITM2A genes on the X chromosome might be associated with AITD in Korean children.


Introduction
Autoimmune thyroid disease (AITD) may occur when genetically susceptible individuals are exposed to environmental triggers such as infection, iodine, or stress [1]. The genetic factor has a major role in AITD etiology and the heritability of Graves' disease (GD) has been reported to be 79% [2]. AITD encompasses GD and Hashimoto's thyroiditis (HD) [3]. HD seems to involve a CD4 Th1 response. The effects of antibodies and effector T cells specific for thyroid antigens lead to the progressive destruction of normal thyroid tissue. The autoimmune response in GD is biased towards a CD4 Th2 progressive destruction of normal thyroid tissue. The autoimmune response in GD is biased towards a CD4 Th2 response and is focused on antibody production. The production of anti-thyroid Stimulating Hormone (TSH) receptor antibodies promotes chronic overproduction of thyroid hormone [3]. However, the fact that GD and HD are commonly observed in the same family tree reflects a similar genetic basis for these diseases [4,5]. In early onset autoimmune disease, genetic susceptibility might be greater concern than in late onset cases [6]. We have reported an increase in allele frequencies of HLA-B*46, -DRB1*08, and -Cw*01 [7]. The statistical significance in our previous study was much higher than observed in other studies conducted on Korean adults [8], which might suggest that early-onset AITD is more influenced by genetic factors than late-onset AITD.
The female predominance in autoimmune disease has long been recognized and the most striking sex differences in prevalence are observed in AITD (>80% women) [9,10]. However, the biology of sexual dimorphism in AITD is not clearly understood. Recently, a great deal of attention has focused on sexual dimorphism in the immune response. Sex dimorphisms in immune response appear to be partially associated with direct genetic differences such as X chromosome and linked genes, and sex hormone and sex-specific regulation of immune-related genes [11]. The X chromosome contains approximately 1000 genes, including many immune-related genes that encode receptors and associated proteins, immune response-related proteins and transcriptional and translational regulator [12]. Some genes located on the X chromosome may play an important role in susceptibility to GD [13]. Furthermore, X chromosome inactivation and skewing might be important contributors to the increased risk for AITD in females [14][15][16].
Previous studies found associations with genes on the X chromosome in patients with AITD. Associations of AITD have been reported with polymorphisms of FOXP3 in Caucasian [17] and Japanese [18], TLR7 in a Chinese Cantonese population [19] and IRAK1 in Chinese [20]. We also reported that polymorphisms of IRAK1 gene on X chromosome is associated with HD in Korean children [21]. Recently, the rs3827440 and rs5912838 single nucleotide polymorphisms (SNPs) in G protein-coupled receptor 174 (GPR174) and integral membrane protein 2A (ITM2A) on the X chromosome were suggested to be GD-susceptible loci after the major histocompatibility complex region [13,22]. However, to the best of our knowledge, there have been no reports on possible associations of GPR174 and ITM2A polymorphisms with AITD in Korean children. In this study, we investigated the role of GPR174 and ITM2A polymorphisms (rs3810712, rs3810711, rs3827440, rs5912838) with AITD in Korean children ( Figure 1A Boxes are colored deep red if the D' values are high, which means LD is more strong. Especially, rs3810712, rs3810711 and rs3827440 were found to be in perfect linkage disequilibrium (D' = 1, r 2 = 1).

Subjects
The present study is a noninterventional registry study. There were 206 patients diagnosed with AITD who agreed to participate in this study between March 2009 and August 2019 in the pediatric endocrine clinic at Seoul St. Mary's and St. Vincent's Hospitals. Among these 206, subjects who had blood sample insufficient for genetic study (n = 91) were excluded. Ultimately, our study included 115 patients (90 females and 25 males) diagnosed with AITD (41 HD and 74 GD cases). The mean age (±SD) of GD patients at enrollment was 14.7 ± 3.6 years and HD patients was 13.4 ± 3.2 years. Among the 74 GD patients, 40 patients had thyroid associated ophthalmopathy (TAO) ( Table 1). For the control group, 204 healthy and genetically unrelated Korean adults (100 females, 104 males) without a history of AITD were included. The control group was mainly comprised of students and staff from the Medical College of the Catholic University of Korea and hematopoietic stem cell transplantation (HSCT) center. In general, the health status of students is considered to be free of special problems. All subjects provided informed consent to participate in a genetic study. The Institutional Review Board of the Catholic University of Korea approved our study (IRB Number: KC09FISI0042, MC13SISI0126).
HD was diagnosed when at least three of Fisher's criteria [26] were met: (1) goiter, (2) diffuse goiter and decreased uptake at thyroid scan, (3) the presence of circulating thyroglobulin and/or microsomal autoantibodies, and (4) hormonal evidence of hypothyroidism. GD diagnosis was based on confirmation of clinical symptoms and the biochemical confirmation of hyperthyroidism, including the observation of goiter, elevated 131 I uptake by the thyroid gland, positive TSH receptor antibodies and elevated thyroid hormone levels. In GD, a remission was defined as consistent with the improvement of clinical features and restoration of euthyroidism or induction of hypothyroidism after ATD therapy. We defined an intractable as hyperthyroidism persistent over 2 years of ATD therapy or relapsed after ATD withdrawal or had been treated ATD for at least 5 years [23][24][25]. Patients who had other autoimmune diseases, hematologic diseases and endocrine diseases were excluded. TAO was diagnosed based on the presence of typical clinical features and classified according to the system recommended by the American Thyroid Association Committee. Patients with no symptoms or only lid lag sign were included in the without-TAO group [27]. Patients with soft tissue changes, proptosis, extraocular muscle dysfunction, or the latter two symptoms, were considered to have TAO [28].

DNA Extraction
Genomic DNA was extracted from 4 mL of peripheral blood mixed with ethylenediaminetetraacetic acid using TIANamp Genomic DNA Extraction Kits (Tiangen Biotech Corporation, Beijing, China), according to the manufacturer's instructions. The concentration of the DNA solution was adjusted to 100 ng/µL, and the solution was stored at −20 • C. Samples were used as a polymerase chain reaction (PCR) template for genotyping [29].

Target Gene Primer Design and Multiplex PCR
Four primers designed for GPR174 (rs3810712: C>T), GPR174 (rs3810711: T>C), GPR174 (rs3827440: T>C), GPR174 and ITM2A (rs5912838: A>C) are listed in Table 2. Genomic DNA was acquired from a variety of samples, and AITD pediatric patient and control groups were amenable to analysis using a 50 ng PCR template, or less. The fist amplicons were made by a multiplex PCR process using a Multiplex kit (Cat: 206143; Qiagen, Hilden, Germany) with other genes. In the first PCR, the extracted genomic DNA was amplified in a ProFlex 96-Well PCR System (Thermo Fisher Scientific Waltham, MA, USA) using the following PCR conditions: 1 cycle at 95 • C for 15 min and 40 cycles of denaturation at 94 • C for 30 s, annealing at 63 • C for 90 s, and extension at 72 • C for 30 s. Final extension was at 60 • C for 30 min. In order to enable MiSeq equipment (Illumina, San Diego, CA, USA) to read nucleotides around the target SNP in the next step [30], the PCR2 was carried out with PCR1 amplicon and Illumina universal primer [31]. The universal primer consists of tag, index and adapter sequences. Using 8 bases for each index, to distinguish each sample of PCR1 amplicon, 8 forward primers and 6 reverse primers were employed as universal primers, resulting in 48 unique combinations for PCR2 (Table 2) [32]. The primer information is provided by Illumina, Inc. for SNP typing. The PCR2 primer amplification was performed in a ProFlex 96-Well PCR System (Thermo Fisher Scientific) using the following conditions: 1 cycle at 95 • C for 15 min and 35 cycles of denaturation at 95 • C for 30 s, annealing at 59 • C for 30 s, and extension at 72 • C for 60 s. Bold-face font indicates PCR1 primer region. Underlined regions indicate universal forward and reverse primer tags sequence. The universal primer consists of tag, index and adapter sequences. Between the tag sequence and the Illumina adapter sequence exists an index sequence to specify each sample. Index sequences used in this study (5 -3 ): forward (TATAGCCT, ATAGAGGC, CCTATCCT, G GCTCTGA, AGGCGAAG, TAATCTTA, CAGGACGT, GTACTGAC) and reverse (ATTACTCG, TCCGGAGA, CGCTCATT, GAGATTCC, ATTCAGAA, GAATTCGT); a span: the distance between forward and reverse primers on target sequence.

Sequencing
We used Illumina MiSeq equipment for typing of target X chromosome genes. A sample sheet was prepared on the MiSeq sequencer (Illumina) to provide run details [33]. A standard flow-cell was inserted into the flow-cell chamber. The pooled sample was diluted with chilled HT1 (Hybridization Buffer) to a concentration of 2 nM, and an equal amount of 0.2 N NaOH was added to denature the sample; the mixture was incubated for 5 min. A PhiX control sample at 2 nM was denatured in the same way. Both the sample and the PhiX were diluted to 8 pmol/L and 1% PhiX was added to the sample. Then, 600 µL of the spiked sample with a final concentration of 8 pmol/L was pipetted into the sample well on the MiSeq consumable cartridge before loading in the cooling section of the MiSeq machine. Sequencing was performed on a MiSeq sequencer using 151 bp paired-end reads, including an index run according to the manufacturer's instructions (MiSeq System user guide part #15027617 Rev. C April 2012, MiSeq Reagent kit 300 cycles, Box1 (ref 15026431) and Box2 (ref 15026432)) [34]. To the validation, Sanger sequencing and Integrative Genomics Viewer (IGV) version 2.5.2 were implemented [35].

Data Analysis
Data analysis was performed using the MiSeq output report binary alignment map (BAM) file mapping Burrows-Wheeler Aligner (BWA) Whole Genome Sequencing v1.0. (Illumina) [36]. The proliferation of web-based integrative analysis frameworks has enabled users to perform complex analyses directly through the web using Galaxy [37]. We performed the following steps: Pre-process Next-generation sequencing (NGS)data: (1) MAP with BWA and add read group reference: hg19 [36,38]. The sequence alignment/map format (BAM) dataset was preprocessed from the Illumina MiSeq instrument. (2) VARIANT ANNOTATOR (Galaxy tool) Options: parse variant call format (VCF) () to extract counts, major, minor alleles and minor allele frequency (MAF); MAF threshold >= 0%; coverage >= 0; do not filter sites.

Statistical Analysis
Allele frequencies were determined using Microsoft Office Excel. Fisher's exact test was applied when the expected frequency was lower than 5. The p value was multiplied by the number of alleles observed to give a corrected p value (Pc), which accounts for the multiple comparisons performed. A corrected p value of <0.05 was considered statistically significant. Haldane's formula correction was used when critical entries were equal to zero. The data from cases and controls were analyzed by separate sex-stratified or all combined. Hardy-Weinberg equilibrium (HWE) of each SNP in GPR174 and ITM2A were analyzed according to the calculation proposed by Graffelman and Weir [25]. All genotyped SNPs fit the HWE (see Supplementary Tables S1 and S2). To evaluate the presence of linkage disequilibrium (LD) and block of haplotypes between polymorphisms on GPR174 and ITM2A, LD and haplotypes were analyzed using the Haploview software, version 4.2 [39].
Based on GPR174 and ITM2A SNPs MAF (minor allele frequency) of 25% in a dominant model and predicted prevalence of AITD at 2% [40], unmatched case-control design, a total of 315 participants is required to yield statistical power of 80% and type 1 error of 5%. The power of our study was calculated based on an available sample size of 115 cases and 204 controls. The power of MAF 25.0 for rs3810712, rs3810711, rs3827440 and rs5912838 were 0.71-0.83 in the odds ratio (OR) from 1.8 to 2.0. In the OR from 0.2 to 0.5, it was 0.99-0.80. In GD cases, the power was 0.58-0.72 in the OR from 1.8 to 2.0. In the OR of 0.5, it was 0.67. Sample size and power were calculated using Quanto 1.2.4 software (preventivemedicine.usc.edu/ Los Angeles, CA, USA).

Comparison of Genotype and Allele Frequencies of GPR174 and ITM2A SNPs on X chromosome in HD Patients and Controls
In patients with HD (n = 41), the genotype frequencies of rs3827440 CC/C (Pc = 0.017), and rs5912838 CC/C (Pc = 0.012) were lower than those of controls (n = 204)

Discussion
GPR174, which is located in the Xq21.1 region, encodes a protein member of the P2Y receptor family. Lyso-phosphatidyl-serine secreted by the immune system acts as a ligand for GPR174. GPR174 is extensively expressed in the immune system and thyroid tissue and inhibits the production of the T-helper 1 cytokine [13]. ITM2A, which is encoded by a gene also located in the Xq21.1 region, is expressed by CD4+ T cells and plays a role in the activation of T cells [41]. The SNP rs3827440 of GPR174 is a nucleotide transition in the single exon of GPR174 that causes an amino acid substitution [13]. The amino acid substitution in GPR174 maps to the second extracellular loop region, which is required for ligand recognition and receptor activation, and this mutation may alter these activities [42]. A nucleotide transition (519T>C) in rs3827440 causes serine [TCT]> proline [CCT] [43]. The SNPs rs3810711 and rs3810712 are both located in the 5 UTR of GPR174. The SNP rs5912838, located between the immune receptor GPR174 and ITM2A, was identified as an important signal on Xq21.1 associated with GC in a Chinese Han population [22]. Two SNPs, rs3810711 and rs3810712, both located in the 5 UTR of GPR174, have been reported in perfect LD with rs3827440 [13]. In the present study, we also found rs3827440 to be in perfect LD with rs3810711 and rs3810712 and in strong LD with rs5912838 ( Figure 1B).
Our study revealed that rs3827440 TT/T and rs5912838 AA/A were disease-susceptible genotypes and rs3827440 CC/C and rs5912838 CC/C were disease-protective genotypes in the overall AITD, HD, GD, GD-TAO and GD-non-TAO patient groups. When cases and controls were analyzed according to sex, rs3827440 TT and rs5912838 AA were disease-susceptible genotypes and rs3827440 CC and rs5912838 CC showed disease-protective genotypes in female AITD, GD, GD-TAO, and GD-non-TAO patients. In male AITD patients, rs3827440 T and rs5912838 A were disease-susceptible genotypes and rs3827440 C and rs5912838 C were disease-protective genotypes. The T allele of rs3827440 in GPR174 has been suggested to be GD susceptible in both Chinese and Polish population [22,44]. Chu et al. found that freshly isolated peripheral blood cells from both female homozygous carriers and male carriers of the risk allele T rs3827440 showed a higher level of GPR174 expression [13]. Ye et al. showed that the transcription level of ITM2A in PBMCs from volunteers was regulated by the different alleles of rs3827440 and its linked SNP rs5912838 [45]. Lyso-phosphatidyl-serine acts via GPR174 and G s α subunit to suppress IL-2 production by activated T cells and limit upregulation of the activation markers CD25 and CD69 [46]. Some studies suggested GPR174 as an abundantly expressed gas-dependent receptor that can negatively regulate naive T-cell activation [46]. Thus, GPR174 could mediate an important regulatory pathway connected to central T cell development and peripheral function [47]. In additions, the GPR174 and ITM2A transcripts in CD4 and CD8 T, Natural killer (NK), and monocyte cells with rs3827440 CC or rs5912838 CC genotypes were lower than those with rs3827440 TT or rs5912838 AA genotypes (https://dice-database.org/eqtls). These results might suggest that rs3827440 TT or rs5912838 AA could be more the causative variants in AITD children in males, females and a combination of the sexes.
In genomic linkage and association studies, the X chromosome is less well studied than autosomes, predominantly because of the higher complexity of analyses [47]. Traits and markers on the X chromosome are different from autosomal markers with respect to HWE [48]. The complicating factor for assessing deviation from HWE is that males are hemizygous, and have only one allele on X-chromosomal markers outside of the pseudoautosomal regions, while females have two alleles on the autosomes [49]. Some ignore male subjects and conduct tests for HWE in females only. However, this reduces the sample size and results in a loss of power, and if males are neglected, deviation from HWE cannot be thoroughly investigated [49]. In this present study, we confirmed that the data were in HWE using the calculation proposed by Graffelman and Weir [25]. We specifically analyzed data from cases and controls by sex-stratification or a combination of the sexes to assess the role of GPR174 and ITM2A in immunopathogenesis of female-predominant AITD. To the best of our knowledge, this is the first report of an association between GPR174 and ITM2A SNPs and children with AITD. However, there are limitations of the small sample size, and the controls were not investigated via laboratory methods to exclude subclinical cases of AITD in this study. To confirm the role of GPR174 and ITM2A genes in AITD, further studies on gene expression of immune cells with ethnically diverse populations including large numbers of patients are necessary.
In conclusion, we found that rs3827440 TT/T and rs5912838 AA/A in GPR174 and ITM2A genes on the X chromosome were disease-susceptible genotypes and rs3827440 CC/C and rs5912838 CC/C were disease-protective genotypes among female, male and combined AITD patient groups. In investigating X chromosome data, full use of samples and detailed analysis of data are necessary for case and control disease association genetic studies. These results suggest that polymorphisms in GPR174 and ITM2A genes on the X chromosome play a role in the immunopathogenesis of female-predominant AITD in Korean children.