Robust evidence for five new Graves' disease risk loci from a staged genome-wide association analysis.

Graves' disease (GD), characterized by autoantibodies targeting antigens specifically expressed in thyroid tissues causing hyperthyroidism, is triggered by a combination of genetic and environmental factors. However, only a few loci for GD risk were confirmed in the various ethnic groups, and additional genetic determinants have to be detected. In this study, we carried out a three-stage study in 9529 patients with GD and 9984 controls to identify new risk loci for GD and found genome-wide significant associations in the overall populations for five novel susceptibility loci: the GPR174-ITM2A at Xq21.1, C1QTNF6-RAC2 at 22q12.3-13.1, SLAMF6 at 1q23.2, ABO at 9q34.2 and an intergenic region harboring two non-coding RNAs at 14q32.2 and one previous indefinite locus, TG at 8q24.22 (Pcombined < 5 × 10(-8)). The genotypes of corresponding variants at 14q32.2 and 8q24.22 were correlated with the expression levels of C14orf64 and a TG transcript skipping exon 46, respectively. This study increased the number of GD loci with compelling evidence and indicated that non-coding RNAs might be potentially involved in the pathogenesis of GD.

Identification of BACH2 as a susceptibility gene for Graves' disease in the Chinese Han population based on a three-stage genome-wide association study.

The BACH2 gene regulates B cell differentiation and function and has been reported to be a shared susceptibility gene for several autoimmune diseases. Our previous genome-wide association study (GWAS) indicated that several single nucleotide polymorphisms (SNPs) in the BACH2 gene are associated with Graves' disease (GD) in the Chinese Han population; however, the association did not achieve genome-wide significance levels. Recently, this association of BACH2 with GD was confirmed in Caucasians in the UK population, but fine mapping in this region has not yet been reported. Here, we provide a refined analysis of a 331-kb region in the BACH2 gene, which harbors 359 SNPs, using GWAS data from 1,442 GD patients and 1,468 controls. The SNPs rs2474619 and rs9344996 were implied as the independent variants associated with GD by forward and two-locus logistic regression analysis. We genotyped eight out of 10 tagSNPs with P < 1 × 10(-3) in 3,508 GD patients and 3,209 controls, the results also showed that rs2474619 was independently associated with GD in the combined population from GWAS and the second stage (P = 1.81 × 10(-5)). The rs2474619 and rs9344996 were further genotyped in the third stage cohorts, and rs2474619 showed evidence of association with GD at genome-wide significance levels in the combined population (P = 3.28 × 10(-8), odds ratio = 1.13). The association of rs9344996 with GD can be explained by its linkage to rs2474619 in the combined population. Our study clearly demonstrated that BACH2 is a susceptibility gene for GD in the Chinese Han population and further supported rs2474619, in intron 2 of BACH2, is the best association signal with GD. However, the mechanism by which BACH2 confers increased risk of GD requires further study.

Dense mapping of IL2RA shows no association with Graves' disease in Chinese Han population.

OBJECTIVE: Associations between IL2RA and various autoimmune diseases have been reported in Caucasians. We investigated whether genetic polymorphisms at the IL2RA locus were associated with Graves' disease (GD) in the Chinese Han population. DESIGN: We performed a genome-wide association study (GWAS) in 1 536 GD patients and 1 516 controls. The 1000 Genomes Project data were adopted as references for imputation analysis. After forward and conditional logistic regressions, we found that rs11256313 was the major risk variant in the CD25/IL2RA region. Thus, we further genotyped rs11256313 in a replication cohort with 3 694 GD patients and 3 510 controls using ABI 7900HT TaqMan Real-Time PCR System. RESULTS: Nine single nucleotide polymorphisms (SNPs) in the IL2RA block were nominally associated with GD in our GWAS (0·01 < P < 0·05). After imputation analysis, 13 imputed SNPs in the IL2RA block were weakly associated with GD (P ≤ 0·05). Logistic regression analysis suggested that the imputed rs11256313 could represent the IL2RA block (P = 0·003). However, we failed to replicate the association of rs11256313 in a larger cohort (P = 0·145). A subphenotype analysis of rs11256313 on thyroid hormone receptor antibody (TRAb) and gender showed that there was no association in any of the subphenotype groups (P > 0·05). CONCLUSIONS: The results suggested that common genetic polymorphisms at IL2RA do not exert a significant genetic effect on the development of GD in the Chinese Han population. Previously reported associations between CD25/IL2RA and autoimmune diseases including GD in Caucasians again imply that heterogeneity exists in different ethnic populations.

Functional study of an aberrant splicing variant of the human luteinizing hormone (LH) receptor.

The luteinizing hormone receptor (LHR) is a member of a subfamily of G protein-coupled receptors that is characterized by its alternative splicing. In a previous study, we identified a splice site mutation of intron 6 (IVS6-3C>A) in a patient suffering from Leydig cell hypoplasia, which leads to aberrant splicing of LHR mRNA. In vitro expression analysis confirmed that this mutation results in the skipping of exon 7 in the mature mRNA of the LHR gene. In this study, we determined the impact of IVS6-3C>A on the RNA secondary structure and function of LHR-Del7. The three-dimensional structure of the leucine-rich repeats in LHR was predicted by molecular modeling. Radioactive ligand-binding assays verified that LHR-Del7 has no binding affinity for hCG. Furthermore, we detected negligible cAMP production in cells transfected with LHR-Del7. Cells co-expressing LHR-WT and LHR-Del7 were able to generate cAMP in response to hCG, but there was no significant difference between cells transfected with LHR-WT/vector and LHR-WT/LHR-Del7, although the variant was able to localize to cell surface, similar to wild-type receptor. These results indicated that LHR-Del7 does not have a dominant negative effect on LHR-WT cell surface expression, and although the pathological splicing variant LHR-Del7 was able to localize to cell membranes it failed to bind hCG and had no effect on wild-type LHR.

The mechanism of mimecan transcription induced by glucocorticoid in pituitary corticotroph cells.

Mimecan, a secretary protein that is expressed in mouse and human pituitary corticotroph cells, is up-regulated by glucocorticoids (GC) in the corticotroph cells via classical glucocorticoid receptor (GR) pathways. In this study, we further explore the GC mechanism for mimecan expression in these cells. Five putative GR response elements (GREs) were identified in ~2 kb of the mimecan promoter by programme analysis. An EMSA assay further indicated that these putative GREs were bound by the GR. Moreover, three proximal GREs are conserved between species. Although luciferase assays showed that the -1474/+43 region of the mimecan promoter achieved the highest expression of mimecan, the -803/+43 mimecan promoter region was sufficient for the GC-mediated expression of mimecan. The mutations of three conserved GREs located in the -1474/+43 mimecan promoter region did not affect mimecan transcription, which suggests that the effects of GC on mimecan are independent of the GREs in the promoter. In addition, cycloheximide, a protein synthesis inhibitor, blocked GC-induced mimecan expression in AtT-20 cells. Taken together, these results suggest that, although there are 3-5 GREs in the mimecan promoter, GC may regulate mimecan transcription indirectly through the synthesis of intermediate proteins and not through the GREs in pituitary corticotroph cells.

Assessment of Molecular Subtypes in Thyrotoxic Periodic Paralysis and Graves Disease Among Chinese Han Adults: A Population-Based Genome-Wide Association Study.

Importance: Thyrotoxic periodic paralysis (TPP) is a potentially lethal complication of hyperthyroidism. However, only 1 specific susceptibility locus for TPP has been identified. Additional genetic determinants should be detected so that a prediction model can be constructed. Objective: To investigate the genetic architecture of TPP and distinguish TPP from Graves disease cohorts. Design, Setting, and Participants: This population-based case-control study used a 2-stage genome-wide association study to investigate the risk loci of TPP and weighted genetic risk score to construct a TPP prediction model with data from a Chinese Han population recruited in hospitals in China from March 2003 to December 2015. The analysis was conducted from November 2014 to August 2016. Main Outcomes and Measures: Loci specifically associated with TPP risk and those shared with Graves disease and prediction model of joint effects of TPP-specific loci. Results: A total of 537 patients with TPP (mean [SD] age, 35 [11] years; 458 male) 1519 patients with Graves disease and no history of TPP (mean [SD] age, 38 [13] years; 366 male), and 3249 healthy participants (mean [SD] age, 46 [10] years; 1648 male) were recruited from the Han population by hospitals throughout China. Two new TPP-specific susceptibility loci were identified: DCHS2 on 4q31.3 (rs1352714: odds ratio [OR], 1.58; 95% CI, 1.35-1.85; P = 1.24 × 10-8) and C11orf67 on 11q14.1 (rs2186564: OR, 1.50; 95% CI, 1.29-1.74; P = 2.80 × 10-7). One previously reported specific locus was confirmed on 17q24.3 near KCNJ2 (rs312729: OR, 2.08; 95% CI, 1.83-2.38; P = 8.02 × 10-29). Meanwhile, 2 risk loci (MHC and Xq21.1) were shared by Graves disease and TPP. After 2 years of treatment, the ratio of persistent thyrotropin receptor antibody positivity was higher in patients with TPP than in patients with Graves disease and no history of TPP (OR, 3.82; 95% CI, 2.04-7.16; P = 7.05 × 10-6). The prediction model using a weighted genetic risk score and 11 candidate TPP-specific single-nucleotide polymorphisms had an area under the curve of 0.80. Conclusions and Relevance: These findings provide evidence that TPP is a novel molecular subtype of Graves disease. The newly identified loci, along with other previously reported loci, demonstrate the growing complexity of the heritable contribution to TPP pathogenesis. A complete genetic architecture will be helpful to understand the pathophysiology of TPP, and a useful prediction model could prevent the onset of TPP.

ITM2A Expands Evidence for Genetic and Environmental Interaction in Graves Disease Pathogenesis.

Context: Graves disease (GD) is a common autoimmune disease triggered by genetic predisposition and environmental factors. However, the mechanisms of interaction between genetic and environmental factors contributing to the development of GD remain unknown. Objective: We aimed to identify GD susceptibility variants and genes on Xq21.1 locus and interpret the contribution of interaction between genetic predisposition on Xq21.1 and environmental factors to GD. Design: We performed refining study on Xq21.1 in a 2-stage study and carried out expression quantitative trait locus analysis of the best association signal with GD. Setting and Participants: A total of 4316 GD patients and 4374 sex-matched controls were collected from the Chinese Han population by cooperation with multiple hospitals. Results: We identified that rs3827440 or its linkage single nucleotide polymorphisms (SNPs) were probably the causal variant in the Xq21.1 locus, with the most substantial association with GD in our combined cohorts (P = 2.45 × 10-15). The genotypes of rs3827440 were correlated with the expression of ITM2A in monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Notably, the expression of ITM2A in monocytes after lipopolysaccharide (LPS) and interferon-γ (INF-γ) stimulation showed substantial difference among the volunteers that carried different genotypes of rs3827440 (P = 9.40 × 10-7 and P = 1.26 × 10-5 for 24 hours' LPS and INF-γ stimulation, respectively). Moreover, ITM2A expression was significantly decreased in PBMCs from untreated GD patients than that from controls. Conclusion: The results suggest that ITM2A might be a susceptibility gene for GD in the Xq21.1 locus, and environmental factors, such as viral and bacterial infections, probably contribute to GD pathogenesis by interacting with the risk SNP rs3827440 mediating the regulation of ITM2A expression.

Refined association of TSH receptor susceptibility locus to Graves' disease in the Chinese Han population.

BACKGROUND: Convincing evidence has demonstrated the association of TSH receptor (TSHR) with Graves' disease (GD) in the Chinese Han population. OBJECTIVE: The aim of this study was to identify the causal variants for GD in the region encompassing TSHR by a refining association study. DESIGN AND METHODS: GD patients (1536) and 1516 sex-matched controls were recruited in the first stage, and an additional 3832 GD patients and 3426 sex-matched controls were recruited in the replication stage. Genotyping was performed using Illumina Human660-Quad BeadChips or TaqMan single nucleotide polymorphism (SNP) Genotyping Assays and the Fluidigm EP1 platform. RESULTS: When the results of regression analysis for 74 genotyped SNPs and 922 imputed SNPs in the first-stage cohort were combined, rs179243 and rs3783949 were the probable susceptibility SNPs associated with GD in TSHR. Eleven SNPs, including rs179243 and rs3783949, were selected to further refine the association in the replication study. Finally, rs12101261 and rs179243 were confirmed as independent GD susceptibility variants in the replication and combined populations. Further, we also found that the rate of persistent TSHR autoantibody positivity (pTRAb+) was significantly higher in the GD patients with the susceptible genotypes rs12101261 or rs179243 than in the GD patients carrying the protective genotypes, after the GD patients had been treated for more than 1 year. CONCLUSIONS: These findings indicate that rs12101261 and rs179243 are the possible causal SNPs for GD susceptibility in the TSHR gene and could serve as genetic markers to predict the outcome of pTRAb+ in GD patients.

Identification of a novel mutation in CYP17A1 gene.

17α-hydroxylase/17,20-lyase deficiency (17OHD) is a rare autosomal recessive genetic disease that is characterized by low-renin hypertension, hypokalemia, and abnormal development of the genitalia. Mutations in the CYP17A1 gene account for this disease. We aim to investigate the CYP17A1 mutation and analyze its possible influence on phenotype in a Chinese patient with 17OHD. Steroid hormones were assayed. The 8 exons of the CYP17A1 gene were amplified and directly sequenced. Wild-type and mutant CYP17A1 cDNA were cloned into pcDNA3.1 expression vectors and transfected into 293T cells. Finally, 17-hydroxylase and 17,20-lyase activity were detected by using progesterone and 17-hydroxypregnenolone as the substrates. A novel missense mutation c.716 G>A located in exon 4 that changed the amino acid from arginine to glutamine (R239Q) was discovered in the patient. Steric model analysis of CYP17A1 showed that R239Q changed the local structure and the electrostatic potential. Functional study indicated that the R239Q mutant caused the complete loss of both 17α-hydroxylase and 17,20-lyase activities. Our study expanded the CYP17A1 mutation spectrum. With a functional study, we confirmed that the novel mutation caused the complete loss of both 17α-hydroxylase and 17,20-lyase activities.

A refined study of FCRL genes from a genome-wide association study for Graves' disease.

To pinpoint the exact location of the etiological variant/s present at 1q21.1 harboring FCRL1-5 and CD5L genes, we carried out a refined association study in the entire FCRL region in 1,536 patients with Graves' disease (GD) and 1,516 sex-matched controls by imputation analysis, logistic regression, and cis-eQTL analysis. Among 516 SNPs with P<0.05 in the initial GWAS scan, the strongest signals associated with GD and correlated to FCRL3 expression were located at a cluster of SNPs including rs7528684 and rs3761959. And the allele-specific effects for rs3761959 and rs7528684 on FCRL3 expression level revealed that the risk alleles A of rs3761959 and C of rs7528684 were correlated with the elevated expression level of FCRL3 whether in PBMCs or its subsets, especially in CD19(+) B cells and CD8(+) T subsets. Next, the combined analysis with 5,300 GD cases and 4,916 control individuals confirmed FCRL3 was a susceptibility gene of GD in Chinese Han populations, and rs3761959 and rs7528684 met the genome-wide association significance level (P(combined) = 2.27×10(-12) and 7.11×10(-13), respectively). Moreover, the haplotypes with the risk allele A of rs3761959 and risk allele C of rs7528684 were associated with GD risk. Finally, our epigenetic analysis suggested the disease-associated C allele of rs7528684 increased affinity for NF-KB transcription factor. Above data indicated that FCRL3 gene and its proxy SNP rs7528684 may be involved in the pathogenesis of GD by excessive inhibiting B cell receptor signaling and the impairment of suppressing function of Tregs.

Mimecan in pituitary corticotroph cells may regulate ACTH secretion and the HPAA.

Mimecan is a protein of unknown function that is expressed in the pituitary tissues of mouse and human. In this study, we observed the function of mimecan on the proopiomelanocortin (POMC) gene in the pituitary and the hypothalamo-pituitary-adrenal axis (HPAA). Incubating pituitary corticotroph AtT-20 cells with recombinant mimecan protein stimulated adrenocorticotrophic hormone (ACTH) secretion without significantly up-regulating POMC gene expression. In addition, pituitary corticotroph AtT-20 cell corticotropin-releasing hormone receptor 1 (CRHR1) gene expression was induced by mimecan. Interestingly, long-term mimecan overexpression in corticotroph cells increased CRHR1 mRNA levels while slightly decreasing POMC mRNA expression and ACTH secretion. Using mimecan knockout mice, we found that, although the serum ACTH concentration was not significantly different between wild type and mimecan knockout mice under basal conditions, the serum ACTH level was relatively lower in mimecan knockout mice after treatment with corticotropin-releasing hormone (CRH). Meanwhile, we observed that POMC and CRHR1 gene expression decreased in primary cultured knockout mouse pituitary cells compared with wild type cells. Taken together, these data suggest that mimecan expressed in pituitary corticotroph cells mainly regulates ACTH secretion in the pituitary and coordinates the HPAA.

A genome-wide association study identifies two new risk loci for Graves' disease.

Graves' disease is a common autoimmune disorder characterized by thyroid stimulating hormone receptor autoantibodies (TRAb) and hyperthyroidism. To investigate the genetic architecture of Graves' disease, we conducted a genome-wide association study in 1,536 individuals with Graves' disease (cases) and 1,516 controls. We further evaluated a group of associated SNPs in a second set of 3,994 cases and 3,510 controls. We confirmed four previously reported loci (in the major histocompatibility complex, TSHR, CTLA4 and FCRL3) and identified two new susceptibility loci (the RNASET2-FGFR1OP-CCR6 region at 6q27 (P(combined) = 6.85 × 10(-10) for rs9355610) and an intergenic region at 4p14 (P(combined) = 1.08 × 10(-13) for rs6832151)). These newly associated SNPs were correlated with the expression levels of RNASET2 at 6q27, of CHRNA9 and of a previously uncharacterized gene at 4p14, respectively. Moreover, we identified strong associations of TSHR and major histocompatibility complex class II variants with persistently TRAb-positive Graves' disease.