Genetic Study in a Large Cohort Supported Different Pathogenesis of Graves' Disease and Hashimoto's Hypothyroidism.

CONTEXT: Hashimoto's thyroiditis (HT) and Graves' disease (GD) are the 2 main autoimmune thyroid diseases that have both similarities and differences. Determining the genetic basis that distinguishes HT from GD is key for a better understanding of the differences between these closely related diseases. OBJECTS: To identify the susceptibility genes for HT in the Chinese cohort and compare susceptibility genes between GD and HT. DESIGN: In the current study, 18 SNPs from 18 established GD risk loci were selected and then genotyped in 2682 patients with HT, 4980 patients with GD, and 3892 controls. The association analysis between HT and controls and heterogeneity analysis between HT and GD were performed on SPSS, with the logistic regression analysis adjusted for sex and age. RESULTS: We identified 11 susceptibility loci for HT in the Chinese Han population, with 4 loci, including the rs1265883 in SLAMF6 locus, rs1024161 in CTLA4, rs1521 in HLA-B, and rs5912838 in GPR174/ ITM2A at X chromosome, reaching genome-wide significance of 5 × 10-8. Five loci were reported to be associated with HT for the first time. We also identified 6 susceptibility loci with heterogeneity between GD and HT. Out of them, 4 loci were associated with GD but not with HT, including HLA-DPB1, CD40, TSHR, and TG; the association of HLA-B with GD was stronger than that with HT, but the association of SLAMF6 was reversed. CONCLUSION: Our findings suggested that the pathogenesis of HT and GD was different.

Potential role of IL-37 signaling pathway in feedback regulation of autoimmune Hashimoto thyroiditis.

IL-37, the anti-inflammatory cytokine of the IL-1 family, plays several key roles in the regulation of autoimmune diseases. Yet, its role in Hashimoto's thyroiditis (HT) is not clear. In the present study, we found that, in tissues from HT patients, most of the follicular epithelial cells were positive for both IL-37 and single Ig IL-1-related receptor (SIGIRR) by immunohistochemical staining, while the infiltrating lymphocytes and other inflammatory cells hardly expressed any. Meanwhile, mRNA expression levels of IL-37 in peripheral blood mononuclear cells (PBMC) of HT patients were significantly higher than those in normal controls measured by quantitative real-time PCR. Finally, we studied the possible role of IL-37 in IFN-γ-stimulated rat FRTL-5 cells. The results showed that IL-1ß, TNF-α, and MCP-1 mRNA levels were significantly decreased, while the expression of IL-4 mRNA was dramatically up-regulated in IFN-γ-stimulated rat thyroid cell line FRTL-5 pre-treated with IL-37. The current study, for the first time, demonstrated that the IL-37 network is involved in Hashimoto's thyroiditis, and IL-37 signaling pathway may ameliorate the excessive autoimmune responses in this chronic lymphocytic thyroiditis.

Uterus globulin associated protein 1 (UGRP1) is a potential marker of progression of Graves' disease into hypothyroidism.

Approximately 20% of Graves' disease (GD) patients may result eventually in hypothyroidism in their natural course. Uterus globulin-associated protein 1 (UGRP1) was associated with GD in our previous study. Here we investigated the role of UGRP1 in the development of autoimmune thyroid disease (AITD). The results showed that UGRP1 was expressed in the thyrocytes of most Hashimoto's thyroiditis (HT) patients and a proportion of GD patients (293 HT and 198 GD). The pathologic features of UGRP1-positive thyrocytes resembled "Hürthle cells", and were surrounded by infiltrated leukocytes. The positivity rate of TPOAb in UGRP1-positive GD patients was much higher than that in -negative GD patients. Moreover, UGRP1 was co-expressed with Fas and HLA-DR in the thyrocytes of AITD patients. We also found IL-1ß but not Th1 or Th2 cytokines was able to upregulate the expression of UGRP1. Our findings indicated that UGRP1 may be a novel marker in thyrocytes to predict GD patients who develop hypothyroidism.

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.

Mimecan, a Hormone Abundantly Expressed in Adipose Tissue, Reduced Food Intake Independently of Leptin Signaling.

Adipokines such as leptin play important roles in the regulation of energy metabolism, particularly in the control of appetite. Here, we describe a hormone, mimecan, which is abundantly expressed in adipose tissue. Mimecan was observed to inhibit food intake and reduce body weight in mice. Intraperitoneal injection of a mimecan-maltose binding protein (-MBP) complex inhibited food intake in C57BL/6J mice, which was attenuated by pretreatment with polyclonal antibody against mimecan. Notably, mimecan-MBP also induced anorexia in A(y)/a and db/db mice. Furthermore, the expression of interleukin (IL)-1ß and IL-6 was up-regulated in the hypothalamus by mimecan-MBP, as well as in N9 microglia cells by recombinant mouse mimecan. Taken together, the results suggest that mimecan is a satiety hormone in adipose tissue, and that mimecan inhibits food intake independently of leptin signaling by inducing IL-1ß and IL-6 expression in the hypothalamus.

Genome-wide association study identifies a novel susceptibility gene for serum TSH levels in Chinese populations.

Thyroid-stimulating hormone (TSH) is a sensitive indicator of thyroid function. High and low TSH levels reflect hypothyroidism and hyperthyroidism, respectively. Even within the normal range, small differences in TSH levels, on the order of 0.5-1.0 mU/l, are associated with significant differences in blood pressure, BMI, dyslipidemia, risk of atrial fibrillation and atherosclerosis. Most of the variance in TSH levels is thought to be genetically influenced. We conducted a genome-wide association study of TSH levels in 1346 Chinese Han individuals. In the replication study, we genotyped four candidate SNPs with the top association signals in an independent isolated Chinese She cohort (n = 3235). We identified a novel serum TSH susceptibility locus within XKR4 at 8q12.1 (rs2622590, Pcombined = 2.21 × 10(-10)), and we confirmed two previously reported TSH susceptibility loci near FOXE1 at 9q22.33 and near CAPZB at 1p36.13, respectively. The rs2622590_T allele at XKR4 and the rs925489_C allele near FOXE1 were correlated with low TSH levels and were found to be nominally associated to patients with papillary thyroid carcinoma (PTC) (OR = 1.41, P= 0.014 for rs2622590_T, and OR = 1.61, P= 0.030 for rs925489_C). The rs2622590 and rs925489 genotypes were also correlated with the expression levels of FOXE1 and XKR4, respectively, in PTC tissues (P = 2.41 × 10(-4) and P= 0.02). Our findings suggest that the SNPs in XKR4 and near FOXE1 are involved in the regulation of TSH levels.

Identifying a novel mutation of CYP17A1 gene from five Chinese 17α-hydroxylase/17, 20-lyase deficiency patients.

Mutations of CYP17A1 gene could cause complete or partial, combined or isolated 17α-hydroxylase/17,20-lyase enzyme deficiencies (17OHD). We intended to investigate the CYP17A1 mutation in five unrelated patients and analyze its possible influence on phenotype of an atypical 17OHD patient presented with micropenis, hypertension and intermittent hypokalemia. Steroid hormones were assayed in these patients. A novel missense mutation (c.1169C>G, p. Thr390Arg) located in exon 7 was detected in one of the patients. Homozygous c. 985_987delinsAA, p. Tyr329fs mutation was found in two patients, while compound heterozygous mutations (c. 985_987delinsAA, p. Tyr329fs/c. 932-939 del, p. Val311fs and c. 287G>A, p. Arg96Gln/c. 985_987delinsAA, p. Tyr329fs) were found in two other patients, respectively. Then, steric model analysis of CYP17A1 showed that the novel mutation T390R changed the local structure as well as the electrostatic potential of the nearby beta sheet. Finally, site-directed mutagenesis and in vitro expression were used to analyze the activity of novel mutant CYP17A1. It indicated the T390R mutant retained part of enzyme activity, which was consistent to the clinical features. In conclusion, we identified a novel missense mutation of CYP17A1 gene from a patient with micropenis, hypertension and intermittent hypokalemia, which varied from other four patients. It also expanded our understanding of genotype-phenotype correlation of the disease.

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.

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.

Glucocorticoid up-regulates mimecan expression in corticotroph cells.

Mimecan is a protein of unknown function that is expressed in the pituitary. The aim of this study is to clarify the regulation and intracellular localisation of mimecan gene expression in the pituitary. With immunohistochemistry, we observed that mimecan protein was co-expressed with ACTH in pituitary corticotroph cells. Northern and Western blot analyses revealed that mimecan expression and secretion in corticotroph cells were up-regulated by treating AtT-20 cells with glucocorticoid. Meanwhile, mimecan expression in rat primary culture pituitary cells was also promoted by glucocorticoid. Co-incubation of AtT-20 cells with RU486 and glucocorticoid completely reversed the induction of mimecan gene expression by glucocorticoid. In addition, luciferase reporter assays showed that the -1474/+43 promoter region of mimecan was sufficient for glucocorticoid-responsive mimecan expression. These data collectively suggest that mimecan expressed in pituitary corticotroph cells is increased by glucocorticoid and that the up-regulation may be mediated by the classical GR pathways.

Identification of steroid biosynthetic defects in genotype-proven heterozygous individuals for 17alpha-hydroxylase/17,20-lyase deficiency.

OBJECTIVE: P450c17 deficiency (17alpha-hydroxylase/17,20-lyase deficiency, 17OHD) is a rare form of congenital adrenal hyperplasia caused by CYP17A1 gene mutations. The D487_F489 deletion in exon 8 and Y329fs in exon 6 are relatively frequent mutations of the CYP17A1 gene in China that completely abolish the enzyme activity of P450c17. However, little remains known about steroid biosynthetic functions in carriers with these mutations in a single allele of the CYP17A1 gene, who are assumed to have 50% P450c17 activity. We investigated adrenal steroidogenic function in genotype-proven heterozygotes carrying such mutations in the CYP17A1 gene in vivo. PATIENTS AND DESIGN: Eight patients and fourteen family members from five families with 17OHD were recruited. The mutations of the CYP17A1 gene in these individuals were screened by sequencing. The hormonal response to cosyntropin (ACTH) was evaluated in the 14 genotype-proven carriers and 45 age- and gender-matched normal controls. RESULTS: Fourteen carriers of the CYP17A1 mutation - seven with the D487_F489 deletion, six with Y329fs and one with H373L - were identified from the five families with 17OHD. Compared with normal controls, carriers showed lower basal and ACTH-stimulated cortisol levels but higher ACTH-stimulated corticosterone levels. The ratios of corticosterone to cortisol in the genotype-proven heterozygotes were higher than those of the normal controls at the baseline and after cosyntropin stimulation. Similarly, the progesterone levels and the ratios of progesterone to 17-hydroxyprogesterone in the male heterozygotes were also higher than those of the normal controls, both before and after ACTH stimulation. CONCLUSION: Genotype-proven carriers of the CYP17A1 mutation who lack apparent clinical symptoms exhibit decreased adrenal 17alpha-hydroxylase activity and altered adrenal gland reserve for steroid biosynthesis.

[Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17a-hydroxylase/17, 20 lyase deficiency].

OBJECTIVE: To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17a-hydroxylase/17,20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. METHODS: Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. RESULTS: Seven patients (5 of them were 46,XX; 2 were 46,XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cortisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. CONCLUSION: The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD.There might be certain frequency of heterozygotes for D487_F489del in Chinese population.