Treatment of Obesity with Thyroid hormones in Europe. Data from the THESIS* Collaboration.

PURPOSE: The use of thyroid hormones (TH) to treat obesity is unsupported by evidence as reflected in international guidelines. We explored views about this practice, and associations with respondent characteristics among European thyroid specialists. METHODS: Specialists from 28 countries were invited to a survey via professional organisations. The relevant question was whether "Thyroid hormones may be indicated in biochemically euthyroid patients with obesity resistant to lifestyle interventions". RESULTS: Of 17,232 invitations 5695 responses were received (33% valid response rate; 65% women; 90% endocrinologists). Of these, 290 (5.1%) stated that TH may be indicated as treatment for obesity in euthyroid patients. This view was commoner among non-endocrinologists (8.7% vs. 4.7%, p < 0.01), private practice (6.5% vs. 4.5%, p < 0.01), and varied geographically (Eastern Europe, 7.3%; Southern Europe, 4.8%; Western Europe, 2.7%; and Northern Europe, 2.5%). Respondents from Northern and Western Europe were less likely to use TH than those from Eastern Europe (p < 0.01). Gross national income (GNI) correlated inversely with this view (OR 0.97, CI: 0.96-0.97; p < 0.001). Having national guidelines on hypothyroidism correlated negatively with treating obesity with TH (OR 0.71, CI: 0.55-0.91). CONCLUSIONS: Despite the lack of evidence, and contrary to guidelines' recommendations, about 5% of respondents stated that TH may be indicated as a treatment for obesity in euthyroid patients resistant to life-style interventions. This opinion was associated with (i) respondent characteristics: being non-endocrinologist, working in private practice, treating a small number of hypothyroid patients annually and (ii) national characteristics: prevalence of obesity, Eastern Europe, low GNI and lack of national hypothyroidism guidelines.

Sexual function in women with androgen excess disorders: classic forms of congenital adrenal hyperplasia and polycystic ovary syndrome.

PURPOSE: We compared the sexual function in women with classic forms of congenital adrenal hyperplasia (CAH) and polycystic ovary syndrome (PCOS) to find if the cause of androgen excess determines sexual functioning. METHODS: Hundred and four women (21 with CAH, 63 with PCOS and 20 healthy controls) aged 18-40 years were included into the study. All participants completed a questionnaire regarding their sociodemographic background and underwent anthropometric and basic biochemical measurements. Plasma levels of total testosterone, androstenedione, and 17-hydroxyprogesterone were measured with immunoassay. To assess the sexual functions, the Female Sexual Function Index (FSFI) questionnaire was applied. RESULTS: Apart from the higher physical activity in PCOS patients (P = 0.017), we found no significant sociodemographic differences between the studied groups. In clinical assessment, women with CAH had a lower incidence of acne (P = 0.006). Their plasma levels of 17OHP (P = 0.005) and insulin resistance index (P = 0.0248) were higher, while total testosterone (P = 0.0495) and glucose (P = 0.0061) was lower compared to the PCOS group. Significantly more women with CAH were homosexual (P = 0.003) and bisexual (P = 0.006). CAH group showed a lower total FSFI score (P = 0.0043) and lower scores in three domains: lubrication (P = 0.0131), sexual satisfaction (P = 0.0006), and dyspareunia (P < 0.0001). Higher physical activity was associated in all women with higher total FSFI score (P = 0.009) and scores in the domain of desire (P = 0.034) and sexual satisfaction (P = 0.01), while in CAH women apart from the total score (P = 0.03) and sexual satisfaction (P = 0.002) also in the domains of orgasm (P = 0.005), and pain (P = 0.03). CONCLUSIONS: CAH women present more often homosexual and bisexual orientation, while their sexual functions are impaired compared to PCOS patients.

High-dose intravenous methylprednisolone therapy in patients with Graves' orbitopathy is associated with the increased activity of factor VIII.

PURPOSE: Venous thromboembolic events (VTE), with their life-threatening manifestation as pulmonary embolism, are potential adverse effects of intravenous methylprednisolone (IVMP) pulse therapy, partially due to a hypercoagulable state. The aim of the study was to analyze the influence of IVMP on selected hemostatic parameters in patients with moderate-to-severe Graves' orbitopathy (GO). METHODS: 26 euthyroid patients with GO were treated with 12 pulses of IVMP (6 × 0.5, 6 × 0.25 g every week). Hemostatic variables [factor (F) II, FV, FVII, FVIII, fibrinogen, antithrombin, activated partial thromboplastin time (aPTT), prothrombin time, international normalized ratio of prothrombin time, platelets and D-dimer] were analysed before, 24 and 48 h after 1st, 6th and 12th pulse. RESULTS: A constant, transient trend in changes of some hemostatic variables was observed after all assessed pulses. We discovered an increase in median activity of FVIII 24 and 48 h after pulses, with a shortening of aPTT 24 h after each IVMP pulse (p < 0.00005). FVII decreased 24 h after each pulse (p < 0.0005 after 1st and 12th, p < 0.00005 after 6th). Fibrinogen level decreased 48 h after each pulse (P < 0.00005). We did not observe any statistically significant changes in hemostatic parameters in the long-term evaluation. Therapy was concluded in one patient after the 9th pulse due to pulmonary embolism. CONCLUSIONS: The increase of FVIII activity is a consequence of treatment with IVMP and occurs after each pulse. In patients with additional risk factors of VTE, anticoagulation prophylaxis should be considered.

DNA methylation in ELOVL2 and C1orf132 correctly predicted chronological age of individuals from three disease groups.

Improving accuracy of the available predictive DNA methods is important for their wider use in routine forensic work. Information on age in the process of identification of an unknown individual may provide important hints that can speed up the process of investigation. DNA methylation markers have been demonstrated to provide accurate age estimation in forensics, but there is growing evidence that DNA methylation can be modified by various factors including diseases. We analyzed DNA methylation profile in five markers from five different genes (ELOVL2, C1orf132, KLF14, FHL2, and TRIM59) used for forensic age prediction in three groups of individuals with diagnosed medical conditions. The obtained results showed that the selected age-related CpG sites have unchanged age prediction capacity in the group of late onset Alzheimer's disease patients. Aberrant hypermethylation and decreased prediction accuracy were found for TRIM59 and KLF14 markers in the group of early onset Alzheimer's disease suggesting accelerated aging of patients. In the Graves' disease patients, altered DNA methylation profile and modified age prediction accuracy were noted for TRIM59 and FHL2 with aberrant hypermethylation observed for the former and aberrant hypomethylation for the latter. Our work emphasizes high utility of the ELOVL2 and C1orf132 markers for prediction of chronological age in forensics by showing unchanged prediction accuracy in individuals affected by three diseases. The study also demonstrates that artificial neural networks could be a convenient alternative for the forensic predictive DNA analyses.

rs3827440, a nonsynonymous single nucleotide polymorphism within GPR174 gene in X chromosome, is associated with Graves' disease in Polish Caucasian population.

Recently Chu et al. conducted a two-stage genome wide association study in Chinese that identified a novel X-linked Graves' disease (GD) susceptibility marker at rs3827440 - a nonsynonymous (P162S) nucleotide transition (519C

The replication of the association of the rs6832151 within chromosomal band 4p14 with Graves' disease in a Polish Caucasian population.

Recently Chu et al. conducted a genome-wide association study in a Chinese Han population and identified two novel Graves' disease (GD) susceptibility loci within 4p14 (rs6832151) and 6q27 (rs9355610). Our purpose was to replicate these associations in a Polish Caucasian population. We analyzed rs6832151 and rs9355610 genotypes in a case-control study based on 560 GD patients and 1475 unrelated controls using TaqMan assays. Our study had the power of 0.8 and 0.6 to detect the effects originally reported for rs6832151 and rs9355610, respectively. We found an association between GD and the rs6832151 G allele (odds ratio OR = 1.27, P = 0.002). Analysis of model of inheritance suggested that the dominant model should be preferred (P(fit) = 0.938, OR = 1.39, P = 0.001). For rs9355610 a formally significant effect was observed assuming a recessive model (OR = 1.24, P = 0.028), whereas analysis of allele distribution showed a trend for association (OR = 1.14,95%, P = 0.082). Our findings are the first to show that rs6832151 and possibly rs9355610 contribute to GD pathogenesis also in Caucasians.

Functionally defective germline variant of sialic acid acetylesterase (Met89Val) is not associated with type 1 diabetes mellitus and Graves' disease in a Polish population.

Recently, rare genotypes encoding defective variants of sialic acid acetylesterase (SIAE), such as homozygous Met89Val substitution, were strongly associated [odds ratio (OR) = 8] with a panel of autoimmune diseases including type 1 diabetes mellitus (T1DM). Our purpose was to replicate this finding in T1DM and explore whether Met89Val predisposes to Graves' diseases (GD). We studied 561 GD patients, 379 T1DM patients and 1822 controls. The prevalence of Met89Val homozygosity was similar among patients (GD: 0.4%, n = 2; T1DM: 0.3%, n = 1) and controls (0.4%, n = 7) yielding OR of 0.93 [95% confidence interval (CI): 0.19-4.48, P = 0.9] and 0.69 (95% CI: 0.08-5.59, P = 0.71) for GD and T1DM, respectively. We conclude that further studies are needed before the proposed strong effect of defective SIAE variants on susceptibility to autoimmunity can be universally accepted.

Vitamin D-binding protein (DBP) gene polymorphism is associated with Graves' disease and the vitamin D status in a Polish population study.

OBJECTIVE: Vitamin D-binding protein (DBP) genetic variants have an influence on vitamin D status and, therefore, they may contribute to the development of autoimmune diseases. In this case-control study, we investigated the association of DBP gene polymorphisms with susceptibility to Graves' disease (GD) in a Polish population. Furthermore, we analyzed the distribution of DBP genotypes in GD patients divided according to the clinical (gender, age of onset, ophthalmopathy, family history, smoking habits) and genetic parameters (CTLA4 49G and HLA-DRB1*03 alleles), as well as the vitamin D serum levels. METHODS: 332 polish patients with GD and 185 healthy controls were genotyped for the DBP gene single nucleotide polymorphisms (SNPs) at codon 420 ACG --> AAG (Thr --> Lys) and at codon 416 GAT --> GAG (Asp --> Glu) by the PCR-RFLP method. The variable (TAAA)N repeat polymorphism in the intron 8 was analyzed in 332 patients and 164 controls by the PCR amplification followed by the PAGE. In addition, 25(OH)D3 serum levels were measured in 110 patients. RESULTS: In patients with GD, the frequency of the Lys allele (34.2% vs. 25.7%, p = 0.005; OR = 1.50; 95% CI: 1.13-1.99) at codon 420 was significantly higher compared to controls. The distribution of codon 420 genotypes also differed significantly (p = 0.01), with the frequency of the Lys/Lys homozygotes (9.3% vs. 5.9%; OR = 1.63; 95% CI: 0.80-3.32) being higher in GD. The distribution of codon 416 alleles and genotypes did not differ in both studied groups (p = 0.59 and p = 0.81, respectively). Analysis of the intron 8 (TAAA)N repeat polymorphism led to the identification of a novel variant in the Polish population, described as 7 repeats, but no association between the intron 8 alleles and GD was observed. The 420 Lys allele was associated with lower 25(OH)D3 serum levels (p = 0.01). No correlation between the DBP genotypes and other susceptibility alleles or the GD clinical phenotype was observed. CONCLUSIONS: (i) The DBP gene Lys allele at codon 420 confers susceptibility to GD in the Polish population, (ii) the codon 416 alleles and intron 8 (TAAA)N variants are not associated with susceptibility to and clinical phenotype of GD, and (iii) the codon 420 Lys allele correlates with lower 25(OH)D3 serum concentration.

Cytokine profiles in eye muscle tissue and orbital fat tissue from patients with thyroid-associated ophthalmopathy.

Eye muscle (EM) and retroorbital fat tissue are two major sites of involvement in thyroid-associated ophthalmopathy (TAO). Lymphocytic infiltration in these tissues is a prominent histological feature of TAO. We have investigated the cytokine gene profiles in EM and orbital fat (OF) tissues from patients with TAO. Total RNA was isolated from EM tissue of 14 patients and from OF tissues of 29 patients with TAO. Cytokine gene expression was assessed by RT-PCR using paired primers for interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, CD4, CD8, and glyceraldehyde-3-phosphate dehydrogenase. IFNgamma, TNFalpha, IL-1beta, and IL-6 messenger RNA (mRNA) were mainly detected in EM tissue, whereas IL-4 and IL-10 mRNA were detected in only one patient. On the other hand, in OF tissue, IL-4 and IL-10 mRNA were detected in 24% and 38% of the patients, respectively, and IFNgamma, IL-1beta, and IL-6 mRNA were less often detected compared with EM tissue. The enlargement of EM tissue as assessed by computed tomography correlated significantly with TNFalpha mRNA expression in EM tissue. The orbital volume was positively correlated with IL-6 mRNA expression and negatively correlated with IL-4 mRNA and IL-10 mRNA expression in OF tissue. These results suggest that T helper (Th) 1-like cytokines predominate in EM tissue in most patients and that the predominant cytokine profile in OF tissue varies from patient to patient. Both Th1-like and Th2-like immune responses may play roles in the development of two components of ophthalmopathy.

T cell interactions with extracellular matrix proteins in patients with thyroid-associated ophthalmopathy.

Although thyroid-associated ophthalmopathy (TAO) is now generally accepted as an autoimmune inflammatory disorder of the extraocular muscles and the orbital connective tissue, its aetiopathogenesis remains poorly understood. Recent data indicate that impaired interactions between T cells and extracellular matrix (ECM) proteins may play an important role in development and maintaining of an inflammatory process. We report here results of the study focusing on interactions between T lymphocytes and collagen-I (Coll-I), collagen-IV (Coll-IV), fibronectin (FN), laminin (LM) in patients with TAO. Using a standard peripheral blood mononuclear cells (PBMC) proliferation assay, we observed a markedly enhanced T cell response to Coll-I in patients with active TAO (mean SI=4.5). The proliferatory response to Coll-I was significantly greater (Wilcoxon test; p < 0.001) than in normal subjects (mean SI=1.88), patients with stable TAO (mean SI=2.05) and patients with thyroid autoimmune diseases (AITD) without ophthalmopathy (mean SI=2.49). PBMC stimulation by Coll-I is likely to be antigen-dependent requiring engagement of the T cell receptor with collagen peptides, rather than mediated via integrins. The percentage of circulating CD29+ (beta1 integrin chain) T cells was not increased in patients with active TAO. Additionally in the assay of costimulation of CD3-mediated proliferation, we found that peripheral blood T cells from patients with TAO and AITD were costimulated only by FN. On the other hand a markedly enhanced costimulation of CD3-mediated proliferative responses by Coll-I, Coll-IV, FN and LM were observed in a retrobulbar T cell line. We conclude that abnormalities in T cell interactions with ECM proteins, especially Coll-I may play a role in the pathogenesis of TAO.

Increased serum soluble Fas in patients with Graves' disease.

We addressed the role of soluble Fas (sFas), which suppresses Fas-mediated apoptosis, in the pathogenesis of Graves' disease (GD). The serum concentration of sFas was measured by enzyme-linked immunosorbent assay and the expression of sFas mRNA in thyroid tissues by reverse transcriptase-polymerase chain reaction. The serum concentration of sFas was significantly increased in untreated GD (mean+/-SD: 1.57+/-0.48 ng/mL) compared to age-matched control subjects (0.77+/-0.46 ng/mL). The serum sFas level tended to decrease after the medication of antithyroid drugs for 6 to 8 weeks and was significantly decreased in patients who were euthyroid for more than 3 years (0.98+/-0.23 ng/mL), compared to that in untreated GD. The concentration of serum sFas was significantly correlated with anti-thyrotropin (TSH) receptor antibody titers, but not with the other clinical parameters (free triiodothyronine [FT3], free thyroxine [FT4], TSH, antithyroglobulin antibody titer, antimicrosomal antibody titer, or 123I uptake). The sFas mRNA was detected in thyroid tissue, cultured thyrocytes, and intrathyroidal lymphocytes. sFas was detected in supernatant of cultured thyrocytes from patients with GD. Its production by thyrocytes was induced by culture with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). The present study confirms serum sFas increases in GD and provides evidence of local production of sFas by thyrocytes and its regulation by cytokines. These data suggest that sFas may play a role in the pathogenesis of GD.

Role of the eye muscles in thyroid eye disease: identification of the principal autoantigens.

Thyroid-associated ophthalmopathy (TAO) is a progressive orbital disorder associated with Graves' hyperthyroidism and, less often, Hashimoto's thyroiditis in which autoantibodies react with orbital antigens and lead to exophthalmos and eye muscle inflammation. Eye muscle (EM) membrane proteins initially reported as 55 and 64 kd are the best markers of ophthalmopathy. The "64-kd protein" is now shown to be the flavoprotein subunit of mitochondrial succinate dehydrogenase and to have a correct molecular weight of 67 kd. We have cloned a fragment of a novel eye muscle protein, which we call G2s, and sequenced 1.4 kb of the full length cDNA. G2s does not share any significant homologies with other reported proteins. The 5.9 kb G2s mRNA, that corresponds to a protein of approximately 220 kd, is expressed in EM, other skeletal muscle and thyroid, but not in other tissues tested. We have also cloned and sequenced a 63-kd eye muscle protein identified as the calcium binding protein calsequestrin. Antibodies against calsequestrin were found in 40% of patients with active ophthalmopathy, but in 0% of normal subjects. Finally, we have sequenced a 19 amino acid fragment of a 55-kd porcine eye muscle membrane protein that exactly matched porcine and human sarcalumenin, a 160-kd glycoprotein localized in the lumen of the longitudinal sarcoplasmic reticulum of the skeletal muscle fiber where it binds calcium. A 53-kd glycoprotein fragment of the molecule corresponds to the 55-kd protein. In a preliminary study, serum antibodies against purified sarcalumenin were detected in 40% of patients with active TAO of less than 1 year duration, but in no controls tested. We porpose that the primary autoantigen in TAO is G2s, which would also explain the association of ophthalmopathy with thyroid autoimmunity, and that antibodies against the intracellular proteins flavoprotein, calsequestrin, and sarcalumenin are secondary markers of an immune-mediated reaction in eye muscle in patients with thyroid autoimmunity.

Autoantibodies reactive with extracellular matrix proteins in patients with thyroid-associated ophthalmopathy.

Autoantibodies reacting with extracellular matrix proteins have been extensively studied in various autoimmune connective tissue diseases. Because of the possibility that such antibodies may play a role in orbital connective tissue inflammation in thyroid-associated ophthalmopathy (TAO), we studied the humoral immune response against specific extracellular matrix (ECM) proteins, namely: collagen types I, III, IV, V (CI, CIII, CIV, CV), fibronectin (FN), and laminin (LM). Anti-ECM antibodies of immunoglobulin G (IgG), IgA, and IgM classes were determined by enzyme linked immunosorbent assay (ELISA). Overall, sera from 50% of patients with TAO contained antibodies reactive against one or more ECM proteins, compared to 27% with Graves' disease (GD) without evident eye involvement, 28% with Hashimoto's thyroiditis (HT), and 9% of normal subjects. Serum anti-CI, anti-CIII, anti-CV and anti-LM levels were significantly (p<0.05) higher in patients with TAO than in normals. Anti-CI, anti-CV and anti-LM reactivity was antigen-specific in most TAO sera, while anti-CIII antibodies cross-reacted with other antigens. Anti-collagen antibodies were mainly of the IgG class. To determine the structural epitopes of these proteins, we performed immunoblotting studies on cyanogenbromide (CNBr)-derived peptides of CI and CV. While sera from 9 of 10 patients with TAO reacted with CI peptides, the response was polyclonal and uniform in all patients. However, only 2 of 10 TAO sera reacted with CV peptides. In conclusion, our study suggests that a variety of ECM proteins (CI, CV, LM) may be secondary autoantigens that are recognized by antibodies in TAO. While these antibodies appear to react with epitopes expressed on both native and denatured proteins, and may therefore have the potential to bind to ECM in vivo, their pathogenic role in TAO remains unknown.

[Pharmacologic effect of excess iodine on type I thyroxine 5'-deiodinase activity in rat thyroid]. / Wplyw farmakologicznych dawek jodu na aktywnosc enzymu tyroksyna-5'-dejodynazy typu I w gruczole tarczowym szczurów.

Many mechanism may participate in the inhibition of thyroid function by excess of iodine. The present study was aimed at answering the question, whether the excess of iodine influences the activity of type I thyroxine 5'-deiodinase. To 40 female rats fed with standard diet a dose of 42 micrograms of iodine daily was administered into the stomach through the gastric tube. Control group consisting of 10 rats received standard diet only. Part of the animals was sacrificed after each of the consecutive time intervals (2, 4, 7 and 14 days of treatment with iodine) and the activity of deiodinase in the thyroid as well as the blood serum levels of hormones, T3 and T4, were determined. A decrease in the activity of deiodinase in comparison with control group was observed during the whole experiment, the highest inhibition occurring after 4 days of treatment with iodine. A fall in the concentration of T3 was observed after the second and fourth dose of iodine, with no change in T4 concentration. The results of the experiments indicate that the excess of iodine inhibits the activity of type I thyroxine 5'-deiodinase in the thyroid.

Association of NFKB1 -94ins/del ATTG promoter polymorphism with susceptibility to and phenotype of Graves' disease.

Recently, a functional polymorphism in the NFKB1 gene promoter (-94ins/del ATTG) has been identified and associated with chronic inflammatory diseases. The aim of this study was to analyze the association of NFKB1 polymorphism with susceptibility to and phenotype of Graves' disease (GD). The initial case-control association study, performed in a Polish-Warsaw cohort (388 GD patients and 688 controls), was followed by the two replication studies performed in Polish-Gliwice and Japanese-Kurume cohorts (198 GD patients and 194 controls, and 424 GD patients and 222 controls, respectively). The frequency of the -94del ATTG (D) allele was increased in GD compared to controls in Warsaw cohort. This finding was replicated in Gliwice cohort. Combining both Polish-Caucasian cohorts showed that the NFKB1 polymorphism was significantly associated with susceptibility to GD with a codominant mode of inheritance (P=0.00005; OR=1.37 (1.18-1.60)). No association with GD was found in Japanese cohort. However, subgroup analysis in Japanese GD patients revealed a correlation between the NFKB1genotype and the development of ophthalmopathy (P=0.009; OR=1.49 (1.10-2.01)), and the age of disease onset (P=0.009; OR=1.45 (1.09-1.91)). Our results suggest that NFKB1 -94ins/del ATTG polymorphism may be associated with susceptibility to and/or phenotype of GD.

IL-18 gene polymorphism confers susceptibility to the development of anti-GAD65 antibody in Graves' disease.

AIMS: This study aimed to investigate whether interleukin-18 (IL-18) gene polymorphisms are associated with the development of antibody against the 65-kDa isoform of recombinant human glutamic acid decarboxylase (GAD65Ab) in patients with Graves' disease. METHODS: A total of 398 unrelated Japanese patients with Graves' disease, with and without GAD65Ab, were recruited. Three single nucleotide polymorphisms in the IL-18 gene were examined and the polymorphic allele and the genotype and haplotype frequencies calculated. RESULTS: The frequency of the GG genotype at position -4675 of the IL-18 gene was significantly lower in Graves' disease patients with GAD65Ab than those without (4% vs. 24%, P = 0.0126). The -4675C allele frequency was significantly greater in patients with GAD65Ab than those without (69% vs. 53%, P = 0.0168). The homozygous -4675G/-607A/-137G haplotype was less common in Graves' disease patients with GAD65Ab than those without (4% vs. 23%, P = 0.0144). CONCLUSIONS: These findings in a Japanese population indicate that Graves' disease patients carrying the GG genotype at position -4657 of the promoter of the IL-18 gene or a gene in linkage disequilibrium with the -4675G/-607A/-137G haplotype have a low risk for the development of GAD65Ab in Graves' disease.

Development of Graves' disease in a patient under immunosuppressive therapy after liver transplantation.

Susceptibility to Graves' disease (GD) is determined by multiple environmental and genetic factors, which are not fully understood. Because of the autoimmune etiology of the disease, recent reports describing the development of GD during long-term immunosuppressive treatment seem quite surprising. We report a second case of GD in a 17-yr old patient, treated with cyclosporin A and prednisone, after liver transplantation. The development of GD despite adequate immunosuppressive therapy may suggest that this patient had a genetic predisposition to autoimmunity and severe immunoregulatory defects. We analyzed the HLA-DRB1 alleles and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene polymorphism (A/G) at position 49 in exon 1. The patient had the HLA-DRB1*03 allele which is known to confer susceptibility to GD. Further studies are necessary to identify genes that may predispose patients specifically to GD during immunosuppressive therapies.

A polymorphism of the 5' flanking region of tumour necrosis factor alpha gene is associated with thyroid-associated ophthalmopathy in Japanese.

OBJECTIVE: We have studied the polymorphism of the 5' flanking region of the tumour necrosis factor (TNF)-alpha gene in order to better understand the genetic background of autoimmune thyroid disorders and thyroid-associated ophthalmopathy. PATIENTS AND METHODS: We studied the polymorphism of the 5' flanking region of the TNF-alpha gene at positions - 1031 (T to C change, termed as - 1031C), - 863 (C to A, - 863 A), - 857 (C to T, - 857T), - 308 (G to A, - 308 A) and - 238 (G to A, - 238 A) in Japanese patients with Graves' disease [n = 173, 62 of whom had associated ophthalmopathy (American Thyroid Association (ATA) class III or greater)] and healthy control subjects (n = 575), using a polymerase chain reaction sequence-specific oligonucleotide probe method. RESULTS: The allele frequency of - 857T in the Graves' disease patients (22. 5% vs. 17.7%, OR = 1.35, P = 0.045, corrected P = 0.23) was slightly greater than in the Japanese healthy subjects, respectively. However, the difference was not statistically significant. In Graves' disease patients with evident ophthalmopathy (ATA class III or greater), the allele frequencies of - 1031C and - 863 A were significantly greater than those with no or mild ophthalmopathy (ATA class 0-II) (31.5% vs. 13.5%, OR =2.94, P < 0.0001, corrected P < 0. 0005; 23.4% vs. 11.7%, OR =2.30, P = 0.0044, corrected P = 0.022, respectively) and in control subjects. The strength of the association of the polymorphism - 1031C increased with the severity of ophthalmopathy, with odds ratios of 2.36 for ATA class III, and 5. 43 for ATA class IV-VI, respectively, compared with Graves' disease with no or mild ophthalmopathy (ATA class 0-II). Although the phenotype frequency of DRB1*0901 was not different among Graves' disease patients with or without ophthalmopathy and control subjects, the phenotype frequency of DRB1*0901(-)/-1031C(+) was significantly increased in Graves' disease patients with ophthalmopathy compared to those with no or mild ophthalmopathy (OR = 4.91, P = 0.0005) or control subjects (OR = 4.59, P < 0.0001). CONCLUSIONS: These results suggest that the - 1031C or - 863 A alleles, or a gene in linkage disequilibrium with the TNF-alpha gene, predispose to the development of ophthalmopathy in Japanese patients with Graves' disease.

Eye muscle antibodies in patients with ocular myasthenia gravis: possible mechanism for eye muscle inflammation in acetylcholine-receptor antibody-negative patients.

Myasthenia gravis is an organ-specific autoimmune disorder generally thought to be caused by an antibody-mediated attack against the skeletal muscle nicotinic acetylcholine (Ach) receptor (AchR) at the neuromuscular junction. Extraocular muscle weakness and double vision are present in about 90% of patients with myasthenia gravis and are the predominant complaints in about 20% of patients, when the condition is called ocular myasthenia gravis (OMG). While serum antibodies against the AchR are detected in most patients with generalized myasthenia gravis (GMG), they are not found in about one-third of patients with the ocular variety, and epidemiological, clinical, and serological studies suggest that OMG and GMG are two separate diseases. Both forms of myasthenia gravis are sometimes associated with thyroid autoimmunity or thyroid-associated ophthalmopathy (TAO). We have therefore tested the sera of patients with GMG and OMG by Western blotting for antibodies against porcine eye muscle membrane proteins in general, and by enzyme-linked immunosorbent assays (ELISA) specifically for reaction with two skeletal muscle antigens which are prominent marker antigens for TAO, namely, the calcium-binding protein calsequestrin and the so-called "64-kDa protein." The 64-kDa protein has recently been identified as the flavoprotein subunit of mitochondrial succinate dehydrogenase. Patients with ophthalmopathy and myasthenia were excluded. Nine of the patients had associated Graves' hyperthyroidism without evident ophthalmopathy and one had Hashimoto's thyroiditis. Antibodies against porcine eye muscle membrane antigens of M(r) 15-110 kDa were detected in patients with GMG or OMG, one or more antibodies being detected in 100% of patients with GMG and in 88% of those with OMG. The most frequently found antibodies were those targeting eye muscle membrane proteins of 15, 67, and 110 kDa. Antibodies reactive with purified calsequestrin (63 kDa) were detected in 21% of patients with OMG but in no patient with GMG. Antibodies recognizing purified succinate dehydrogenase (67 kDa) were found in 42% of patients with OMG, in 100% (5 of 5) of patients with GMG, and in 48% of all patients with myasthenia gravis not associated with Graves' hyperthyroidism. There was no close correlation between any eye muscle-reactive antibody and antibodies against the AchR in either group of myasthenic patients. The findings support the notion that immunoreactivity against skeletal muscle proteins other than the AchR may play a role in the development of the muscle weakness in AchR antibody-negative patients with OMG and GMG, although it is unlikely that any of the antibodies demonstrated in this study are directly implicated. Similarly, while the demonstration of antibodies reactive with eye muscle antigens associated with TAO in patients with OMG raises the possibility that the link between the ocular lesions of myasthenia gravis and Graves' disease may be autoimmunity against a common antigen(s), it is more likely that both disorders are mediated by cytotoxic T cells recognizing another cell membrane antigen, such as the novel thyroid and eye muscle shared protein G2s, and that serum antibodies reactive with succinate dehydrogenase Fp subunit and calsequestrin are markers of an immune-mediated eye muscle reaction.