Methylation levels of the IL10 gene in peripheral blood are related to the intractability of Graves' disease.

The prognosis of autoimmune thyroid diseases (AITDs), including Hashimoto's disease (HD) and Graves' disease (GD), is difficult to predict. DNA methylation regulates gene expression of immune mediating factors. Interleukin (IL)-10 is a Th2 cytokine that downregulates inflammatory cytokines produced by Th1 cells. To clarify the role of methylation of the IL10 gene in the prognosis of AITD, we evaluated the methylation levels of two CpG sites in the IL10 promoter using pyrosequencing. The methylation levels of the -185 CpG site of the IL10 gene were related to age and GD intractability in GD patients. Furthermore, the C carrier of the IL10-592 A/C polymorphism was related to low methylation levels of the -185 CpG site. The methylation levels of the IL10-185 CpG site of the IL10 gene were related to the intractability of GD and were lower in individuals with the C allele of the IL10-592 A/C polymorphism.

Rigorous evaluation of genetic and epigenetic effects on clinical laboratory measurements using Japanese monozygotic twins.

The investigation of environmental effects on clinical measurements using individual samples is challenging because their genetic and environmental factors are different. However, using monozygotic twins (MZ) makes it possible to investigate the influence of environmental factors as they have the same genetic factors within pairs because the difference in the clinical traits within the MZ mostly reflect the influence of environmental factors. We hypothesized that the within-pair differences in the traits that are strongly affected by genetic factors become larger after genetic risk score (GRS) correction. Using 278 Japanese MZ pairs, we compared the change in within-pair differences in each of the 45 normalized clinical measurements before and after GRS correction, and we also attempted to correct for the effects of genetic factors to identify Cytosine-phosphodiester-Guanine (CpG) sites in DNA sequences with epigenetic effects that are regulated by genetic factors. Five traits were classified into the high heritability group, which was strongly affected by genetic factors. CpG sites could be classified into three groups: regulated only by environmental factors, regulated by environmental factors masked by genetic factors, and regulated only by genetic factors. Our method has the potential to identify trait-related methylation sites that have not yet been discovered.

Intraindividual variation in histone acetylation and its impact on autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs), such as Graves' disease (GD) and Hashimoto's disease (HD), are organ-specific autoimmune diseases. Histone acetylation, especially that of histone H3, is an epigenetic mechanism that regulates gene expression and is associated with the development of autoimmune diseases. However, physiological variations in histone acetylation are not yet clear, and we believe that physiological variations should be examined prior to analysis of the role of histone H3 in the pathogenesis of AITDs. In this study, we analyzed histone H3 acetylation levels in peripheral blood mononuclear cells (PBMCs) using a histone H3 total acetylation detection fast kit. Blood samples were collected before meals, between 8:30-9:00 am, daily for 10 weeks to evaluate the daily variation. At 4 days, blood was also collected before meals three times a day (at 8:30-9:00, 12:30-13:00, and 16:30-17:00) to evaluate circadian variation. Then, histone H3 acetylation levels were evaluated in AITD patients to clarify the association with the pathogenesis of AITD. Although we could not find a common pattern of circadian variance, we observed daily variation in histone H3 acetylation levels, and their coefficient of variances (CVs) were approximately 48.3%. Then, we found that histone H3 acetylation levels were significantly lower in GD and HD patients than in control subjects and these differences were larger than the daily variation in histone acetylation. In conclusion, histone H3 acetylation levels were associated with the development of AITD, even allowing for daily variation.

Association of B7H3 and B7H4 gene polymorphisms and protein expression with the development and prognosis of autoimmune thyroid diseases.

BACKGROUND: The prognosis of autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and Hashimoto's disease (HD), varies among patients. B7-H3 and B7-H4, members of the B7 family of proteins, regulate immune response. To clarify the association of B7-H3 and B7-H4 with the pathogenesis and prognosis of AITDs, we examined the expression of the soluble and membrane form of B7-H3 and B7-H4 and genotyped single nucleotide polymorphisms (SNPs) in the B7H3 and B7H4 genes. METHODS: We examined the expression of the membrane form of B7-H3 and B7-H4 by flow cytometry and their soluble forms by enzyme-linked immunosorbent assay. We genotyped SNPs in B7H3 and B7H4 in 187 GD patients, 217 HD patients, and 110 healthy volunteers using the PCR-RFLP method. RESULTS: The frequency of the B7H3 rs3816661 CC genotype was higher in patients with severe HD. G carriers of B7H4 rs10754339 A/G and B7H4 rs13505 T/G were more frequent in patients with AITD. A carrier of B7H4 rs10158166 A/G and C carriers of B7H4 rs3806373 C/T were more frequent in patients with intractable GD. The proportion of B7-H3+ monocytes was higher in the CC genotype of B7H3 rs3816661 C/T than in the other genotypes and was lower in patients with GD and HD than in healthy controls. The concentration of soluble B7-H4 was lower in the TG genotype of B7H4 rs13505 T/G than in the TT genotype and was higher in patients with AITD than in healthy controls. CONCLUSION: B7H3 and B7H4 are associated with AITD susceptibility and prognosis.

PD-1 gene polymorphisms and thyroid expression of PD-1 ligands differ between Graves' and Hashimoto's diseases.

The programmed cell death-1 (PD-1)/PD ligand pathway plays a key role in the maintenance of peripheral tolerance by enhancing the suppressive activity of regulatory T (Treg) cells. The promoter activity of the A allele of PD1 rs36084323 G/A polymorphism is lower than that of the G allele. We examined the association of PD1 gene polymorphisms, PD-1 expression on Treg cells, and thyroid PD-1/PD-1 ligand (PD-L1) expression with the pathogenesis of autoimmune thyroid disease (AITD). We classified patients and genotyped PD-1 polymorphisms by using the PCR-RFLP method in a total of 176 Graves' disease (GD) patients, 150 Hashimoto's disease (HD) patients with different disease severities and 99 healthy controls. PD-1 expression on Treg cells was analysed by flow cytometry. Indirect immunofluorescence staining was performed in thyroid tissue to detect PD-1, PD-L1, and PD-L2. The frequencies of the A allele and the AA + AG genotypes of the PD1 rs36084323 polymorphism were lower in HD patients than in GD patients, and the frequencies of the AA genotype of the PD1 rs36084323 and of the TT genotype of the PD1 rs2227982 were lower in mild HD patients than in severe HD patients. In patients with severe HD, the titres of TgAb at the onset were higher in patients with the PD1 rs36084323 AA genotype than in patients with the GG genotype. Peripheral PD1+ Treg cells tended to decrease in individuals with the PD1 rs36084323 AA genotype than with the G carrier genotype. Peripheral PD-1+ Treg cells were increased in HD, especially in mild HD. PD-1, PD-L1, and PD-L2 were expressed in thyroid-infiltrating mononuclear cells (TIMCs), and PD-L1 and PD-L2 were expressed in thyroid epithelial cells (TECs) in AITD patients but not in normal controls. Expression of PD-L1 in TIMCs and expression of PD-L2 in TECs were predominant in HD and GD patients, respectively. In conclusion, the functional PD1 rs36084323 polymorphism and the thyroid PD-1/ PD-L1s expression which may enhance the suppressive activity of Treg cells differ between GD and HD, and the PD1 rs36084323 and rs2227982 polymorphisms and PD1+ Treg cells are related to the severity of HD.

Polymorphisms in Vitamin A-Related Genes and Their Functions in Autoimmune Thyroid Disease.

Background: Vitamin A is a factor that suppresses immune responses, including T helper (Th)1 and Th17 responses. However, there has been no report showing the association between vitamin A-related genes (CYP26B1, RARB, and RARG) and the prognosis of autoimmune thyroid disease (AITD). The objective of this study was to clarify the association between vitamin A-related genes and the susceptibility and prognosis of AITD. Methods: We genotyped polymorphisms in genes encoding vitamin A-related molecules using the polymerase chain reaction-restriction fragment length polymorphism method. The proportion of T helper cells was analyzed by flow cytometry. Serum interleukin (IL)-17 and interferon (IFN)-γ were examined by enzyme-linked immunosorbent assay. Results:CYP26B1 rs3768641 GG genotype and G allele were significantly more frequent in patients with mild Hashimoto's thyroiditis (HT) than in those with severe HT (p = 0.0013 and 0.0024, respectively). The RARB rs1997352 CC genotype was significantly more frequent in HT patients than in controls (p = 0.0207). The proportion of Th17 cells was significantly higher in CYP26B1 rs2241057 TT genotype than C carrier (CC+CT genotypes) (p = 0.0385), in RARB rs1997352 A carrier (AA+AC genotypes) than those with CC genotype (p = 0.0246), and in RARG rs7398676 G carrier (GG+GT genotypes) than in TT genotype (p = 0.0249). In the RARB rs1997352 polymorphism, HT patients with a high concentration of IFN-γ (≥150 ng/mL) were more frequent in the CC genotype than in A carriers (AA+AC genotypes) (p = 0.0226). Serum levels of IL-17 were significantly elevated in subjects with the TT genotype of the CYP26B1 rs2241057 single nucleotide polymorphism (SNP) (p = 0.0026) and in subjects with the GG genotype of the CYP26B1 rs3798641 SNP (p = 0.030). Subjects with a high concentration of IL-17 (≥0.71 pg/mL) were more frequent in RARG 7398676 G carriers (GG+GT genotypes) than in TT genotype (p = 0.0218). Conclusions: Polymorphisms in the CYP26B1 gene were related to the proportion of Th17 cells, the level of IL-17 and the severity of HT. Polymorphisms in RAR were related to the proportion of Th17 cells, concentrations of IFN-γ and IL-17, and susceptibility to HT.

Relationship between Nutrient Intake and Human Gut Microbiota in Monozygotic Twins.

Background and Objectives: The gut microbiota is associated with human health and dietary nutrition. Various studies have been reported in this regard, but it is difficult to clearly analyze human gut microbiota as individual differences are significant. The causes of these individual differences in intestinal microflora are genetic and/or environmental. In this study, we focused on differences between identical twins in Japan to clarify the effects of nutrients consumed on the entire gut microbiome, while excluding genetic differences. Materials and Methods: We selected healthy Japanese monozygotic twins for the study and confirmed their zygosity by matching 15 short tandem repeat loci. Their fecal samples were subjected to 16S rRNA sequencing and bioinformatics analyses to identify and compare the fluctuations in intestinal bacteria. Results: We identified 12 genera sensitive to environmental factors, and found that Lactobacillus was relatively unaffected by environmental factors. Moreover, we identified protein, fat, and some nutrient intake that can affect 12 genera, which have been identified to be more sensitive to environmental factors. Among the 12 genera, Bacteroides had a positive correlation with retinol equivalent intake (rs = 0.38), Lachnospira had a significantly negative correlation with protein, sodium, iron, vitamin D, vitamin B6, and vitamin B12 intake (rs = -0.38, -0.41, -0.39, -0.63, -0.42, -0.49, respectively), Lachnospiraceae ND3007 group had a positive correlation with fat intake (rs = 0.39), and Lachnospiraceae UCG-008 group had a negative correlation with the saturated fatty acid intake (rs = -0.45). Conclusions: Our study is the first to focus on the relationship between human gut microbiota and nutrient intake using samples from Japanese twins to exclude the effects of genetic factors. These findings will broaden our understanding of the more intuitive relationship between nutrient intake and the gut microbiota and can be a useful basis for finding useful biomarkers that contribute to human health.

Gene polymorphisms of VEGF and VEGFR2 are associated with the severity of Hashimoto's disease and the intractability of Graves' disease, respectively.

Vascular endothelial growth factor (VEGF) is one of main regulators of angiogenesis that functions by binding to its receptors, including VEGF receptor (VEGFR) 2. There are few data available regarding the association between VEGF and VEGFR polymorphisms and the susceptibility to and prognosis of autoimmune thyroid diseases (AITDs). To elucidate this association, we genotyped four functional VEGF and two VEGFR2 polymorphisms and measured serum VEGF levels. In the four functional VEGF polymorphisms, the frequencies of the I carrier and I allele of VEGF -2549 I/D, which has lower activity, were higher in patients with severe HD than in those with mild HD. In the two functional VEGFR2 polymorphisms, the frequency of the rs2071559 CC genotype, which has higher activity, was higher in patients with intractable GD than in controls, and the proportion of GD patients with larger goiters was higher in those with the CC genotype. Moreover, the frequency of the rs1870377 TT genotype with higher activity was higher in patients with intractable GD than in those with GD in remission. Combinations of VEGF and VEGFR2 polymorphisms with stronger interactions were associated with the intractability of GD. Serum VEGF levels were higher in HD and AITD patients than those in controls. In conclusion, VEGF polymorphisms with lower activity were associated with the severity of HD, while VEGFR2 polymorphisms and the combinations of VEGF and VEGFR2 polymorphisms, which have stronger interactions, were associated with the intractability of GD. VEGF and VEGFR2 polymorphisms were associated with HD severity and GD intractability, respectively.

Association of CD58 Polymorphisms and its Protein Expression with the Development and Prognosis of Autoimmune Thyroid Diseases.

The prognosis of autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and Hashimoto's disease (HD), varies among patients. The interaction of CD58 and its ligand (CD2) promotes the differentiation of regulatory T cells and suppresses the immune response. To clarify the association of CD58 expression with the pathogenesis and prognosis of AITDs, we genotyped polymorphisms in the CD58 gene including rs12044852A/C (SNP1), rs2300747A/G (SNP2), rs1335532C/T (SNP3), rs1016140G/T (SNP4), rs1414275C/T (SNP5) and rs11588376C/T (SNP6). The CD58 SNPs were genotyped in 177 GD patients, 193 HD patients and 116 healthy volunteers (control subjects). We used the Polymerase chain reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method for the genotyping of SNP1 and SNPs3-6 and the TaqMan® SNP genotyping assay for the genotyping of SNP2. The frequencies of the AA genotype in SNP1 tend to be high in all patients with AITDs than in control subjects, although it was not significant. The GG genotype of SNP2, the CC genotype of SNP3, the TT genotype of SNP4, the CC genotype of SNP5 and the CC genotype of SNP6 were all significantly more frequent in patients with AITDs than in control subjects. The proportion of CD58+ cells in monocytes was significantly lower in healthy individuals with each of these risk genotypes of AITDs and lower in GD and HD patients than that in healthy controls. In conclusion, CD58 SNPs are involved in AITD susceptibility through the reduction in CD58 expression, which probably suppresses regulatory T cells.

Increases of CD80 and CD86 Expression on Peripheral Blood Cells and their Gene Polymorphisms in Autoimmune Thyroid Disease.

The prognosis of autoimmune thyroid diseases (AITDs), such as Graves' disease (GD) and Hashimoto's disease (HD), are difficult to predict. Both CD80 and CD86 costimulatory signals promote T cell activation in cooperation with T cell receptor signal. To clarify whether any association between CD80 and CD86 and the pathogenesis of AITD exist, we examined the expressions and gene polymorphisms of CD80 and CD86. We examined the expressions of CD80 and CD86 proteins on peripheral blood cells by flowcytometry and genotyped CD80 and CD86 gene polymorphisms by PCR-RFLP and Taqman PCR methods. In the analysis of the Blymphocytes elevated CD80+ cells (>8%) were found more often in the patients than in control subjects, and also it was more frequent in patients with intractable GD than in those with GD in remission (p= .0176). The mean fluorescence intensity of CD86 expression on monocytes was higher in GD and HD patients than in control subjects (p= <0.0001 and p= .0017, respectively). CD80 rs1599795 T allele carriers were more frequent in patients with severe HD than in those with mild HD. CD86 rs2715267 AA genotype was more frequent in HD patients than in controls. In conclusion, the expressions of CD80 on Bcells and of CD86 on monocytes were increased in peripheral blood from patients with AITD, especially in severe cases, and their gene polymorphisms are associated with the susceptibility and the severity of HD.

Association of IL6 gene methylation in peripheral blood cells with the development and prognosis of autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs), including Hashimoto's disease (HD) and Graves' disease (GD), are archetypes of organ-specific autoimmune diseases, but the prognosis of patients with AITD varies. Autoimmune diseases, including AITDs, are believed to develop in response to both genetic and environmental factors. Interleukin (IL)-6 plays a major role in B cell differentiation and T cell proliferation, and methylation of the IL6 gene is associated with IL-6 production. To clarify the role of IL6 gene methylation in the pathogenesis and prognosis of AITDs, we measured the methylation levels of -666, -664, -610, -491 and -426 CpG sites in the IL6 gene. We measured the methylation levels of 5 CpG sites in 29 patients with HD, 31 patients with GD and 16 healthy volunteers using pyrosequencing. The methylation level at each of the -664, -491 and -426 CpG sites was negatively correlated with the age at the time of sampling. Multiple regression analysis indicated that patients with HD, including severe or mild HD, showed higher methylation levels at the -426 CpG site than control subjects. Patients with intractable GD showed lower methylation levels at the -664 and -666 CpG sites than patients with GD in remission. In conclusion, IL6 gene methylation levels were related to the susceptibility to HD and the intractability of GD.

Association of IFNG gene methylation in peripheral blood cells with the development and prognosis of autoimmune thyroid diseases.

The intractability of Graves' disease (GD) and the severity of Hashimoto's disease (HD) vary among patients. Both genetic and environmental factors may be associated with their prognoses. To clarify the role of methylation of the IFNG gene in the pathogenesis and prognosis of (AITDs), we examined interferon gamma (IFNG) methylation levels at various CpG sites and genotyped IFNG +874 A/T and +2109 C/T polymorphisms. We analyzed methylation 59 patients with HD, 57 patients with GD and 26 healthy volunteers by pyrosequencing. We genotyped IFNG gene polymorphisms from 207 patients with GD, 208 patients with HD, and 102 healthy controls. The methylation levels of IFNG -54 CpG were higher in patients with intractable GD than in those with GD in remission, but there was no difference between patients with severe and mild HD. In carriers of IFNG +2109 T (CT + TT) (85.5% in controls), the -54 CpG methylation levels were significantly higher in patients with intractable GD than in those with GD in remission. On the other hand, in carriers of IFNG +2109 CC, the -4293 CpG methylation levels were higher in intractable GD patients. The methylation levels of IFNG -54 CpG and -4293 CpG were negatively correlated with the age in HD, especially severe HD, patients and GD patients, respectively. There was no circadian variation but considerable daily variation in the methylation levels of IFNG -54 CpG. In conclusion, both the methylation levels of CpG sites and the functional polymorphisms in the IFNG gene were associated with the pathogenesis and prognosis of AITD, especially with GD intractability.

Toll-like receptor 4 promoter polymorphisms in a familial mediterranean fever patient with asymptomatic bacteriuria.

A periodic fever, due to inherited inflammatory disorders, can be misdiagnosed as a common infection, when a possible pathogen is detected from a patient. TLR4 SNPs that are responsible for asymptomatic bacteriuria might disturb the pathophysiology of familial Mediterranean fever without MEFV mutations.

Polymorphisms in Th17-related genes and the pathogenesis of autoimmune thyroid disease.

The prognosis of autoimmune thyroid disease (AITD) including Graves' disease (GD) and Hashimoto's disease (HD) is difficult to predict. We previously suggested that Th17 cells may be associated with the pathogenesis of AITD. However, the association between gene polymorphisms in Th17-related genes and the prognosis of AITD was not clarified. To clarify this association, we genotyped 12 polymorphisms in 11 Th17-related genes (IL1Ra, IL6R, IL17R, IL21R, IL23R, CCR6, SOCS3, RORC, IL17A, IL17F and IL21) in 142 HD patients including 58 patients with severe HD and 48 patients with mild HD, 170 patients with GD including 81 patients with intractable GD and 49 patients with GD in remission, and 84 healthy volunteers. The frequency of the IL17F rs763780 T allele was higher in patients with severe HD than in patients with mild HD (p = .008). The frequency of the IL17R rs9606615 T allele was higher in patients with HD than in normal subjects (p = .011). The frequencies of the SOCS3 rs4969170 AA genotype, CCR6 rs3093024 AA genotype, and IL21 rs907715 AA genotype were higher in patients with intractable GD than in patients with GD in remission (p = .035, p = .002 and p = .030, respectively). In conclusion, IL17R rs9607715 and IL17F rs763780 polymorphisms are associated with the susceptibility and severity of HD, respectively. IL21 rs907715, SOCS3 rs4969170 and CCR6 rs3093024 polymorphisms are associated with the intractability of GD.

Association of current and former smoking with body mass index: A study of smoking discordant twin pairs from 21 twin cohorts.

BACKGROUND: Smokers tend to weigh less than never smokers, while successful quitting leads to an increase in body weight. Because smokers and non-smokers may differ in genetic and environmental family background, we analysed data from twin pairs in which the co-twins differed by their smoking behaviour to evaluate if the association between smoking and body mass index (BMI) remains after controlling for family background. METHODS AND FINDINGS: The international CODATwins database includes information on smoking and BMI measured between 1960 and 2012 from 156,593 twin individuals 18-69 years of age. Individual-based data (230,378 measurements) and data of smoking discordant twin pairs (altogether 30,014 pairwise measurements, 36% from monozygotic [MZ] pairs) were analysed with linear fixed-effects regression models by 10-year periods. In MZ pairs, the smoking co-twin had, on average, 0.57 kg/m2 lower BMI in men (95% confidence interval (CI): 0.49, 0.70) and 0.65 kg/m2 lower BMI in women (95% CI: 0.52, 0.79) than the never smoking co-twin. Former smokers had 0.70 kg/m2 higher BMI among men (95% CI: 0.63, 0.78) and 0.62 kg/m2 higher BMI among women (95% CI: 0.51, 0.73) than their currently smoking MZ co-twins. Little difference in BMI was observed when comparing former smoking co-twins with their never smoking MZ co-twins (0.13 kg/m2, 95% CI 0.04, 0.23 among men; -0.04 kg/m2, 95% CI -0.16, 0.09 among women). The associations were similar within dizygotic pairs and when analysing twins as individuals. The observed series of cross-sectional associations were independent of sex, age, and measurement decade. CONCLUSIONS: Smoking is associated with lower BMI and smoking cessation with higher BMI. However, the net effect of smoking and subsequent cessation on weight development appears to be minimal, i.e. never more than an average of 0.7 kg/m2.

Association of the polymorphisms in Th2 chemotaxis-related genes with the development and prognosis of autoimmune thyroid diseases.

The prognosis of autoimmune thyroid disease (AITD) is difficult to predict. Th2 cells suppress the differentiation of Th1 and Th17 cells, which are associated with the prognosis of AITD. However, there are few reports as to whether Th2 chemotaxis-related genes, such as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), IL-25, TARC/CCL17 (Thymus and activation regulated chemokine/chemokine ligand 17) or STAT6 (Signal transducer and activator of transcription 6), affect the pathology of and/or susceptibility to AITD. Therefore, in this study, we genotyped functional SNPs in these genes to clarify the association of the genetic differences of genes related to Th2 differentiation and chemotaxis with the development and the prognosis of AITDs. The frequencies of the AA genotype of the CRTH2 rs545659 SNP and the CC genotype and the C allele of the CRTH2 rs634681 SNP were higher in patients with severe HD than in patients with mild HD. The frequency of the CC genotype in the TARC rs223828 SNP was higher in patients with intractable GD than in patients with GD in remission. In conclusion, the CRTH2 rs545659 and rs634681 SNPs were associated with the severity of HD, and the TARC/CCL17 rs223828 SNP was associated with the intractability of GD.

Functional Polymorphisms of the Type 1 and Type 2 Iodothyronine Deiodinase Genes in Autoimmune Thyroid Diseases.

Graves' disease (GD) and Hashimoto's disease (HD) are major autoimmune thyroid diseases (AITDs), and their pathological conditions vary among patients. Type 1 iodothyronine deiodinase (D1) and type 2 iodothyronine deiodinase (D2) convert from thyroxine (T4) to triiodothyronine (T3). However, few findings have been described concerning the association between polymorphisms in D1 and D2 genes and AITD. Therefore, we genotyped D1 rs11206244, D2 rs225014, and rs12885300 polymorphisms in 134 GD patients, including 54 patients with intractable GD and 44 patients with GD in remission and 132 HD patients, including 57 patients with severe HD, 45 patients with mild HD, and 84 healthy controls using PCR-RFLP. In the D2 rs225014 polymorphism, the TT genotype, which was correlated with higher D2 activity, was less frequent in AITD, especially in HD, than in control subjects (P = 0.0032 and 0.0002, respectively). Moreover, they were also less frequent in HD than in GD (P = 0.0199). The TT genotype and T allele were less frequent in severe HD and mild HD than in control subjects (P = 0.0003, 0.0006, 0.0432, and 0.0427, respectively). In conclusion, the low frequency of the TT genotype D2 rs225014 polymorphism was associated with the development of AITD and severity of HD.

Methylation levels of the TNFA gene are different between Graves' and Hashimoto's diseases and influenced by the TNFA polymorphism.

Graves' disease (GD) and Hashimoto's disease (HD) are different pathological types of autoimmune thyroid diseases (AITDs). However, the epigenetic differences between these diseases have not been elucidated. DNA methylation is one of the primary epigenetic modifications that reflect environmental influences on gene expression. In this study, we evaluated the methylation status of six CpG sites in the TNFA promoter using pyrosequencing and analyzed the data in combination with functional polymorphisms (-1031 T/C and +123 C/T) in the TNFA gene to clarify the role of gene methylation on the prognosis of AITDs. We examined the methylation pattern in 52 patients with GD, 60 patients with HD, and 29 healthy controls by pyrosequencing. Additionally, we also genotyped the polymorphisms from 163 patients with GD, 152 patients with HD, and 94 healthy controls using the restriction fragment length polymorphism (RFLP) method. Each proportion of subjects with low methylation of the -72 CpG site (≤11.9%), low methylation of the -49 CpG site (≤15.5%), and low methylation of the -38 CpG site (≤8.9%) was significantly increased in the groups with high concentration of TNF-α (≥0.134 pg/mL). The methylation level of the -72 CpG site was significantly higher in GD cases (10.7 ± 4.9%) than in healthy controls (6.8 ± 3.9%). The methylation level of the -49 and -38 CpG sites were significantly higher in patients with GD in remission (20.5 ± 9.5%, 17.6 ± 8.0%, respectively) than in healthy controls (13.0 ± 7.6%, 7.9 ± 7.3%, respectively). The frequency of the TNFA - 1031C carrier (CT + CC) is correlated with higher TNF-α production and was significantly higher in GD (35.0%) and HD (39.5%) cases than in controls (19.1%). In the subjects with the TNFA - 1031C carrier (CT + CC), the methylation level of the -72 CpG site was significantly higher in GD (11.5 ± 5.7%) than in HD (6.0 ± 3.4%). However, there was no difference between GD and HD in patients with the TT genotype. Cumulatively, our data indicate the methylation levels of CpG sites in the TNFA gene may be related to the difference between GD and HD in AITDs and may be influenced by the TNFA gene polymorphism.

Mean platelet component and mean platelet volume as useful screening markers for myelodysplastic syndrome.

BACKGROUND: Hematologic disorders, including myelodysplastic syndrome (MDS), are difficult to identify in routine hematologic examinations using automated hematology analyzers. However, the practical uses of mean platelet component and mean platelet volume (MPV) measured by these analyzers as screening markers for MDS, remain unclear. METHODS: Mean platelet component and MPV values were measured in the peripheral blood of patients with MDS, aplastic anemia, idiopathic thrombocytopenic purpura, myeloproliferative neoplasms, and in healthy controls using an automated hematologic analyzer. Cutoff values for discriminating between the MDS group and healthy controls were determined by recursive partitioning analysis. RESULTS: Mean platelet component was significantly lower in MDS patients compared with controls, while MPV was significantly higher. Combined cutoff values for MDS diagnosis of <25.3 g/dL for mean platelet component and >10.0 fL for MPV showed a specificity and positive predictive value of 99.9% and 99.1%, respectively. These cutoff values also differentiated between MDS and diagnoses of aplastic anemia, idiopathic thrombocytopenic purpura, and myeloproliferative neoplasms. CONCLUSION: Mean platelet component and MPV may, thus, be useful and convenient screening markers for MDS.