IL2RA Genetic Heterogeneity in Multiple Sclerosis and Type 1 Diabetes Susceptibility and Soluble Interleukin-2 Receptor Production

Multiple sclerosis (MS) and type 1 diabetes (T1D) are organ-specific autoimmune disorders with significant heritability, part of which is conferred by shared alleles. For decades, the Human Leukocyte Antigen (HLA) complex was the only known susceptibility locus for both T1D and MS, but loci outside the HLA complex harboring risk alleles have been discovered and fully replicated. A genome-wide association scan for MS risk genes and candidate gene association studies have previously described the IL2RA gene region as a shared autoimmune locus. In order to investigate whether autoimmunity risk at IL2RA was due to distinct or shared alleles, we performed a genetic association study of three IL2RA variants in a DNA collection of up to 9,407 healthy controls, 2,420 MS, and 6,425 T1D subjects as well as 1,303 MS parent/child trios. Here, we report “allelic heterogeneity” at the IL2RA region between MS and T1D. We observe an allele associated with susceptibility to one disease and risk to the other, an allele that confers susceptibility to both diseases, and an allele that may only confer susceptibility to T1D. In addition, we tested the levels of soluble interleukin-2 receptor (sIL-2RA) in the serum from up to 69 healthy control subjects, 285 MS, and 1,317 T1D subjects. We demonstrate that multiple variants independently correlate with sIL-2RA levels.     

IL12RB2 Gene Is Associated with the Age of Type 1 Diabetes Onset in Croatian Family Trios

Background

Common complex diseases are influenced by both genetic and environmental factors. Many genetic factors overlap between various autoimmune diseases. The aim of the present study is to determine whether four genetic variants known to be risk variants for several autoimmune diseases could be associated with an increased susceptibility to type 1 diabetes mellitus.

Methods and Findings

We genotyped four genetic variants (rs2358817, rs1049550, rs6679356, rs9865818) within VTCN1, ANXA11, IL12RB2 and LPP genes respectively, in 265 T1DM family trios in Croatian population. We did not detect association of these polymorphisms with T1DM. However, quantitative transmission disequilibrium test (QTDT, orthogonal model) revealed a significant association between the age of onset of T1DM and IL12RB2 rs6679356 variant. An earlier onset of T1DM was associated with the rs6679356 minor dominant allele C (p = 0.005). The association remained significant even after the Bonferroni correction for multiple testing and permutation.

Conclusions

Variants originally associated with juvenile idiopathic arthritis (VTCN1 gene), sarcoidosis (ANXA11 gene), primary biliary cirrhosis (IL12RB2 gene) and celiac disease (LPP gene) were not associated with type 1 diabetes in our dataset. Nevertheless, association of IL12RB2 rs6679356 polymorphism with the age of T1DM onset suggests that this gene plays a role in defining the time of disease onset.     

Genetic association of IL2RA,IL17RA,IL23R,and IL31RA single nucleotide polymorphisms with alopecia areata

The signalling of cytokine receptors plays a crucial role in regulating tolerance and immunity. Impaired immunological processes result in autoimmune inflammation that target the hair follicles, causing many hair disorders, mainly alopecia areata (AA). Therefore, polymorphisms in cytokine receptor genes are suggested to have a significant impact on the pathogenesis of AA, a disease with a multifactorial basis and uncertain etiology. In the present study, 152 AA patients of the Jordanian population were investigated for their genetic susceptibility to develop AA compared to 150 control subjects. Genomic DNA extraction and genotyping had conducted for IL17RA (rs879575, rs2229151, and rs4819554), IL2RA (rs3118470), IL23R (rs10889677), and IL31RA (rs161704) using the Sequenom MassARRAY® system. The allele frequency of IL17RA rs879575 is significantly higher in patients, while no statistical differences were found for IL2RA, IL23R, and IL31RA SNPs. Also, the recessive model of IL31RA rs161704 showing that AA genotype is significantly associated with AA development. To date, there is no published data regarding the association between AA and the selected genetic variants in our population. However, this study's findings assert that SNPs of IL17RA and IL31RA are linked to AA susceptibility in Jordanian patients.     

IL2RA/CD25 Gene Polymorphisms: Uneven Association with Multiple Sclerosis (MS) and Type 1 Diabetes (T1D)

Background

IL-2 receptor (IL2R) alpha is the specific component of the high affinity IL2R system involved in the immune response and in the control of autoimmunity.

Methods and Results

Here we perform a replication and fine mapping of the IL2RA gene region analyzing 3 SNPs previously associated with multiple sclerosis (MS) and 5 SNPs associated with type 1 diabetes (T1D) in a collection of 798 MS patients and 927 matched Caucasian controls from the south of Spain. We observed association with MS in 6 of 8 SNPs. The rs1570538, at the 3′- UTR extreme of the gene, previously reported to have a weak association with MS, is replicated here (P = 0.032). The most associated T1D SNP (rs41295061) was not associated with MS in the present study. However, the rs35285258, belonging to another independent group of SNPs associated with T1D, showed the maximal association in this study but different risk allele. We replicated the association of only one (rs2104286) of the two IL2RA SNPs identified in the recently performed genome-wide association study of MS.

Conclusions

These findings confirm and extend the association of this gene with MS and reveal a genetic heterogeneity of the associated polymorphisms and risk alleles between MS and T1D suggesting different immunopathological roles of IL2RA in these two diseases.     

Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.     

Dynamic demethylation of the IL2RA promoter during in vitro CD4+ T cell activation in association with IL2RA expression

IL2RA, a subunit of the high affinity receptor for interleukin-2 (IL2), plays a crucial role in immune homeostasis. Notably, IL2RA expression is induced in CD4+ T cells in response to various stimuli and is constitutive in regulatory T cells (Tregs). We selected for our study 18 CpGs located within cognate regulatory regions of the IL2RA locus and characterized their methylation in naive, regulatory, and memory CD4+ T cells. We found that 5/18 CpGs (notably CpG + 3502) show dynamic, active demethylation during the in vitro activation of naive CD4+ T cells. Demethylation of these CpGs correlates with appearance of IL2RA protein at the cell surface. We found no influence of cis located SNP alleles upon CpG methylation. Treg cells show constitutive demethylation at all studied CpGs. Methylation of 9/18 CpGs, including CpG +3502, decreases with age. Our data thus identify CpG +3502 and a few other CpGs at the IL2RA locus as coordinated epigenetic regulators of IL2RA expression in CD4+ T cells. This may contribute to unravel how the IL2RA locus can be involved in immune physiology and pathology.     

Methylation at CpG islands in intron 1 of EGR2 confers enhancer-like activity

We previously demonstrated several lines of evidence indicating that early growth response 2 (EGR2) functions as a tumor suppressor, partly on the basis that its expression was often decreased in human tumors and cancer cell lines. Here we report a possible molecular mechanism to account for down-regulation of EGR2 in tumor cells. Although no genetic mutations in the gene or alterations in methylation status of its promoter were detected, we found a high degree of methylation at CpG islands in intron 1 of EGR2 in cell lines that were expressing this gene at a high level. Moreover, reporter gene experiments revealed that methylated intron 1 had somehow conferred enhancer-like activity. The data imply the existence of a previously unsuspected mechanism of gene expression regulation.     

Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications

Gene MAGEA1 belongs to a group of human germline-specific genes that rely on DNA methylation for repression in somatic tissues. Many of these genes, termed cancer-germline (CG) genes, become demethylated and activated in a wide variety of tumors, where they encode tumor-specific antigens. The process leading to DNA demethylation of CG genes in tumors remains unclear. Previous data suggested that histone acetylation might be involved. Here, we investigated the relative contribution of DNA methylation and histone acetylation in the epigenetic regulation of gene MAGEA1. We show that MAGEA1 DNA hypomethylation in expressing melanoma cells is indeed correlated with local increases in histone H3 acetylation (H3ac). However, when MAGEA1-negative cells were exposed to a histone deacetylase inhibitor (TSA), we observed only short-term activation of the gene and detected no demethylation of its promoter. As a more sensitive assay, we used a cell clone harboring a methylated MAGEA1/hph construct, which confers resistance to hygromycin upon stable re-activation. TSA induced only transient de-repression of the transgene, and did not lead to the emergence of hygromycin-resistant cells. In striking contrast, transient depletion of DNA-methyltransferase-1 in the reporter cell clone gave rise to a hygromycin-resistant population, in which the re-activated MAGEA1/hph transgene displayed not only marked DNA hypomethylation, but also significant reversal of histone marks, including gains in H3ac and H3K4me2, and losses of H3K9me2. Collectively, our results indicate that DNA methylation has a dominant role in the epigenetic hierarchy governing MAGEA1 expression.     

The Interactive Effect of SIRT1 Promoter Region Polymorphism on Type 2 Diabetes Susceptibility in the North Indian Population

Our previous studies have implicated genes mainly involved in the activity of pancreatic β cells in type 2 diabetes (T2D) susceptibility in the North Indian population. Recent literature on the role of SIRT1 as a potential master switch modulating insulin secretion and regulating gene expression in pancreatic β cells has warranted an evaluation of SIRT1 promoter region polymorphisms in the North Indian population, which is the main focus of the present study. 1542 samples (692 T2D patients and 850 controls) were sequenced for the 1.46 kb region upstream the translation start site of the SIRT1 gene. We performed a functional characterization of the SIRT1 promoter region polymorphisms using luciferase assay and observed a single-nucleotide polymorphism (SNP), rs12778366, in association with SIRT1 expression. We propose that TT, the high-expressing genotype of SNP rs12778366 in the SIRT1 promoter region and present in >80% of the North Indian population, was favored under conditions of feast-famine cycles in evolution, which has turned out to be a cause of concern in the present sedentary lifestyle under ad libitum conditions. Case-control association analysis did not implicate rs12778366 in T2DM per se in the studied population. However, our earlier reported risk genotype combinations of mt-ND3, PGC1α, and UCP2-866, when compared with the protective genotype combinations, in the background of the high-expressing TT genotype of SIRT1 SNP rs12778366, showed a very high additive risk [corrected odd ratio (OR) = 8.91; p = 6.5×10⁻¹¹]. The risk level was considerably low in the genotype backgrounds of TX (OR = 6.68; p = 2.71×10⁻¹²) and CX (OR = 3.74; p = 4.0×10⁻³). In addition, we screened other reported T2D-associated polymorphisms: PIK3R1 rs3730089, IRS1 rs1801278, and PPP1R3 rs1799999, which did not show any significant association in North Indian population. The present paper emphasizes the importance of gene interactions in the biological pathways of T2D, a complex lifestyle disease.     

The Changes in MGMT Promoter Methylation Status in Initial and Recurrent Glioblastomas

To evaluate the mechanism of the development of therapeutic resistance after temozolomide treatment, we focused on changes in O⁶-methylguanine DNA methyltransferase (MGMT) and mismatch repair (MMR) between initial and recurrent glioblastomas. Tissue samples obtained from 24 paired histologically confirmed initial and recurrent adult glioblastoma patients who were initially treated with temozolomide were used for MGMT and MMR gene promoter methylation status and protein expression analysis using methylation-specific multiplex ligation probe amplification (MS-MLPA), methylation-specific polymerase chain reaction (MSP), and immunohistochemical staining. There was a significant decrease in the methylation ratio of the MGMT promoter determined by MS-MLPA, which was not detectable with MSP, and MGMT protein expression changes were not remarkable. However, there was no epigenetic variability in MMR genes, and a relatively homogeneous expression of MMR proteins was observed in initial and recurrent tumors. We conclude that the development of reduced methylation in the MGMT promoter is one of the mechanisms for acquiring therapeutic resistance after temozolomide treatment in glioblastomas.     

Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter

Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.     

IL2RA is associated with persistence of rheumatoid arthritis

IntroductionAlthough rheumatoid arthritis (RA) is generally a chronic disease, a proportion of RA-patients achieve disease-modifying antirheumatic drug (DMARD)-free sustained remission, reflecting loss of disease-persistence. To explore mechanisms underlying RA-persistence, we performed a candidate gene study. We hypothesized that variants associating with lack of radiographic progression also associate with DMARD-free sustained remission.Methods645 Dutch RA-patients were studied on DMARD-free sustained remission during a maximal follow-up duration of 10-years. Variants associated with radiographic progression under an additive model in the total RA-population (Human Leukocyte Antigens (HLA)-DRB1-shared epitope (SE), Dickkopf-1 (DKK1)-rs1896368, DKK1-rs1896367, DKK1-rs1528873, C5Orf30-rs26232, Interleukin-2 receptor-α (IL2RA)-rs2104286, Matrix metalloproteinase-9 (MMP-9)-rs11908352, rs451066 and Osteoprotegerin (OPG)-rs1485305) were studied. Cox-regression analyses were performed and Bonferroni correction applied. Soluble IL2Rα (sIL2Rα)-levels were studied. For replication, 622 RA-patients included in the French Evaluation et Suivi de POlyarthrites Indifférenciées Récentes cohort (ESPOIR)-cohort were investigated. Results were combined in inverse-variance weighted meta-analysis.ResultsSimilar as previously reported, the SE-alleles associated with less remission (hazard ratio (HR) = 0.57, 95 % confidence interval (95 % CI) = 0.42-0.77, p = 2.72×10⁻⁴). Variants in DKK-1, C5orf30, MMP-9 and OPG were not associated with remission. The IL2RA-rs2104286 minor allele associated with a higher chance on remission (HR = 1.52, 95 % CI = 1.16-1.99, p = 2.44×10⁻³). The rs2104286 minor allele associated with lower sIL2Rα-levels (p = 1.44×10⁻³); lower sIL2Rα-levels associated with a higher chance on remission (HR per 100 pg/L = 0.81, 95 % CI = 0.68-0.95, p = 0.012). When including rs2104286 and sIL2Rα-levels in one analysis, the HR for rs2104286 was 2.27 (95 % CI = 1.06-4.84, p = 0.034) and for sIL2Rα 0.83 (95 % CI = 0.70-0.98, p = 0.026). Within ESPOIR, the HR of rs2104286 was 1.31 (95 % CI = 0.90-1.90). The meta-analysis revealed a p-value of 1.01×10⁻³.ConclusionIL2RA-rs2104286 and sIL2Rα-level associated with RA-persistence. IL2RA variants are known to protect against multiple sclerosis, diabetes mellitus and RA. Besides HLA-SE, IL2RA-rs2104286 is thus far the only known genetic variant associated with both joint destruction and RA-persistence. This underlines the relevance of IL2RA for RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0739-6) contains supplementary material, which is available to authorized users.     

The 5-Year Onset and Regression of Diabetic Retinopathy in Chinese Type 2 Diabetes Patients

Purpose

To determine the rate and risk factors of diabetic retinopathy (DR) onset and regression in Chinese type 2 diabetes mellitus patients.

Methods

This is a 5-year community-based prospective study. The demographic information, systemic examination results and ophthalmological test results of each participant were collected. The study outcomes were DR incidence, defined as the onset of DR in at least one eye, and DR regression, defined as full regression from existing DR to no retinopathy without invasive treatments. The associations between each potential risk factor and the outcomes were studied.

Results

In total, 778 participants were enrolled. There were 322 patients without DR at baseline, of which 151 participants developed DR during follow-up (DR incidence rate = 46.89%). Baseline hyperglycemia and high blood pressure were two independent risk factors associated with DR incidence. Among the 456 participants with existing DR at entry, 110 fully recovered after 5 years (DR regression rate = 24.12%). Low baseline glucose and low serum triglyceride were two independent factors associated with DR regression.

Conclusions

DR incidence occurred more frequently in patients with hyperglycemia and high blood pressure. DR regression occurred mostly in patients with lower glucose and lower serum triglyceride levels among Chinese type 2 diabetes patients.     

Risk for Coexistent Autoimmune Diseases in Familial and Sporadic Type 1 Diabetes is Related to Age at Diabetes Onset

ObjectiveType 1 diabetes (T1D) is frequently associated with other autoimmune diseases (AIDs). Although most of T1D patients are sporadic cases (S-T1D), 10% to 15% have a familial form (F-T1D) involving 2 or more first-degree relatives. This study evaluated the effect of T1D family aggregation and age onset on AIDs occurrence.MethodsIn this observational, cross-sectional, case-control, single center study, we enrolled 115 F-T1D and 115 S-T1D patients matched for gender, age, T1D age onset, and duration. With respect to T1D age onset (before or after 18 years), both groups were further subdivided into young- or adult-onset F-T1D and young- or adult-onset S-T1D. The presence of organ-specific antibodies and/or overt AIDs was evaluated.ResultsThe F-T1D group had a higher percentage of AIDs (29.8% vs 18.4%, P = .04) and a significant earlier onset of AIDs at Cox regression analysis (P = .04) than the S-T1D group. Based on multivariate analysis, the adult-onset F-T1D subgroup had the highest prevalence of both additional organ-specific antibodies (60.5%) and overt AIDs (34.9%), whereas the adult S-T1D subgroup was the least frequently involved (29.1% and 12.7%, respectively). In F-T1D patients, offsprings develop T1D and AIDs earlier than their parents do.ConclusionsIn T1D patients, familial aggregation and adult-onset of T1D increase the risk for coexistent AIDs. These clinical predictors could guide clinicians to address T1D patients for the screening of T1D-related AIDs.     

Prognostic Value of PLAGL1-Specific CpG Site Methylation in Soft-Tissue Sarcomas

Soft tissue sarcomas (STS) are rare, complex tumors with a poor prognosis. The identification of new prognostic biomarkers is needed to improve patient management. Our aim was to determine the methylation status of the 118 CpG sites in the PLAGL1 tumor-suppressor gene P1 CpG island promoter and study the potential prognostic impact of PLAGL1 promoter methylation CpG sites in STS. Training cohorts constituted of 28 undifferentiated sarcomas (US) and 35 leiomyosarcomas (LMS) were studied. PLAGL1 mRNA expression was investigated by microarray analysis and validated by RT-qPCR. Pyrosequencing was used to analyze quantitative methylation of the PLAGL1 promoter. Associations between global promoter or specific CpG site methylation and mRNA expression were evaluated using Pearson’s product moment correlation coefficient. Cox univariate and multivariate proportional hazard models were used to assess the predictive power of CpG site methylation status. Sixteen CpG sites associated with PLAGL1 mRNA expression were identified in US and 6 in LMS. Statistical analyses revealed an association between CpG107 methylation status and both overall and metastasis-free survival in US, which was confirmed in a validation cohort of 37 US. The exhaustive study of P1 PLAGL1 promoter methylation identified a specific CpG site methylation correlated with mRNA expression, which was predictive for both metastasis-free and overall survival and may constitute the first US-specific biomarker. Such a biomarker may be relevant for identifying patients likely to derive greater benefit from treatment.     

贵州矮马IGF-I启动子-529 CpG甲基化及个别核苷酸变异与贵州矮马矮小表型有关

胰岛素样生长因子（insulin-like growth factors,IGFs）和生长（激）素（growth hormone,GH）是动物机体主要的促生长因子,它们的表达水平直接决定成熟个体的高度。贵州矮马的体高明显低于伊犁马等大型马,但其原因尚不清楚。本研究从贵州矮马基因组DNA中克隆了GH和IGF-I基因5′-侧翼序列,分别为239 bp和817 bp,包括部分启动子结构;进而采用生物信息学、比较基因组学方法对比分析了贵州矮马与伊犁马两个基因5′-侧翼序列/启动子的转录因子结合位点及潜在甲基化位点（CpG岛）分布。亚硫酸氢盐PCR测序法（bisulfite sequencing PCR,BSP）显示,两个马群的GH基因5′-侧翼区域（239 bp）内的6个CpG位点均发生了甲基化,甲基化频率无明显差异。然而,在IGF-I基因5′-侧翼区的148 bp片段内含有4个CpG位点中,贵州矮马的-529 bp处CpG位点的甲基化程度明显高于伊犁马（P < 0.01）,且该甲基化位点处于基本启动子邻近3′端;此外,两个马群IGF-I基因5′-侧翼区的-561 bp处检测到T、C碱基改变,导致贵州矮马的顺式调控元件/转录因子结合位点较伊犁马少1个,有可能影响IGF-I基因的转录效率。血清IGF-I浓度测定揭示,贵州矮马血清IGF-I含量极显著低于伊犁马（P< 0.01）。Spearman相关性结果显示,贵州矮马及伊犁马的IGF-Ⅰ基因甲基化频率与血清IGF-I浓度呈中度负相关（r=-0.468）,提示IGF-Ⅰ基因甲基化抑制其编码蛋白质的表达。结果证明,IGF-I启动子高甲基化及某些核苷酸（碱基）序列变异可能是贵州矮马个体矮小的部分原因。