Maternal genome-wide DNA methylation profiling in gestational diabetes shows distinctive disease-associated changes relative to matched healthy pregnancies.

Several recent reports have described associations between gestational diabetes (GDM) and changes to the epigenomic landscape where the DNA samples were derived from either cord or placental sources. We employed genome-wide 450K array analysis to determine changes to the epigenome in a unique cohort of maternal blood DNA from 11 pregnant women prior to GDM development relative to matched controls. Hierarchical clustering segregated the samples into 2 distinct clusters comprising GDM and healthy pregnancies. Screening identified 100 CpGs with a mean ß-value difference of ≥0.2 between cases and controls. Using stringent criteria, 5 CpGs (within COPS8, PIK3R5, HAAO, CCDC124, and C5orf34 genes) demonstrated potentials to be clinical biomarkers as revealed by differential methylation in 8 of 11 women who developed GDM relative to matched controls. We identified, for the first time, maternal methylation changes prior to the onset of GDM that may prove useful as biomarkers for early therapeutic intervention.

Epigenome-wide association study of rheumatoid arthritis identifies differentially methylated loci in B cells.

Epigenetic regulation of immune cell types could be critical for the development and maintenance of autoimmune diseases like rheumatoid arthritis (RA). B cells are highly relevant in RA, since patients express autoantibodies and depleting this cell type is a successful therapeutic approach. Epigenetic variation, such as DNA methylation, may mediate the pathogenic activity of B cells. In this study, we performed an epigenome-wide association study (EWAS) for RA with three different replication cohorts, to identify disease-specific alterations in DNA methylation in B cells. CpG methylation in isolated B lymphocytes was assayed on the Illumina HumanMethylation450 BeadChip in a discovery cohort of RA patients (N = 50) and controls (N = 75). Differential methylation was observed in 64 CpG sites (q < 0.05). Six biological pathways were also differentially methylated in RA B cells. Analysis in an independent cohort of patients (N = 15) and controls (N = 15) validated the association of 10 CpG sites located on 8 genes CD1C, TNFSF10, PARVG, NID1, DHRS12, ITPK1, ACSF3 and TNFRSF13C, and 2 intergenic regions. Differential methylation at the CBL signaling pathway was replicated. Using an additional case-control cohort (N = 24), the association between RA risk and CpGs cg18972751 at CD1C (P = 2.26 × 10-9) and cg03055671 at TNFSF10 (P = 1.67 × 10-8) genes was further validated. Differential methylation at genes CD1C, TNFSF10, PARVG, NID1, DHRS12, ITPK1, ACSF3, TNFRSF13C and intergenic region chr10p12.31 was replicated in a cohort of systemic lupus erythematosus (SLE) patients (N = 47) and controls (N = 56). Our results highlight genes that may drive the pathogenic activity of B cells in RA and suggest shared methylation patterns with SLE.

DNA methylation at diagnosis is associated with response to disease-modifying drugs in early rheumatoid arthritis.

AIM: A proof-of-concept study to explore whether DNA methylation at first diagnosis is associated with response to disease-modifying antirheumatic drugs (DMARDs) in patients with early rheumatoid arthritis (RA). PATIENTS & METHODS: DNA methylation was quantified in T-lymphocytes from 46 treatment-naive patients using HumanMethylation450 BeadChips. Treatment response was determined in 6 months using the European League Against Rheumatism (EULAR) response criteria. RESULTS: Initial filtering identified 21 cytosine-phosphate-guanines (CpGs) that were differentially methylated between responders and nonresponders. After conservative adjustment for multiple testing, six sites remained statistically significant, of which four showed high sensitivity and/or specificity (≥75%) for response to treatment. Moreover, methylation at two sites in combination was the strongest factor associated with response (80.0% sensitivity, 90.9% specificity, AUC 0.85). CONCLUSION: DNA methylation at diagnosis is associated with disease-modifying antirheumatic drug treatment response in early RA.

Quantitative genome-wide methylation analysis of high-grade non-muscle invasive bladder cancer.

High-grade non-muscle invasive bladder cancer (HG-NMIBC) is a clinically unpredictable disease with greater risks of recurrence and progression relative to their low-intermediate-grade counterparts. The molecular events, including those affecting the epigenome, that characterize this disease entity in the context of tumor development, recurrence, and progression, are incompletely understood. We therefore interrogated genome-wide DNA methylation using HumanMethylation450 BeadChip arrays in 21 primary HG-NMIBC tumors relative to normal bladder controls. Using strict inclusion-exclusion criteria we identified 1,057 hypermethylated CpGs within gene promoter-associated CpG islands, representing 256 genes. We validated the array data by bisulphite pyrosequencing and examined 25 array-identified candidate genes in an independent cohort of 30 HG-NMIBC and 18 low-intermediate-grade NMIBC. These analyses revealed significantly higher methylation frequencies in high-grade tumors relative to low-intermediate-grade tumors for the ATP5G2, IRX1 and VAX2 genes (P<0.05), and similarly significant increases in mean levels of methylation in high-grade tumors for the ATP5G2, VAX2, INSRR, PRDM14, VSX1, TFAP2b, PRRX1, and HIST1H4F genes (P<0.05). Although inappropriate promoter methylation was not invariantly associated with reduced transcript expression, a significant association was apparent for the ARHGEF4, PON3, STAT5a, and VAX2 gene transcripts (P<0.05). Herein, we present the first genome-wide DNA methylation analysis in a unique HG-NMIBC cohort, showing extensive and discrete methylation changes relative to normal bladder and low-intermediate-grade tumors. The genes we identified hold significant potential as targets for novel therapeutic intervention either alone, or in combination, with more conventional therapeutic options in the treatment of this clinically unpredictable disease.

Genome-wide profiling in treatment-naive early rheumatoid arthritis reveals DNA methylome changes in T and B lymphocytes.

AIM: Although aberrant DNA methylation has been described in rheumatoid arthritis (RA), no studies have interrogated this epigenetic modification in early disease. Following recent investigations of T and B lymphocytes in established disease, we now characterize in these cell populations genome-wide DNA methylation in treatment-naive patients with early RA. PATIENTS & METHODS: HumanMethylation450 BeadChips were used to examine genome-wide DNA methylation in lymphocyte populations from 23 early RA patients and 11 healthy individuals. RESULTS: Approximately 2000 CpGs in each cell type were differentially methylated in early RA. Clustering analysis identified a novel methylation signature in each cell type (150 sites in T lymphocytes, 113 sites in B lymphocytes) that clustered all patients separately from controls. A subset of sites differentially methylated in early RA displayed similar changes in established disease. CONCLUSION: Treatment-naive early RA patients display novel disease-specific DNA methylation aberrations, supporting a potential role for these changes in the development of RA.

DNA methylation profiling of synovial fluid FLS in rheumatoid arthritis reveals changes common with tissue-derived FLS.

AIM: Alterations in DNA methylation contribute to the abnormal phenotype of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). We profiled genome-wide DNA methylation in these cells from synovial fluid, a more readily accessible source of disease-associated cells. PATIENTS & METHODS: Genome-wide DNA methylation was interrogated in fluid-derived FLS from five RA and six osteoarthritis patients using Human Methylation 450 Bead Chip and bisulfite pyrosequencing. RESULTS: Array analysis identified 328 CpGs, representing 195 genes, that were differentially methylated between RA and osteoarthritis fluid-derived FLS. Comparison with the genes identified in two independent studies of tissue-derived FLS revealed 73 genes in common (~40%), of which 22 shared identity with both studies. Pyrosequencing confirmed altered methylation of these genes. CONCLUSION: Synovial fluid-derived RA FLS show methylation changes common with tissue-derived FLS, supporting the use of fluid-derived FLS for future investigations.

An in vitro model of mesenchymal stem cell targeting using magnetic particle labelling.

The specific targeting of cells to sites of tissue damage in vivo is a major challenge precluding the success of stem cell-based therapies. Magnetic particle-based targeting may provide a solution. Our aim was to provide a model system to study the trapping and potential targeting of human mesenchymal stem cells (MSCs) during in vitro fluid flow, which ultimately will inform cell targeting in vivo. In this system magnet arrays were used to trap superparamagnetic iron oxide particle-doped MSCs. The in vitro experiments demonstrated successful cell trapping, where the volume of cells trapped increased with magnetic particle concentration and decreased with increasing flow rate. Analysis of gene expression revealed significant increases in COL1A2 and SOX9. Using principles established in vitro, a proof-of-concept in vivo experiment demonstrated that magnetic particle-doped, luciferase-expressing MSCs were trapped by an implanted magnet in a subcutaneous wound model in nude mice. Our results demonstrate the effectiveness of using an in vitro model for testing superparamagnetic iron oxide particles to develop successful MSC targeting strategies during fluid flow, which ultimately can be translated to in vivo targeted delivery of cells via the circulation in a variety of tissue-repair models.

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations.

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.

Epigenome-wide profiling identifies significant differences in DNA methylation between matched-pairs of T- and B-lymphocytes from healthy individuals.

Multiple reports now describe changes to the DNA methylome in rheumatoid arthritis and in many cases have analyzed methylation in mixed cell populations from whole blood. However, these approaches may preclude the identification of cell type-specific methylation, which may subsequently bias identification of disease-specific changes. To address this possibility, we conducted genome-wide DNA methylation profiling using HumanMethylation450 BeadChips to identify differences within matched pairs of T-lymphocytes and B-lymphocytes isolated from the peripheral blood of 10 healthy females. Array data were processed and differential methylation identified using NIMBL software. Validation of array data was performed by bisulfite pyrosequencing. Genome-wide DNA methylation was initially determined by analysis of LINE-1 sequences and was higher in B-lymphocytes than matched T-lymphocytes (69.8% vs. 65.2%, P ≤ 0.01). Pairwise analysis identified 679 CpGs, representing 250 genes, which were differentially methylated between T-lymphocytes and B-lymphocytes. The majority of sites (76.6%) were hypermethylated in B-lymphocytes. Pyrosequencing of selected candidates confirmed the array data in all cases. Hierarchical clustering revealed perfect segregation of samples into two distinct clusters based on cell type. Differentially methylated genes showed enrichment for biological functions/pathways associated with leukocytes and T-lymphocytes. Our work for the first time shows that T-lymphocytes and B-lymphocytes possess intrinsic differences in DNA methylation within a restricted set of functionally related genes. These data provide a foundation for investigating DNA methylation in diseases in which these cell types play important and distinct roles.

Cyclic tensile strain upon human mesenchymal stem cells in 2D and 3D culture differentially influences CCNL2, WDR61 and BAHCC1 gene expression levels.

It has been shown that tensile strain can alter cell behaviour. Evidence exists to confirm that human mesenchymal stem cells can be encouraged to differentiate in response to tensile loading forces. We have investigated the short-term effects of cyclic tensile strain (3%, 1 Hz) on gene expression in primary human mesenchymal stem cells in monolayer and whilst encapsulated in a self-assembled peptide hydrogel. The main aims of the project were to gain the following novel information: (1) to determine if the genes CCNL2, WDR61 and BAHCC1 are potentially important mechanosensitive genes in monolayer, (2) to determine if these genes showed the same differential expression in a 3D environment (either tethered to RGD or simply encapsulated within a hydrogel (with RGE motif)) and (3) to determine whether the mesenchymal stem cells would survive within the hydrogels over several days whilst enduring dynamic culture. In the monolayer system, real-time PCR confirmed CCNL2 was significantly downregulated after 1 h strain and 2 h latency (post strain). BAHCC1 was significantly downregulated after 1 h strain (both 2 h and 24 h latency). WDR61 followed the same trend in 2D culture. After 24 h strain and 2 h latency, BAHCC1 was significantly upregulated. We found that both types of peptide hydrogel supported viable mesenchymal stem cells over 48 h. Results of the 3D dynamic culture did not correspond with those of the 2D dynamic culture, where the BAHCC1 gene was not expressed in the 3D experiments. The disparity in the differential gene expression observed between the 2D and 3D culture systems may partly be a result of the different cellular environments in each. It is likely that cells cultured within an intricate 3D architecture respond to mechanical cues in a different and more complex manner than do cells in 2D monolayer, as is illustrated by our gene expression data.

Tensile strain and magnetic particle force application do not induce MAP3K8 and IL-1B differential gene expression in a similar manner to fluid shear stress in human mesenchymal stem cells.

Mechanical forces, important in a variety of cellular processes, including proliferation, differentiation and gene expression, are also key in the development, remodelling and maintenance of load-bearing tissues such as cartilage and bone. Thus, there is great interest in using in vitro mechanical conditioning of mesenchymal stem cells (MSCs), multipotent adult stem cells, for tissue engineering of these tissues. In a previous gene expression study, we reported a potentially important role for mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and interleukin-1ß (IL-1B) in MAPK signalling in MSCs exposed to fluid shear stress. In this follow-up study, we examined the expression of these genes in MSCs exposed to other types of mechanical force: uniaxial tensile strain (3% cell elongation) and forces generated through the exposure of magnetic particle-labelled MSCs to an oscillating magnetic field (maximum field strength 90 mT). Exposure to both types of mechanical force for 1 h did not significantly alter the gene expression of MAP3K8 or IL-1B over the 24 h period subsequent to force exposure. These data demonstrate that uniaxial tensile strain and magnetic particle-based forces do not induce MAP3K8-related MAPK signalling in the same manner as does fluid flow-induced shear stress. This illustrates divergence in the process of mechanotransduction in mechanically stimulated MSCs.

Effect of fluid flow-induced shear stress on human mesenchymal stem cells: differential gene expression of IL1B and MAP3K8 in MAPK signaling.

Human bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into numerous cell lineages, making them ideal for tissue engineering. Mechanical forces and mechanotransduction are important factors influencing cell responses, although such data are limited for MSCs. We investigated the effect of different profiles of fluid flow-induced shear stress on mitogen-activated protein kinase (MAPK) signaling pathway gene expression in MSCs using DNA microarray and quantitative real-time reverse transcription-PCR analysis. In response to different magnitudes and durations of fluid flow-induced shear stress, we observed significant differential gene expression for various genes in the MAPK signaling pathway. Independent of magnitude and duration, shear stress induced consistent and marked up-regulation of MAP kinase kinase kinase 8 (MAP3K8) and interleukin-1 beta (IL1B) [2-fold to >35-fold, and 4-fold to >50-fold, respectively]. We also observed consistent up-regulation of dual specificity phosphatase 5 and 6, growth arrest and DNA-damage-inducible alpha and beta, nuclear factor kappa-B subunit 1, Jun oncogene, fibroblast growth factor 1, and platelet-derived growth factor alpha. Our data support MAP3K8-induced activation of different MAPK signaling pathways in response to different profiles of shear stress, possibly as a consequence of shear-induced IL1B expression. Thus, MAP3K8 may be an important mediator of intracellular mechanotransduction in human MSCs.

Circulating levels of tumor necrosis factor receptors are highly predictive of mortality in patients with rheumatoid arthritis.

OBJECTIVE: To investigate whether circulating levels of soluble tumor necrosis factor receptors (sTNFR) are predictive of mortality in rheumatoid arthritis (RA). METHODS: Levels of sTNFRI and sTNFRII at study entry were quantified using enzyme-linked immunosorbent assays in sera from 401 white patients with RA followed up for 13 years. Patients were tracked via the National Health Service Central Register, and the relationship between sTNFR levels and mortality was analyzed using a Cox proportional hazards regression model. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: At the end of the followup period, 132 (32.9%) of 401 patients had died. Of these, 64 (48.5%) died of cardiovascular disease (CVD). Significant associations between all-cause mortality and baseline levels of sTNFRI and sTNFRII were identified in men (HR 1.7 [95% CI 1.2-2.4] and HR 1.18 [95% CI 1.05-1.32], respectively) and women (HR 1.33 [95% CI 0.99-1.8] and HR 1.14 [95% CI 1.02-1.28], respectively). Analysis including levels of both sTNFRI and sTNFRII indicated that the sTNFRII level was the best overall predictor of mortality. Multivariate analysis also revealed that the sTNFRII level was a predictor of all-cause and CVD mortality independently of age, sex, disease duration, C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor, nodular disease, modified Health Assessment Questionnaire score, taking CVD drugs, and smoking. CONCLUSION: Our data indicate that serum levels of sTNFR are powerful predictors of mortality in RA. Elevated levels are particularly associated with mortality due to CVD and may be useful for identifying patients at increased risk of premature death.

Polymorphism in the tumour necrosis factor receptor II gene is associated with circulating levels of soluble tumour necrosis factor receptors in rheumatoid arthritis.

Levels of soluble tumour necrosis factor receptors (sTNFRs) are elevated in the circulation of patients with rheumatoid arthritis (RA). Although these receptors can act as natural inhibitors of tumour necrosis factor-alpha, levels of sTNFRs in RA appear to be insufficient to prevent tumour necrosis factor-alpha induced inflammation. The factors that regulate circulating levels of sTNFRs are unclear, but polymorphisms in the tumour necrosis factor receptor genes may play a role. We investigated the relationship between polymorphisms in the tumour necrosis factor receptor I (TNF-RI) and II (TNF-RII) genes and levels of sTNFRs in two groups of Caucasian RA patients: one with early (disease duration < or = 2 years; n = 103) and one with established disease (disease duration > or = 5 years; n = 151). PCR restriction fragment length polymorphism analysis was used to genotype patients for the A36G polymorphism in the TNF-RI gene and the T676G polymorphism in TNF-RII. Levels of sTNFRs were measured using ELISA. We also isolated T cells from peripheral blood of 58 patients with established RA with known TNF-R genotypes, and release of sTNFRs into the culture medium was measured in cells incubated with or without phytohaemagglutinin. Serum levels of the two sTNFRs (sTNF-RI and sTNF-RII) were positively correlated in both populations, and the level of each sTNFR was significantly higher in the patients with established disease (P < 0.0001). Multiple regression analyses corrected for age, sex and disease duration revealed a significant trend toward decreasing sTNF-RI and sTNF-RII levels across the TNF-RII genotypes (TT > TG > GG) of patients with established disease (P for trend = 0.01 and P for trend = 0.03, respectively). A similar nonsignificant trend was seen for early disease. No relationship with the TNF-RI A36G polymorphism was observed. sTNFRs released by isolated T cells exhibited a similar trend toward decreasing levels according to TNF-RII genotype, although only the association with levels of sTNF-RII was significant. Strong correlations were found between levels of circulating sTNFRs and levels released by T cells in vitro. Our data indicate that the T676G polymorphism in TNF-RII is associated with levels of sTNFRs released from peripheral blood T cells, and with circulating levels of sTNFR in patients with RA.

Anemia in rheumatoid arthritis: association with polymorphism in the tumor necrosis factor receptor I and II genes.

OBJECTIVE: To investigate whether polymorphisms in the tumor necrosis factor receptor I (TNFRSF1A) and receptor II (TNFRSF1B) genes are associated with the anemia observed in rheumatoid arthritis (RA). METHODS: We studied a group of Caucasian patients (n = 160) with established RA on whom longitudinal data of hemoglobin (Hb) levels over 5 years were recorded. A second group of patients (n = 102) with early RA was used for a confirmation study. Polymerase chain reaction restriction fragment length polymorphism analysis was used to genotype patients for the A36G polymorphism in the TNFRSF1A gene, and the T676G polymorphism in TNFRSF1B. Serum levels of ferritin were determined by ELISA and used to differentiate between iron deficiency anemia (IDA) and anemia of chronic disease (ACD). Data were analyzed by Kruskal-Wallis analysis of variance and logistic regression analysis. RESULTS: The TNFRSF1A GG genotype was associated with lower 5-year mean area under the curve Hb levels compared with other genotypes (p = 0.01). Analysis of anemic status showed an increased frequency of anemia in patients carrying a G allele, with the highest frequency in GG homozygotes. The TNFRSF1A GG genotype was significantly associated with IDA in established RA (OR 4.3, p = 0.01), and this was confirmed in a group of patients with early RA (OR 4.8, p = 0.04). Analysis of the combined groups also showed a weak association of the G allele with ACD (OR 2.2, p = 0.04). No association was found between TNFRSF1B variants and anemia when the cohorts were analyzed separately, but an association between carriage of the T allele and ACD was found when the 2 groups were combined (OR 11.5, p = 0.01). CONCLUSION: Our data suggest that polymorphisms within the TNFRSF1A and TNFRSF1B genes are associated with IDA and/or ACD in patients with RA.

No association of polymorphisms in the tumor necrosis factor receptor I and receptor II genes with disease severity in rheumatoid arthritis.

OBJECTIVE: A recent Italian study found that homozygosity for the G allele of the +196 single nucleotide polymorphism (SNP) of the tumor necrosis factor receptor II (TNFRSF1B) gene was more prevalent in patients with severe rheumatoid arthritis (RA). We investigated whether this particular SNP, and also one at position +36 in exon 1 of the TNF receptor I (TNFRSF1A) gene, are associated with disease severity. METHODS: A group of 181 Caucasian patients with RA was studied. DNA was isolated from patient blood samples and subsequently used to genotype both the exon 1 TNFRSF1A SNP and the exon 6 TNFRSF1B SNP by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Radiographic damage was measured by the Larsen score, and functional outcome was assessed by the Health Assessment Questionnaire (HAQ). Data were analyzed by multiple regression analysis, with correction for age, sex, and disease duration. RESULTS: The mean Larsen and HAQ scores did not differ significantly between each of the genotypes from the 2 TNFR SNP. No significant associations between the +36 TNFRSF1A SNP or the +196 TNFRSF1B SNP genotypes and disease severity were found after correcting for age, sex, and disease duration. CONCLUSION: Our data suggest that neither the +36 TNFRSF1A SNP nor the +196 TNFRSF1B SNP is associated with RA severity in a population of Caucasian patients with RA.