To identify differentially methylated positions (DMPs) and regions (DMRs) that predict response t... more To identify differentially methylated positions (DMPs) and regions (DMRs) that predict response to Methotrexate (MTX) in early rheumatoid arthritis (RA) patients. Materials and methods DNA from baseline peripheral blood mononuclear cells was extracted from 72 RA patients. DNA methylation, quantified using the Infinium MethylationEPIC, was assessed in relation to response to MTX (combination) therapy over the first 3 months. Results Baseline DMPs associated with response were identified; including hits previously described in RA. Additionally, 1309 DMR regions were observed. However, none of these findings were genome-wide significant. Likewise, no specific pathways were related to response, nor could we replicate associations with previously identified DMPs. Conclusion No baseline genome-wide significant differences were identified as biomarker for MTX (combination) therapy response; hence meta-analyses are required.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
595 (ADA) or CZP. Clinical and demographic data were collected at baseline (T0) and after 6 month... more 595 (ADA) or CZP. Clinical and demographic data were collected at baseline (T0) and after 6 months (T6) of treatment. RF titers and serum drug levels were measured at T0 and T6 using nephelometry and ELISA respectively. Association between baseline RF titers and drug levels was assessed using non-parametric test (Mann-Whitney). Results: 168 patients were evaluated: 90 received IFX, 48 ADA and 32 CZP. Characteristics at T0 are shown in Table 1. All patients had active disease at baseline and 76% were RF positive: ADA subgroup had lower percentage of positive RF than IFX and CZP subgroups. Patients were stratified into quartiles based on baseline RF titers: low (20-57 IU/ml), medium (57-380 IU/ml), high (>380 IU/ml) and seronegative (<20 IU/ml). Drug levels of IFX and ADA at T6 were significantly lower in those patients who had higher RF titers at T0 compared to seronegative. In contrast, CZP levels remained stable irrespectively of baseline RF titers, without significant differences among quartiles (Figure 1). Conclusion: Higher baseline RF titers are associated with lower IFX and ADA levels at T6 in a cohort of RA patients. A concentration-response association has been clearly established for TNFi, and baseline RF levels appear to influence drug levels. Reduced immune complexes formation with CZP may result in a limited impact of baseline RF titers on drug levels.
Objective: To improve a previously developed prediction model that could assist in the triage of ... more Objective: To improve a previously developed prediction model that could assist in the triage of individual case safety reports using the addition of features designed from free text fields using natural language processing.Methods: Structured features and natural language processing (NLP) features were used to train a bagging classifier model. NLP features were extracted from free text fields. A bag-of-words model was applied. Stop words were deleted and words that were significantly differently distributed among the case and non-case reports were used for the training data. Besides NLP features from free-text fields, the data also consisted of a list of signal words deemed important by expert report assessors. Lastly, variables with multiple categories were transformed to numerical variables using the weight of evidence method.Results: the model, a bagging classifier of decision trees had an AUC of 0.921 (95% CI = 0.918–0.925). Generic drug name, info text length, ATC code, BMI an...
Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhi... more Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhibiting IL-6R signal transduction to downstream inflammatory mediators. TCZ has shown to be effective as monotherapy in early rheumatoid arthritis (RA) patients (1). However, approximately one third of patients inadequately respond to therapy and the biological mechanisms underlying lack of efficacy for TCZ remain elusive (1). Here we report gene expression differences, in both whole blood and peripheral blood mononuclear cells (PBMC) RNA samples between early RA patients, categorized by clinical TCZ response (reaching DAS28 < 3.2 at 6 months). These findings could lead to identification of predictive biomarkers for TCZ response and improve RA treatment strategies.To identify potential baseline gene expression markers for TCZ response in early RA patients using an RNA-sequencing approach.Two cohorts of RA patients were included and blood was collected at baseline, before initiating TCZ...
Mechanism of action of MTX MTX is an antifolate and shares a similar chemical structure with fola... more Mechanism of action of MTX MTX is an antifolate and shares a similar chemical structure with folate, containing one glutamate group. Circulating MTX is transported into tissue mainly through the solute carrier family 19 member 1/reduced folate carrier (SLC19A1/RFC) [26,27] , for which MTX has highest affinity (Km= 1-10 μM). In the intestine, MTX is mainly transported through the solute carrier family 46 member 1/proton coupled folate transporter (SLC46A1/PCFT), which functions well in an acidic environment [28]. Furthermore, MTX is transported through folate receptors (FOLR) 1 and 2 via endocytosis and for which MTX has much lower binding affinity (Kd = 10−300 nM) (Figure 1) [29,30]. Intracellularly, the enzyme folylpolyglutamate synthetase (FPGS) attaches additional polyglutamate groups to MTX in a process called polyglutamylation, while gamma-glutamyl hydrolase (GGH) reverts this action through de-polyglutamylation. Polyglutamylation of MTX is required to remain intracellular, because longer MTX-PG chains (MTX-PG 4-5) are no targets for the ATP binding cassette (ABC) efflux transporter. In RA patients, polyglutamylation of up to 5 MTX-PGs has been observed [24,31-34]. Longchain MTX-PGs have been associated with better response [24,34] and have shown to have higher affinity to its downstream targets. Yet, large longitudinal studies on cellular MTX pharmacokinetics and pharmacodynamics are lacking and large inter-patient variability in erythrocyte MTX-PGs has been observed, which complicates its use for therapeutic drug monitoring. Intracellular downstream targets of MTX include: thymidylate synthase (TS) and phosphoribosylglycinamide formyltransferase (GART), which are required for DNA synthesis. Additionally, it inhibits dihydrofolate reductase (DHFR) in the folate pathway, which is involved in donation of methyl groups for (DNA) methylation reactions [35,36]. Another target is the 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (ATIC), resulting in increased AICAR and enhanced adenosine release, which has anti-inflammatory effects [37,38]. Folic acid is prescribed along (but not on the same day as) MTX to reduce adverse events, which does not reduce the efficacy of MTX [39,40]. As folic acid restores the intracellular folate pool required for DNA synthesis and is not related to the release of adenosine, the adenosine pathway is proposed to be the most likely mechanism of anti-inflammatory action of MTX [26]. PREDICTION OF MTX INEFFICACY Clinical predictors of response to MTX Clinical parameters would be easiest to use as predictors for response, since these are often readily available without the need for laboratory assessments. Several clinical variables have been associated with response to MTX such as symptom duration, baseline Health Assessment Questionnaire (HAQ), baseline DAS28 (components), smoking, Body Mass Index (BMI), and psychological predictors (i.e. higher Hospital Anxiety and Depression Scale anxiety score) [41-43]. However, not all of these predictors have been validated or could General introduction
Table S1. Global DNA methylation and hydroxymethylation levels before MTX and three months after ... more Table S1. Global DNA methylation and hydroxymethylation levels before MTX and three months after MTX therapy. Table S2. Linear regression models of %methylation in 6 LINE-1 CpG sites in relation to Î DAS28 over three months of MTX therapy. Figure S1. Pearson correlation between global DNA methylation quantified using the LC-ESI-MS/MS and LINE-1 technique. (DOCX 153â kb)
Methotrexate (MTX) constitutes the first-line therapy in rheumatoid arthritis (RA), yet approxima... more Methotrexate (MTX) constitutes the first-line therapy in rheumatoid arthritis (RA), yet approximately 30% of the patients do not benefit from MTX. Recently, we reported a prognostic multivariable prediction model for insufficient clinical response to MTX at 3 months of treatment in the treatment in the Rotterdam Early Arthritis Cohort (tREACH), including baseline predictors: Disease activity score 28 (DAS28), Health Assessment Questionnaire (HAQ), erythrocyte folate, single-nucleotide polymorphisms (SNPs; ABCB1, ABCC3), smoking, and BMI. The purpose of the current study was (1) to externally validate the model and (2) to enhance the model’s clinical applicability. Erythrocyte folate and SNPs were assessed in 91 early disease-modifying antirheumatic drug (DMARD)-naïve RA patients starting MTX in the external validation cohort (U-Act-Early). Insufficient response (DAS28 > 3.2) was determined after 3 months and non-response after 6 months of therapy. The previously developed predict...
This study aimed to identify baseline metabolic biomarkers for response to methotrexate (MTX) the... more This study aimed to identify baseline metabolic biomarkers for response to methotrexate (MTX) therapy in rheumatoid arthritis (RA) using an untargeted method. In total, 82 baseline plasma samples (41 insufficient responders and 41 sufficient responders to MTX) were selected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, trial number: ISRCTN26791028) based on patients’ EULAR response at 3 months. Metabolites were assessed using high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Differences in metabolite concentrations between insufficient and sufficient responders were assessed using partial least square regression discriminant analysis (PLS-DA) and Welch’s t-test. The predictive performance of the most significant findings was assessed in a receiver operating characteristic plot with area under the curve (AUC), sensitivity and specificity. Finally, overrepresentation analysis was performed to assess if the best discriminating met...
The goals of this study were to examine whether machine-learning algorithms outperform multivaria... more The goals of this study were to examine whether machine-learning algorithms outperform multivariable logistic regression in the prediction of insufficient response to methotrexate (MTX); secondly, to examine which features are essential for correct prediction; and finally, to investigate whether the best performing model specifically identifies insufficient responders to MTX (combination) therapy. The prediction of insufficient response (3-month Disease Activity Score 28-Erythrocyte-sedimentation rate (DAS28-ESR) > 3.2) was assessed using logistic regression, least absolute shrinkage and selection operator (LASSO), random forest, and extreme gradient boosting (XGBoost). The baseline features of 355 rheumatoid arthritis (RA) patients from the “treatment in the Rotterdam Early Arthritis CoHort” (tREACH) and the U-Act-Early trial were combined for analyses. The model performances were compared using area under the curve (AUC) of receiver operating characteristic (ROC) curves, 95% co...
Background: Epigenetic markers are often quantified and related to disease in stored samples. Whi... more Background: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. Methods: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, −20°C and −80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. Results: global DNA methylation was stable over 18 months in blood at −20°C and −80°C and DNA at 4°C and −80°C. However, at 18 months DNA methylation from DNA stored at −20°C relatively decreased −6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze-thaw cycles. Conclusion: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of small differences occuring during storage depend on the expected effect size and research question.
Background: Low-dose methotrexate (MTX) is the first-line therapy in early rheumatoid arthritis (... more Background: Low-dose methotrexate (MTX) is the first-line therapy in early rheumatoid arthritis (eRA). Up to 40% of eRA patients do not benefit from MTX therapy. MTX has been shown to inhibit one-carbon metabolism, which is involved in the donation of methyl groups. In this study, we investigate baseline global DNA methylation and changes in DNA methylation during treatment in relation to clinical non-response after 3 months of MTX treatment. Methods: Two hundred ninety-four blood samples were collected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, ISRCTN26791028), a multicenter, stratified single-blind clinical trial of eRA patients. Global DNA (hydroxy) methylation was quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/ MS) and validated with a global DNA LINE-1 methylation technique. MTX response was determined as ΔDAS28. Additionally, patients were stratified into two response groups according to the European League Against Rheumatism (EULAR) response criteria. Associations between global DNA methylation and response were examined using univariate regression models adjusted for baseline DAS28, baseline erythrocyte folate levels, and body mass index (BMI). Results: Higher baseline global DNA methylation was associated with less decrease of DAS28 (β = 0.15, p = 0.013) and with MTX non-response (OR = 0.010, 95% CI = 0.001-0.188). This result was validated in LINE-1 elements (β = 0.22, p = 0.026). Changes in global DNA (hydroxy)methylation were not associated with MTX response over 3 months. Conclusions: These results show that higher baseline global DNA methylation in treatment naïve eRA patients is associated with decreased clinical response after 3 months of treatment of eRA patients and can be further evaluated as a predictor for MTX therapy non-response.
Aim To identify differentially methylated positions (DMPs) and regions (DMRs) that predict respon... more Aim To identify differentially methylated positions (DMPs) and regions (DMRs) that predict response to Methotrexate (MTX) in early rheumatoid arthritis (RA) patients. Materials and methods DNA from baseline peripheral blood mononuclear cells was extracted from 72 RA patients. DNA methylation, quantified using the Infinium MethylationEPIC, was assessed in relation to response to MTX (combination) therapy over the first 3 months. Results Baseline DMPs associated with response were identified; including hits previously described in RA. Additionally, 1309 DMR regions were observed. However, none of these findings were genome-wide significant. Likewise, no specific pathways were related to response, nor could we replicate associations with previously identified DMPs. Conclusion No baseline genome-wide significant differences were identified as biomarker for MTX (combination) therapy response; hence meta-analyses are required.
To identify differentially methylated positions (DMPs) and regions (DMRs) that predict response t... more To identify differentially methylated positions (DMPs) and regions (DMRs) that predict response to Methotrexate (MTX) in early rheumatoid arthritis (RA) patients. Materials and methods DNA from baseline peripheral blood mononuclear cells was extracted from 72 RA patients. DNA methylation, quantified using the Infinium MethylationEPIC, was assessed in relation to response to MTX (combination) therapy over the first 3 months. Results Baseline DMPs associated with response were identified; including hits previously described in RA. Additionally, 1309 DMR regions were observed. However, none of these findings were genome-wide significant. Likewise, no specific pathways were related to response, nor could we replicate associations with previously identified DMPs. Conclusion No baseline genome-wide significant differences were identified as biomarker for MTX (combination) therapy response; hence meta-analyses are required.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
595 (ADA) or CZP. Clinical and demographic data were collected at baseline (T0) and after 6 month... more 595 (ADA) or CZP. Clinical and demographic data were collected at baseline (T0) and after 6 months (T6) of treatment. RF titers and serum drug levels were measured at T0 and T6 using nephelometry and ELISA respectively. Association between baseline RF titers and drug levels was assessed using non-parametric test (Mann-Whitney). Results: 168 patients were evaluated: 90 received IFX, 48 ADA and 32 CZP. Characteristics at T0 are shown in Table 1. All patients had active disease at baseline and 76% were RF positive: ADA subgroup had lower percentage of positive RF than IFX and CZP subgroups. Patients were stratified into quartiles based on baseline RF titers: low (20-57 IU/ml), medium (57-380 IU/ml), high (>380 IU/ml) and seronegative (<20 IU/ml). Drug levels of IFX and ADA at T6 were significantly lower in those patients who had higher RF titers at T0 compared to seronegative. In contrast, CZP levels remained stable irrespectively of baseline RF titers, without significant differences among quartiles (Figure 1). Conclusion: Higher baseline RF titers are associated with lower IFX and ADA levels at T6 in a cohort of RA patients. A concentration-response association has been clearly established for TNFi, and baseline RF levels appear to influence drug levels. Reduced immune complexes formation with CZP may result in a limited impact of baseline RF titers on drug levels.
Objective: To improve a previously developed prediction model that could assist in the triage of ... more Objective: To improve a previously developed prediction model that could assist in the triage of individual case safety reports using the addition of features designed from free text fields using natural language processing.Methods: Structured features and natural language processing (NLP) features were used to train a bagging classifier model. NLP features were extracted from free text fields. A bag-of-words model was applied. Stop words were deleted and words that were significantly differently distributed among the case and non-case reports were used for the training data. Besides NLP features from free-text fields, the data also consisted of a list of signal words deemed important by expert report assessors. Lastly, variables with multiple categories were transformed to numerical variables using the weight of evidence method.Results: the model, a bagging classifier of decision trees had an AUC of 0.921 (95% CI = 0.918–0.925). Generic drug name, info text length, ATC code, BMI an...
Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhi... more Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhibiting IL-6R signal transduction to downstream inflammatory mediators. TCZ has shown to be effective as monotherapy in early rheumatoid arthritis (RA) patients (1). However, approximately one third of patients inadequately respond to therapy and the biological mechanisms underlying lack of efficacy for TCZ remain elusive (1). Here we report gene expression differences, in both whole blood and peripheral blood mononuclear cells (PBMC) RNA samples between early RA patients, categorized by clinical TCZ response (reaching DAS28 < 3.2 at 6 months). These findings could lead to identification of predictive biomarkers for TCZ response and improve RA treatment strategies.To identify potential baseline gene expression markers for TCZ response in early RA patients using an RNA-sequencing approach.Two cohorts of RA patients were included and blood was collected at baseline, before initiating TCZ...
Mechanism of action of MTX MTX is an antifolate and shares a similar chemical structure with fola... more Mechanism of action of MTX MTX is an antifolate and shares a similar chemical structure with folate, containing one glutamate group. Circulating MTX is transported into tissue mainly through the solute carrier family 19 member 1/reduced folate carrier (SLC19A1/RFC) [26,27] , for which MTX has highest affinity (Km= 1-10 μM). In the intestine, MTX is mainly transported through the solute carrier family 46 member 1/proton coupled folate transporter (SLC46A1/PCFT), which functions well in an acidic environment [28]. Furthermore, MTX is transported through folate receptors (FOLR) 1 and 2 via endocytosis and for which MTX has much lower binding affinity (Kd = 10−300 nM) (Figure 1) [29,30]. Intracellularly, the enzyme folylpolyglutamate synthetase (FPGS) attaches additional polyglutamate groups to MTX in a process called polyglutamylation, while gamma-glutamyl hydrolase (GGH) reverts this action through de-polyglutamylation. Polyglutamylation of MTX is required to remain intracellular, because longer MTX-PG chains (MTX-PG 4-5) are no targets for the ATP binding cassette (ABC) efflux transporter. In RA patients, polyglutamylation of up to 5 MTX-PGs has been observed [24,31-34]. Longchain MTX-PGs have been associated with better response [24,34] and have shown to have higher affinity to its downstream targets. Yet, large longitudinal studies on cellular MTX pharmacokinetics and pharmacodynamics are lacking and large inter-patient variability in erythrocyte MTX-PGs has been observed, which complicates its use for therapeutic drug monitoring. Intracellular downstream targets of MTX include: thymidylate synthase (TS) and phosphoribosylglycinamide formyltransferase (GART), which are required for DNA synthesis. Additionally, it inhibits dihydrofolate reductase (DHFR) in the folate pathway, which is involved in donation of methyl groups for (DNA) methylation reactions [35,36]. Another target is the 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (ATIC), resulting in increased AICAR and enhanced adenosine release, which has anti-inflammatory effects [37,38]. Folic acid is prescribed along (but not on the same day as) MTX to reduce adverse events, which does not reduce the efficacy of MTX [39,40]. As folic acid restores the intracellular folate pool required for DNA synthesis and is not related to the release of adenosine, the adenosine pathway is proposed to be the most likely mechanism of anti-inflammatory action of MTX [26]. PREDICTION OF MTX INEFFICACY Clinical predictors of response to MTX Clinical parameters would be easiest to use as predictors for response, since these are often readily available without the need for laboratory assessments. Several clinical variables have been associated with response to MTX such as symptom duration, baseline Health Assessment Questionnaire (HAQ), baseline DAS28 (components), smoking, Body Mass Index (BMI), and psychological predictors (i.e. higher Hospital Anxiety and Depression Scale anxiety score) [41-43]. However, not all of these predictors have been validated or could General introduction
Table S1. Global DNA methylation and hydroxymethylation levels before MTX and three months after ... more Table S1. Global DNA methylation and hydroxymethylation levels before MTX and three months after MTX therapy. Table S2. Linear regression models of %methylation in 6 LINE-1 CpG sites in relation to Î DAS28 over three months of MTX therapy. Figure S1. Pearson correlation between global DNA methylation quantified using the LC-ESI-MS/MS and LINE-1 technique. (DOCX 153â kb)
Methotrexate (MTX) constitutes the first-line therapy in rheumatoid arthritis (RA), yet approxima... more Methotrexate (MTX) constitutes the first-line therapy in rheumatoid arthritis (RA), yet approximately 30% of the patients do not benefit from MTX. Recently, we reported a prognostic multivariable prediction model for insufficient clinical response to MTX at 3 months of treatment in the treatment in the Rotterdam Early Arthritis Cohort (tREACH), including baseline predictors: Disease activity score 28 (DAS28), Health Assessment Questionnaire (HAQ), erythrocyte folate, single-nucleotide polymorphisms (SNPs; ABCB1, ABCC3), smoking, and BMI. The purpose of the current study was (1) to externally validate the model and (2) to enhance the model’s clinical applicability. Erythrocyte folate and SNPs were assessed in 91 early disease-modifying antirheumatic drug (DMARD)-naïve RA patients starting MTX in the external validation cohort (U-Act-Early). Insufficient response (DAS28 > 3.2) was determined after 3 months and non-response after 6 months of therapy. The previously developed predict...
This study aimed to identify baseline metabolic biomarkers for response to methotrexate (MTX) the... more This study aimed to identify baseline metabolic biomarkers for response to methotrexate (MTX) therapy in rheumatoid arthritis (RA) using an untargeted method. In total, 82 baseline plasma samples (41 insufficient responders and 41 sufficient responders to MTX) were selected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, trial number: ISRCTN26791028) based on patients’ EULAR response at 3 months. Metabolites were assessed using high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Differences in metabolite concentrations between insufficient and sufficient responders were assessed using partial least square regression discriminant analysis (PLS-DA) and Welch’s t-test. The predictive performance of the most significant findings was assessed in a receiver operating characteristic plot with area under the curve (AUC), sensitivity and specificity. Finally, overrepresentation analysis was performed to assess if the best discriminating met...
The goals of this study were to examine whether machine-learning algorithms outperform multivaria... more The goals of this study were to examine whether machine-learning algorithms outperform multivariable logistic regression in the prediction of insufficient response to methotrexate (MTX); secondly, to examine which features are essential for correct prediction; and finally, to investigate whether the best performing model specifically identifies insufficient responders to MTX (combination) therapy. The prediction of insufficient response (3-month Disease Activity Score 28-Erythrocyte-sedimentation rate (DAS28-ESR) > 3.2) was assessed using logistic regression, least absolute shrinkage and selection operator (LASSO), random forest, and extreme gradient boosting (XGBoost). The baseline features of 355 rheumatoid arthritis (RA) patients from the “treatment in the Rotterdam Early Arthritis CoHort” (tREACH) and the U-Act-Early trial were combined for analyses. The model performances were compared using area under the curve (AUC) of receiver operating characteristic (ROC) curves, 95% co...
Background: Epigenetic markers are often quantified and related to disease in stored samples. Whi... more Background: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. Methods: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, −20°C and −80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. Results: global DNA methylation was stable over 18 months in blood at −20°C and −80°C and DNA at 4°C and −80°C. However, at 18 months DNA methylation from DNA stored at −20°C relatively decreased −6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze-thaw cycles. Conclusion: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of small differences occuring during storage depend on the expected effect size and research question.
Background: Low-dose methotrexate (MTX) is the first-line therapy in early rheumatoid arthritis (... more Background: Low-dose methotrexate (MTX) is the first-line therapy in early rheumatoid arthritis (eRA). Up to 40% of eRA patients do not benefit from MTX therapy. MTX has been shown to inhibit one-carbon metabolism, which is involved in the donation of methyl groups. In this study, we investigate baseline global DNA methylation and changes in DNA methylation during treatment in relation to clinical non-response after 3 months of MTX treatment. Methods: Two hundred ninety-four blood samples were collected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, ISRCTN26791028), a multicenter, stratified single-blind clinical trial of eRA patients. Global DNA (hydroxy) methylation was quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/ MS) and validated with a global DNA LINE-1 methylation technique. MTX response was determined as ΔDAS28. Additionally, patients were stratified into two response groups according to the European League Against Rheumatism (EULAR) response criteria. Associations between global DNA methylation and response were examined using univariate regression models adjusted for baseline DAS28, baseline erythrocyte folate levels, and body mass index (BMI). Results: Higher baseline global DNA methylation was associated with less decrease of DAS28 (β = 0.15, p = 0.013) and with MTX non-response (OR = 0.010, 95% CI = 0.001-0.188). This result was validated in LINE-1 elements (β = 0.22, p = 0.026). Changes in global DNA (hydroxy)methylation were not associated with MTX response over 3 months. Conclusions: These results show that higher baseline global DNA methylation in treatment naïve eRA patients is associated with decreased clinical response after 3 months of treatment of eRA patients and can be further evaluated as a predictor for MTX therapy non-response.
Aim To identify differentially methylated positions (DMPs) and regions (DMRs) that predict respon... more Aim To identify differentially methylated positions (DMPs) and regions (DMRs) that predict response to Methotrexate (MTX) in early rheumatoid arthritis (RA) patients. Materials and methods DNA from baseline peripheral blood mononuclear cells was extracted from 72 RA patients. DNA methylation, quantified using the Infinium MethylationEPIC, was assessed in relation to response to MTX (combination) therapy over the first 3 months. Results Baseline DMPs associated with response were identified; including hits previously described in RA. Additionally, 1309 DMR regions were observed. However, none of these findings were genome-wide significant. Likewise, no specific pathways were related to response, nor could we replicate associations with previously identified DMPs. Conclusion No baseline genome-wide significant differences were identified as biomarker for MTX (combination) therapy response; hence meta-analyses are required.
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