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Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate.

de Andres MC, Perez-Pampin E, Calaza M, Santaclara FJ, Ortea I, Gomez-Reino JJ, Gonzalez A - Arthritis Res. Ther. (2015)

Bottom Line: There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells.In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigacion 10 and Rheumatology Unit, Instituto de Investigación Sanitaria-Hospital Clínico Universitario de Santiago, Travesia de Choupana, s/n, 15706, Santiago de Compostela, Spain. maydeandres@gmail.com.

ABSTRACT

Introduction: DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results.

Method: Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR).

Results: Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.

Conclusions: Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

No MeSH data available.


Related in: MedlinePlus

Expression of methylation enzymes in blood cells of healthy controls (HC) and early rheumatoid arthritis (RA) patients. Normalized relative expression of DNMT1 obtained by quantitative polymerase chain reaction (qPCR) in a T cells, b B cells, and c monocytes, DNMT3A in B cells (d) and of GADD45A in T cells (e) and B cells (f) is shown. Other conventions are as in Fig. 1. All these comparisons were significant. No comparison in other cells subpopulation and none of the DNMT3B analyses were significant
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Fig2: Expression of methylation enzymes in blood cells of healthy controls (HC) and early rheumatoid arthritis (RA) patients. Normalized relative expression of DNMT1 obtained by quantitative polymerase chain reaction (qPCR) in a T cells, b B cells, and c monocytes, DNMT3A in B cells (d) and of GADD45A in T cells (e) and B cells (f) is shown. Other conventions are as in Fig. 1. All these comparisons were significant. No comparison in other cells subpopulation and none of the DNMT3B analyses were significant

Mentions: Levels of global 5mC were similar to those previously reported [21, 22]. Comparison of the global 5mC level between patients and controls was done with the samples of RA patients before MTX treatment. Main effects ANCOVA with sex and age as covariates was used for these comparisons. T lymphocytes showed significant DNA hypomethylation in early RA patients compared with healthy subjects (Fig. 1a; mean = 3.89 %, 95 % confidence interval (CI) = 3.80–3.99 vs. 4.15 %, 95 % CI = 3.96–4.38, respectively, P = 0.011). The multivariate analysis also showed significant DNA hypomethylation in the monocytes of RA patients (Fig. 1c; mean = 3.96 %, 95 % CI = 3.87–4.07 vs. 4.13, 95 % CI = 3.98–4.33, respectively, P = 0.047). Although significant, these differences were small. No significant differences were detected in any of the other blood cell subpopulations: B lymphocytes, NK cells and polymorphonuclear (PMN) cells (Fig. 1b and not shown). No significant correlation between the total methylprednisolone dose received until blood drawing and levels of 5mC in any of the five blood subpopulations were observed in RA patients (not shown). In the same assay, 5hmC was also determined without any significant difference between patients and healthy controls, showing very low levels in all cell subpopulations (around 0.02 % of total cytosine) as is characteristic of most adult tissues [21, 28]. DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3 and GADD45A relative expression was determined by qPCR analysis in each of the blood cell subpopulations. A significant decrease in patients with RA was observed for some enzymes: DNMT1 expression was decreased in T cells (Fig. 2a; mean = 5.6, 95 % CI = 4.3–7.2 vs. 10.0, 95 % CI = 7.7–13.1; P = 0.0022), B cells (Fig. 2b; mean = 3.4, 95 % CI = 2.6–4.3 vs. 6.0, 95 % CI = 4.6–7.4; P = 0.00046) and monocytes (Fig. 2c; mean = 5.6, 95 % CI = 4.5–6.7 vs. 8.4, 95 % CI = 7.4–9.4; P = 0.00020); DNMT3A was decreased, although less markedly, in B cells (Fig. 2d; mean = 27.5, 95 % CI = 17.9–42.2 vs. 46.9, 95 % CI = 32.4–67.9; P = 0.044); no difference was found in the expression of DNMT3B in any of the blood cell subpopulations (not shown); and GADD45A expression was also moderately reduced in T cells (Fig. 2e; mean = 12.2, 95 % CI = 8.7–17.3 vs. 20.0, 95 % CI = 13.9–28.8; P = 0.048) and in B cells (Fig. 2f; mean = 9.6, 95 % CI = 5.8–45.9 vs. 18.3, 95 % CI = 12.9–25.9; P = 0.021). On the contrary, the relative expression of TET enzymes was increased in some cell populations. Monocytes showed the largest increases, with the three TET enzyme genes showing higher relative levels of expression in RA patients than in controls. TET1 and TET3 showed the most marked difference between RA patients and controls (TET1, Fig. 3a; mean = 2.9, 95 % CI = 2.1–4.8 vs. 1.9, 95 % CI = 1.6–2.3; P = 0.0044; and TET3, Fig. 3c; mean = 2.0, 95 % CI = 1.7–2.3 vs. 1.35, 95 % CI = 1.15–1.6; P = 0.0014), but also TET2 (Fig. 3b; mean = 2.5, 95 % CI = 1.9–3.2 vs. 1.8, 95 % CI = 1.5–2.2; P = 0.019) was increased in monocytes of the patients with RA. TET2 was also modestly increased in the T cells of RA patients in relation with the healthy controls (Fig. 3d; mean = 3.4, 95 % CI = 2.4–5.2 vs. 2.2, 95 % CI = 1.8–3.0; P = 0.045). No other differences were detected.Fig. 1


Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate.

de Andres MC, Perez-Pampin E, Calaza M, Santaclara FJ, Ortea I, Gomez-Reino JJ, Gonzalez A - Arthritis Res. Ther. (2015)

Expression of methylation enzymes in blood cells of healthy controls (HC) and early rheumatoid arthritis (RA) patients. Normalized relative expression of DNMT1 obtained by quantitative polymerase chain reaction (qPCR) in a T cells, b B cells, and c monocytes, DNMT3A in B cells (d) and of GADD45A in T cells (e) and B cells (f) is shown. Other conventions are as in Fig. 1. All these comparisons were significant. No comparison in other cells subpopulation and none of the DNMT3B analyses were significant
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Related In: Results  -  Collection

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Fig2: Expression of methylation enzymes in blood cells of healthy controls (HC) and early rheumatoid arthritis (RA) patients. Normalized relative expression of DNMT1 obtained by quantitative polymerase chain reaction (qPCR) in a T cells, b B cells, and c monocytes, DNMT3A in B cells (d) and of GADD45A in T cells (e) and B cells (f) is shown. Other conventions are as in Fig. 1. All these comparisons were significant. No comparison in other cells subpopulation and none of the DNMT3B analyses were significant
Mentions: Levels of global 5mC were similar to those previously reported [21, 22]. Comparison of the global 5mC level between patients and controls was done with the samples of RA patients before MTX treatment. Main effects ANCOVA with sex and age as covariates was used for these comparisons. T lymphocytes showed significant DNA hypomethylation in early RA patients compared with healthy subjects (Fig. 1a; mean = 3.89 %, 95 % confidence interval (CI) = 3.80–3.99 vs. 4.15 %, 95 % CI = 3.96–4.38, respectively, P = 0.011). The multivariate analysis also showed significant DNA hypomethylation in the monocytes of RA patients (Fig. 1c; mean = 3.96 %, 95 % CI = 3.87–4.07 vs. 4.13, 95 % CI = 3.98–4.33, respectively, P = 0.047). Although significant, these differences were small. No significant differences were detected in any of the other blood cell subpopulations: B lymphocytes, NK cells and polymorphonuclear (PMN) cells (Fig. 1b and not shown). No significant correlation between the total methylprednisolone dose received until blood drawing and levels of 5mC in any of the five blood subpopulations were observed in RA patients (not shown). In the same assay, 5hmC was also determined without any significant difference between patients and healthy controls, showing very low levels in all cell subpopulations (around 0.02 % of total cytosine) as is characteristic of most adult tissues [21, 28]. DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3 and GADD45A relative expression was determined by qPCR analysis in each of the blood cell subpopulations. A significant decrease in patients with RA was observed for some enzymes: DNMT1 expression was decreased in T cells (Fig. 2a; mean = 5.6, 95 % CI = 4.3–7.2 vs. 10.0, 95 % CI = 7.7–13.1; P = 0.0022), B cells (Fig. 2b; mean = 3.4, 95 % CI = 2.6–4.3 vs. 6.0, 95 % CI = 4.6–7.4; P = 0.00046) and monocytes (Fig. 2c; mean = 5.6, 95 % CI = 4.5–6.7 vs. 8.4, 95 % CI = 7.4–9.4; P = 0.00020); DNMT3A was decreased, although less markedly, in B cells (Fig. 2d; mean = 27.5, 95 % CI = 17.9–42.2 vs. 46.9, 95 % CI = 32.4–67.9; P = 0.044); no difference was found in the expression of DNMT3B in any of the blood cell subpopulations (not shown); and GADD45A expression was also moderately reduced in T cells (Fig. 2e; mean = 12.2, 95 % CI = 8.7–17.3 vs. 20.0, 95 % CI = 13.9–28.8; P = 0.048) and in B cells (Fig. 2f; mean = 9.6, 95 % CI = 5.8–45.9 vs. 18.3, 95 % CI = 12.9–25.9; P = 0.021). On the contrary, the relative expression of TET enzymes was increased in some cell populations. Monocytes showed the largest increases, with the three TET enzyme genes showing higher relative levels of expression in RA patients than in controls. TET1 and TET3 showed the most marked difference between RA patients and controls (TET1, Fig. 3a; mean = 2.9, 95 % CI = 2.1–4.8 vs. 1.9, 95 % CI = 1.6–2.3; P = 0.0044; and TET3, Fig. 3c; mean = 2.0, 95 % CI = 1.7–2.3 vs. 1.35, 95 % CI = 1.15–1.6; P = 0.0014), but also TET2 (Fig. 3b; mean = 2.5, 95 % CI = 1.9–3.2 vs. 1.8, 95 % CI = 1.5–2.2; P = 0.019) was increased in monocytes of the patients with RA. TET2 was also modestly increased in the T cells of RA patients in relation with the healthy controls (Fig. 3d; mean = 3.4, 95 % CI = 2.4–5.2 vs. 2.2, 95 % CI = 1.8–3.0; P = 0.045). No other differences were detected.Fig. 1

Bottom Line: There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells.In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigacion 10 and Rheumatology Unit, Instituto de Investigación Sanitaria-Hospital Clínico Universitario de Santiago, Travesia de Choupana, s/n, 15706, Santiago de Compostela, Spain. maydeandres@gmail.com.

ABSTRACT

Introduction: DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results.

Method: Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR).

Results: Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.

Conclusions: Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

No MeSH data available.


Related in: MedlinePlus