Contributions
Abstract: P967
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - 14 Genetics/Epigenetics
Background and aims: microRNA (miRNA) are short, non-coding RNAs that can significantly affect gene expression. The role of the immune system in Secondary Progressive MS (SPMS) is not well characterised, and identifying changes in miRNA expression patterns and associated gene expression changes in SPMS immune cells will provide insight into disease mechanisms at this stage. Previous work identified broad down-regulation of miRNAs (97%) in SPMS CD4+ T-cells, including miR-29b. Overall, this was linked with increased expression of SOCS6, a negative regulator of T-cell activation. Considering these results, further research on SPMS CD4+ T-cells was carried out to: determine mRNA levels of key miRNA biogenesis genes; and investigate DNA methylation and the expression of DNA methyltransferases targeted by miR-29b.
Methods: 21 SPMS patients and 21 age and gender-matched healthy controls (HC) participated in this study. Total RNA and DNA was extracted from isolated CD4+ T-cells (purity >95%) using miRNeasy Mini and DNA Micro kits (Qiagen) respectively. Expression of miR-29b, miRNA biogenesis mRNAs (DROSHA, DGCR8 and DICER), and miR-29b targets (DNMT3a and DNMT3b), with RNU44 and GAPDH as endogenous controls, were analysed using TaqMan assays (Applied Biosystems). In addition, 450k BeadChip arrays (Illumina) were used to establish DNA methylation profiles and identify differentially methylated regions (DMRs) between SPMS and HC cohorts.
Results: miRNA biogenesis molecule DROSHA and its cofactor, DGCR8, were co-down-regulated (r=0.89, pDROSHA=0.0006, pDGCR8=0.012) in SPMS, which rationalises our previous finding of broad miRNA down-regulation. miR-29b down-regulation (p=0.03) was also validated in these samples. Direct miR-29b target DNMT3b demonstrated a 2.6-fold up-regulation (p=0.002) in SPMS. The role of DNMT3b in de novo methylation prompted investigation into the DNA methylation status of SPMS CD4+ T-cells. SPMS patients demonstrated DNA hypermethylation in 75% of DMRs compared to HC.
Conclusions: Decreased expression of miRNA biogenesis molecules corresponds to our previous finding of broad miRNA down-regulation in SPMS CD4+ T-cells. Furthermore, de novo hypermethylation driven by miR-29b-associated up-regulation of DNMT3b contrasts strongly with only 51% hypermethylated DMRs previously reported in relapsing-remitting MS. These findings indicate a diminished role of CD4+ T-cells in the disease mechanisms of SPMS.
Disclosure:
Conjoint Professor Jeanette Lechner-Scott's institution receives non-directed funding, as well as honoraria for presentations and membership on advisory boards from Sanofi Aventis, Biogen Idec, Bayer Health Care, Merck Serono, Teva and Novartis Australia.
Katherine A Sanders, Vicki E Maltby, Rod A Lea, Miles C Benton, Rodney J Scott, and Lotti Tajouri: nothing to disclose.
Abstract: P967
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - 14 Genetics/Epigenetics
Background and aims: microRNA (miRNA) are short, non-coding RNAs that can significantly affect gene expression. The role of the immune system in Secondary Progressive MS (SPMS) is not well characterised, and identifying changes in miRNA expression patterns and associated gene expression changes in SPMS immune cells will provide insight into disease mechanisms at this stage. Previous work identified broad down-regulation of miRNAs (97%) in SPMS CD4+ T-cells, including miR-29b. Overall, this was linked with increased expression of SOCS6, a negative regulator of T-cell activation. Considering these results, further research on SPMS CD4+ T-cells was carried out to: determine mRNA levels of key miRNA biogenesis genes; and investigate DNA methylation and the expression of DNA methyltransferases targeted by miR-29b.
Methods: 21 SPMS patients and 21 age and gender-matched healthy controls (HC) participated in this study. Total RNA and DNA was extracted from isolated CD4+ T-cells (purity >95%) using miRNeasy Mini and DNA Micro kits (Qiagen) respectively. Expression of miR-29b, miRNA biogenesis mRNAs (DROSHA, DGCR8 and DICER), and miR-29b targets (DNMT3a and DNMT3b), with RNU44 and GAPDH as endogenous controls, were analysed using TaqMan assays (Applied Biosystems). In addition, 450k BeadChip arrays (Illumina) were used to establish DNA methylation profiles and identify differentially methylated regions (DMRs) between SPMS and HC cohorts.
Results: miRNA biogenesis molecule DROSHA and its cofactor, DGCR8, were co-down-regulated (r=0.89, pDROSHA=0.0006, pDGCR8=0.012) in SPMS, which rationalises our previous finding of broad miRNA down-regulation. miR-29b down-regulation (p=0.03) was also validated in these samples. Direct miR-29b target DNMT3b demonstrated a 2.6-fold up-regulation (p=0.002) in SPMS. The role of DNMT3b in de novo methylation prompted investigation into the DNA methylation status of SPMS CD4+ T-cells. SPMS patients demonstrated DNA hypermethylation in 75% of DMRs compared to HC.
Conclusions: Decreased expression of miRNA biogenesis molecules corresponds to our previous finding of broad miRNA down-regulation in SPMS CD4+ T-cells. Furthermore, de novo hypermethylation driven by miR-29b-associated up-regulation of DNMT3b contrasts strongly with only 51% hypermethylated DMRs previously reported in relapsing-remitting MS. These findings indicate a diminished role of CD4+ T-cells in the disease mechanisms of SPMS.
Disclosure:
Conjoint Professor Jeanette Lechner-Scott's institution receives non-directed funding, as well as honoraria for presentations and membership on advisory boards from Sanofi Aventis, Biogen Idec, Bayer Health Care, Merck Serono, Teva and Novartis Australia.
Katherine A Sanders, Vicki E Maltby, Rod A Lea, Miles C Benton, Rodney J Scott, and Lotti Tajouri: nothing to disclose.