Hypermethylation and Reduced Carnitine Availability - Possible Mechanisms for Downregulation of Microsomal Carnitine Palmitoyltransferase (mCPT) in Myelodysplastic Syndromes (MDS).

Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation of genes involved in cellular proliferation and differentiation and represents, in addition to genetic aberrations, an important mechanism of transcriptional silencing in the pathogenesis of age-associated diseases such as myelodysplastic syndromes (MDS). We have previously reported downregulation of enzymes associated with carnitine-metabolism (including the carnitine transporter OCTN2, which is responsible for carnitine uptake into cells) in healthy elderly persons, as well as a specific downregulation of microsomal carnitine palmitoyltransferase (mCPT; also known as glucose regulated protein GRP58 or endoplasmatic reticulum protein ERp61) a marker for proliferative potential of cells in MDS patients (J.Mol.Med, 81:435ff 2003).

Aim of this study was to elucidate the mechanism of this downregulation by analysing the methylation status of the mCPT promoter in 14 MDS patients (2 RA, 5 RARS, 6 RAEB and one CMML). First, mRNA levels of mCPT, as well as a mitochondrial CPT (CPT1A) and OCTN2 were analysed from peripheral blood mononuclear cells using RTQ-PCR (reverse transcriptase quantitative real time PCR). Bisulfite modifications of genomic DNA were performed with commercially available kits (Epigentek, Chemicon, including methylated and unmethylated DNAs as controls) and subsequently analysed by real-time PCR with specific primers for methylated and unmethylated promoter sequences of the mCPT gene.

Confirming earlier data, gene-expression of mCPT was reduced in all cases. Association of this downregulation with promoter hypermethylation was detected in 10/14 samples of MDS patients. 2 patients with unmethylated mCPT promoter had characteristic distinct MDS subtype-diagnoses: one RAEB (FAB) with single del(5)(q) and one CMML, thus suggesting different mechanisms of gene silencing in heterogeneous MDS. In two more patients with unmethylated mCPT (one RA and one RARS), relative OCTN2-mRNA-expression was elevated 10 fold as compared to hypermethylated samples, which were characterised by extremely low OCTN2-expression. Since L-carnitine has the potential to induce histone acetylation (

In conclusion, our data confirm downregulation of mCPT in MDS. Besides methylation, other factors may also contribute to this phenomenon, because mCPT is located in the microsomal membrane where additionally multiple pre- and postranslational modifications such as prenylation, proteolysis and palmitoylation take place. The exploration of the epigenetic alterations mentioned above may help to develop novel strategies for therapy monitoring as clinical trials using epigenetically targeted therapies have yielded promising results for myelodysplastic syndromes.