Abstract

BackgroundFragile X syndrome is caused by the loss of FMRP expression due to methylation of the FMR1 promoter. Treatment of fragile X syndrome patients’ lymphoblastoid cells with 5-azadeoxycytidine results in demethylation of the promoter and reactivation of the gene. The aim of the study was to analyze if methotrexate, an agent which also reduces DNA methylation but with less toxicity than 5-azadeoxycytidine, has therapeutic potential in fragile X syndrome.MethodsFibroblasts of fragile X syndrome patients were treated with methotrexate in concentrations ranging from 1 to 4 μg/ml for up to 14 days. FMR1 and FMRP expression were analyzed by quantitative PCR and western blotting.ResultsFMR1 mRNA was detected and levels correlated positively with methotrexate concentrations and time of treatment, but western blotting did not show detectable FMRP levels.ConclusionsWe show that it is possible to reactivate FMR1 transcription in fibroblasts of fragile X syndrome patients by treatment with methotrexate. However, we were not able to show FMRP expression, possibly due to the reduced translation efficacy caused by the triplet repeat extension. Unless FMR1 reactivation is more effective in vivo our results indicate that methotrexate has no role in the treatment of fragile X syndrome.

Highlights

  • Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP) expression due to methylation of the fragile X mental retardation 1 (FMR1) promoter

  • Fragile X syndrome (FXS, OMIM #300624) is the most common monogenic cause of mental retardation. It is caused by the expansion of a polymorphic CGG triplet repeat in the 5′-untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene located on the long arm of the X-chromosome (Xq27.3) [1]

  • We show that treatment with methotrexate (MTX), an agent used for the longterm treatment of patients with rheumatoid arthritis and found to decrease cellular methylation including DNA methylation, results in FMR1 expression but no detectable FMRP levels [16,17,18]

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Summary

Introduction

Fragile X syndrome is caused by the loss of FMRP expression due to methylation of the FMR1 promoter. Fragile X syndrome (FXS, OMIM #300624) is the most common monogenic cause of mental retardation It is caused by the expansion of a polymorphic CGG triplet repeat in the 5′-untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene located on the long arm of the X-chromosome (Xq27.3) [1]. The full mutation leads to the loss of a DNA-methylation boundary 650 to 800 nucleotides upstream of the CGG repeat in the first exon of the FMR1 gene [4]. In healthy individuals this boundary protects therapy, is very toxic, making treatment of FXS patients not feasible [15]. We show that treatment with methotrexate (MTX), an agent used for the longterm treatment of patients with rheumatoid arthritis and found to decrease cellular methylation including DNA methylation, results in FMR1 expression but no detectable FMRP levels [16,17,18]

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