The mGluR5 antagonist AFQ056 does not affect methylation and transcription of the mutant FMR1 gene in vitro

Elisabetta Tabolacci,Fabrizio Gasparini,Baltazar Gomez-Mancilla,Giovanni Neri,Filomena Pirozzi

doi:10.1186/1471-2350-13-13

Elisabetta Tabolacci, Fabrizio Gasparini + Show 3 more

Open Access

https://doi.org/10.1186/1471-2350-13-13

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Abstract

BackgroundFragile X syndrome (FXS), the leading cause of inherited mental retardation, is due to expansion and methylation of a CGG sequence in the FMR1 gene, which result in its silencing and consequent absence of FMRP protein. This absence causes loss of repression of metabotropic glutamate receptor 5 (mGluR5)-mediated pathways resulting in the behavioral and cognitive impairments associated with FXS. In a randomized, double-blind trial it was recently demonstrated a beneficial effect of AFQ056, a selective inhibitor of metabotrobic glutamate receptor type 5 (mGluR5), on fully methylated FXS patients respect to partially methylated FXS ones.MethodsTo determine whether AFQ056 may have secondary effects on the methylation and transcription of FMR1, here we treated three FXS lymphoblastoid cell lines and one normal control male line. A quantitative RT-PCR was performed to assess transcriptional reactivation of the FMR1 gene. To assess the methylation status of the FMR1 gene promoter it was carried out a bisulphite sequencing analysis.ResultsBoth FMR1-mRNA levels and DNA methylation were unmodified with respect to untreated controls.ConclusionsThese results demonstrate that the AFQ056 effect on fully methylated FXS patients is not due to a secondary effect on DNA methylation and consequent transcriptional activation of FMR1.

Highlights

Fragile X syndrome (FXS), the leading cause of inherited mental retardation, is due to expansion and methylation of a CGG sequence in the FMR1 gene, which result in its silencing and consequent absence of FMRP protein
FMRP is an RNA-binding protein, which inhibits the translation of messenger RNAs, especially within postsynaptic vesicles of the dendritic spines of hippocampal neurons [2]
To answer this question we studied the effect of AFQ056 on FMR1 promoter methylation and messenger RNAs (mRNAs) production in three FXS lymphoblastoid cell lines with different degrees of DNA methylation and in one normal control line

Summary

Introduction

Fragile X syndrome (FXS), the leading cause of inherited mental retardation, is due to expansion and methylation of a CGG sequence in the FMR1 gene, which result in its silencing and consequent absence of FMRP protein. This absence causes loss of repression of metabotropic glutamate receptor 5 (mGluR5)-mediated pathways resulting in the behavioral and cognitive impairments associated with FXS. A clinical trial was recently performed to assess the safety and tolerability of AFQ056 in FXS patients, as well as its possible beneficial effect on the behavioural phenotype [7] This randomized, double-blind, placebo-controlled, crossover study was performed on 30 FXS male subjects. This unanticipated finding begs the question whether AFQ056 may have an indirect or secondary effect on methylation of the mutant FMR1 gene and, on its transcription

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Conclusion