The fragile X syndrome (FXS, MIM #300624), the most common cause of inherited mental retardation, is due to the amplification (> 200 repeats) of a sequence of CGG triplets in the 5' UTR of the FMR1 gene, followed by methylation of cytosines, including those of the promoter upstream [1]. Although the coding region of the gene remains intact, the two changes, one structural and one epigenetic, lead to transcriptional silencing, and consequent absence of the FMRP protein, responsible for the manifestations of the syndrome. FMRP is an RNA-binding protein, which inhibits the translation of messenger RNAs (mRNAs), especially within post-synaptic vesicles of the dendritic spines of hippocampal neurons [2]. It has been demonstrated that the absence of FMRP causes an upregulation of metabotropic glutamate receptors 5 (mGluR5)-mediated signalling pathways, which is the probable cause of the behavioural and cognitive impairments observed in FXS patients [3]. In FXS animal models it was demonstrated that many aspects of the phenotype (behavioural abnormalities, learning deficit, altered dendritic spines, macroorchidism) may be due to excessive mGluR5 signalling. Indeed, crossing Fmr1 KO mice with heterozygous Grm5 KO mice, expressing reduced amounts of glutamate receptors, rescues many of the FXS phenotypes, except for macroorchidism [4]. Consequently, the use of mGluR5 antagonists may represent an effective treatment for many FXS symptoms. The use of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a prototypic mGluR5 inhibitor, rescues hyperactivity and audiogenic seizures in Fmr1 KO [5]. Recently, in a study performed on Fmr1 KO mice, AFQ056, a subtype-selective inhibitor of mGluR5, rescued the inhibition of the startle response after a prepulse, while cultured hippocampal neurons showed shortened dendritic spines [6]. 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, cross-over study was performed on 30 FXS male subjects. Seven of these, who were carriers of a fully methylated FMR1 mutation, with no detectable production of FMR1-mRNA, showed a significant improvement of their behaviour, as measured with the ABC-C scale, after treatment with AFQ056, compared to the placebo-treated controls. No response was detected in FXS subjects who carried a partially methylated FMR1 full mutation. This unanticipated finding begs the question whether AFQ056 may have an indirect or secondary effect on methylation of the mutant FMR1 gene and, consequently, on its transcription.
To answer this question we studied the effect of AFQ056 on FMR1 promoter methylation and mRNA production in three FXS lymphoblastoid cell lines with different degrees of DNA methylation (two fully and one partially methylated) and in one normal control line. No demethylation was induced by the treatment with AFQ056 and the levels of FMR1-mRNA remained unmodified. These findings support the conclusion that the AFQ056 effect observed in fully methylated patients is not due to a change in the methylation on the FMR1 gene, but may result from the interaction of AFQ056 with other, yet unknown, target proteins.