Modulation of methylation in the FMR1 promoter region after long term treatment with L-carnitine and acetyl-L-carnitine.

Fragile X syndrome (FXS) is a triplet repeat disorder caused by a large expansion of the CGG repeat in the 5′-untranslated region (UTR) of the fragile X mental retardation (FMR1) gene. 2 Full mutation alleles are almost always associated with extensive hypermethylation of the repeat and of the upstream CpG island, which correlates with gene silencing and absence of the FMR1 protein. Cognitive function ranging from severe mental retardation to learning disabilities are found in affected people of both sexes. Many mildly affected people show “mosaic” methylation at the FMR1 promoter. 4 Unusual alleles carrying a completely or partially unmethylated full mutation have been described. 6 It was shown that in male patients with FXS with unmethylated alleles in the full mutation range, the FMR1 mRNA level is higher than in normal controls. This finding shows that upregulation of the FMR1 gene occurs in cells with unmethylated full mutation alleles and that the CGG triplet expansion does not suppress transcription directly. Thus, abnormal hypermethylation of the FMR1 promoter suppresses gene transcription. This hypothesis is also supported by the ability of 5-azadeoxycytidine (5-azadC) to restore the FMR1 gene expression in lymphoblastoid cell lines from patients with non-mosaic full mutation FXS by inducing DNA demethylation. 9 The silencing of the hypermethylated FMR1 gene is consistent with a model in which methylation is coupled with the histone acetylation state. It has been found that the 5′ end of the FMR1 gene of patients with FXS is associated with deacetylated histones H3 and H4 and that the treatment of fragile X cells with 5-azadC results in the reassociation of acetylated histones with the FMR1 promoter and transcriptional reactivation. This finding suggests that both methylation and histone deacetylation are linked to transcriptional inactivity. 11 In fact, it has been shown that fragile X cell lines treated with histone hyperacetylating drugs can markedly potentiate the effect of 5-azadC on FMR1 gene expression. However, when used alone, such drugs induce only a modest reactivation of the FMR1 gene. The same pattern of dominance of DNA methylation over histone acetylation has also been reported for other genes, the promoter of which resides in a CpG island. Changes of the methylation patterns over a five year period of alleles from five brothers variably affected by FXS indicate that methylation of individual CpG cytosines is strikingly variable in hypermethylated genotypes obtained from an individual patient. A reduced frequency of hypermethylated alleles occurred in the leucocytes of the two mildly affected brothers. These findings suggest that maintenance of cytosine methylation is a dynamic process that favours unmethylated alleles. It is conceivable that some compounds can be identified that may modulate this process and achieve gene reactivation. Carnitine is a well known naturally occurring compound with an essential role in intermediary metabolism, mainly at the mitochondrial level. Acetyl-L-carnitine (γ-trimethyl-βacetyl-butyrrobetaine) is the carnitine ester naturally present in the central nervous system, differently distributed in the various areas. The enzyme carnitine acetyltransferase catalyses both the formation of acetyl-L-carnitine from carnitine and acetyl-coenzyme A (acetyl-CoA) and the reversible reaction. The modulation of the intracellular concentration of free CoA and acetyl-CoA is recognised to be a common mechanism for the various physiological activities of acetyl-Lcarnitine, 17 such as the acetylation of H4 histones. The chemical structure of acetyl-L-carnitine is similar to that of the acetylating agent butyrate. It has been shown that acetylL-carnitine, as well as butyrate, inhibits cytogenetic expression of the fragile X site in cultured lymphocytes of patients, suggesting that the interaction of these substances with the chromatin structure at the fragile site was present. Carnitine was also shown to suppress position effect variegation in