Effect of in vitro promoter methylation and CGG repeat expansion on FMR- 1 expression

Fragile X syndrome is associated with a CGG repeat expansion in the 5'-untranslated region of the FMR-1 gene. Within the FMR-1 promoter a CpG island is frequently methylated in fragile X patients. To identify the effect of methylation on FMR-1 expression, we transfected methylated and unmethylated constructs containing the FMR-1 promoter in front of the CAT gene (pFXCAT) into COS-1 cells. No difference between methylated and unmethylated DNA was observed initially, whereas reduced CAT mRNA levels were observed 48 h post-transfection of the methylated construct and increased CAT activity from unmethylated DNA was observed at 72 h. To determine the effect of a CGG repeat expansion on gene expression, we inserted >200 CGG repeats between the SV40 promoter and the CAT gene (pSV2CAT). A 3-fold reduction in CAT activity was observed 24-48 h post-transfection. To study the correlation between CGG repeat expansion and FMR-1 transcription, we inserted 200 CGG trinucleotide repeats into the pFXCAT construct. Only a slight difference in mRNA levels was found between cells transfected with pFX(CGG)200CAT or pFXCAT, but a complete lack of CAT activity was observed with introduction of the repeat. We conclude that a moderate size repeat markedly reduces translation. We propose that the presence of a repeat expansion per se is the major factor influencing FMR-1 function in fragile X syndrome.