Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

Abstract

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.

Fig. 1

PA gel with PCR products of an expanded CGG repeat litter and the breeding couple. The CGG repeat shows both expansion and contraction upon transmission to the next generation.

Fig.2

Histogram summarizing the observed instability of CGG repeat alleles upon transmission to the next generation. Bars represent the percentage of times a transmitted allele fits in an instability category (x=change in repeat length), within a transmission group (male offspring, maternal origin, or female offspring with either maternal or paternal origin). Fisher’s Exact Test revealed no statistically significant differences in frequencies of instability categories amongst the transmission groups (P=12.86, sd=0.09).

Fig. 3

Brain Fmr1 mRNA levels are shown relative to 25-week old male mice. Bars represent two male mice of roughly the same repeat length (SEM were 0.9, 0.8 and 0.5 respectively for 170, 190 and >200 CGGs), with exception of >230, which only represents one male mouse.

Fig.4

Western blot of brain tissue showing Fmrp isoforms (at 70-80kD) of wt (A), Fmr1 KO male mouse (B) and male mice with approximately 230 CGGs (C, D and E). Mouse C,D and E show clearly reduced Fmrp levels: 21%, 5% and 51% of wt levels respectively (corrected for input material). The asterisk marks a background band. Synapthophysin (at 38 kD) was used as a loading control.

Fig.5

Mouse brains from wt (A and B), Fmr1 KO (C and D) and the mouse with >230 CGG repeats (E and F) were stained with antibodies against Fmrp. Photos A,C and E show hippocampus and B,D, and F show cortex tissue. DG: dentate gyrus