341 Repeats Is Not Enough for Methylation in a New Fragile X Mouse Model

Fragile X syndrome (FXS) is a leading monogenic cause of intellectual disability and autism spectrum disorders, spurring decades of intense research and a multitude of mouse models. So far, these models do not recapitulate the genetic underpinning of classical FXS-CGG repeat-induced methylation of the Fmr1 locus-and their findings have failed to translate into the clinic. We sought to answer whether this disparity was because of low repeat length and generated a novel mouse line with 341 repeats, Fmr1(hs341), which is the largest allele in mice reported to date. This repeat length is significantly longer than the 200 repeats generally required for methylation of the repeat tract and promoter region in FXS patients, which leads to silencing of the FMR1 gene. Bisulfite sequencing fails to detect the robust methylation expected of FXS in Fmr1(hs341) mice. Quantitative real-time PCR and Western blotting results also do not resemble FXS and instead produce a biochemical profile consistent with the fragile X-associated premutation disorders. These findings suggest that repeat length is unlikely to be the core determinant preventing methylation in mice, and other organisms phylogenetically closer to humans may be required to effectively model FXS. Significance Statement It is critical for the study of disease, and the translatability of findings into the clinic, that the model used exhibits close homology to the human condition. There remains uncertainty whether knock-in mouse models can replicate the core etiology of fragile X syndrome (FXS) and methylate the Fmr1 gene. We therefore generated a new mouse line with a repeat size that far exceeds the established boundary for human methylation, and we report the continued absence of methylation. Our characterization of this line affirms that alternative models may be required for the comprehensive study of FXS, while these new mice may offer a valuable tool for the study of unmethylated fragile X-associated disorders.

Automated summary

This study suggests that the length of CGG repeat in mouse models is unlikely to be the main factor preventing methylation and alternative models closer to humans may be required to effectively study FXS.