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

Abstract

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.

Figure 1.

FMR^hs341 design and validation. A, Sequence alignmnt. Annotated comparison between Mus musculus (M.m.) and Homo sapiens (H.s.) of the region surrounding the fragile X repeat tract. The repeat tract is highlighted in orange, with the # symbol representing variable CGG repeat length sizes in the human population. Guide RNA sequences and their associated PAM (Protospacer Adjacent Motif) sequence are indicated by their position above the M.m. sequence, with the SNPs incorporated by the template to prevent recutting shown in red (note: Cas9 cuts several nucleotides before the PAM sequence). The green box denotes the coding region of exon 1. Sequences are written 5′ to 3′. B, Illustration of knock-in strategy. Embryos are injected with a mixture containing a single-stranded DNA template generated from human patient DNA, Cas9 protein, and two single-guide RNAs flanking the FMR1 CGG repeat tract. The mouse embryo DNA is cut by Cas9 and repaired through homologous recombination with the patient mutation template. The exchange is irreversible because of SNPs in the corresponding guide sequence on the human allele. Purple, 5′ UTR; blue, CGG repeat tract of mouse; red, CGG repeat tract of human expansion; green, coding region of exon 1. C, Confirmation of repeat size by gel electrophoresis. The FMR1^hs341 amplicon was predicted to be 1353 bp in length: 190 bp upstream, 1023 bp repeat tract (341 * 3), and 140 bp downstream. Lane 1 provides a DNA ladder (1 kb Plus DNA Ladder; catalog #N3200L, New England BioLabs), lanes 3 and 5 were identified as wild type, lane 7 was identified as FMR1^hs341. D, E, Sanger sequencing of upstream and downstream regions, respectively. Red box denotes guide RNA recognition sequence; blue box denotes PAM sequence. The upstream region shows strong incorporation of the human template including several SNPs located 14 bp before the predicted cut site. Upstream sequencing penetrated up to 59 CGG repeats. The downstream region demonstrates precise integration of the human template, and sequencing penetrated up to 74 CGG repeats. Two nucleotides were manually annotated for the downstream sequence. Off-target traces can be found in Extended Data Figure 1-1.

Figure 2.

FMR^hs341 does not exhibit methylation. Bisulfite sequencing of male mouse DNA. Each row represents a single animal, and each circle represents one of the 13 CG cytosines within the sequencing region. Open circle, unmethylated; closed circle, methylated; split circle, partial methylation across multiple reads; no circle, missing in reads. All animals are referenced by F<litter>.<pup>. A, Bisulfite sequencing of wild-type littermates. B, Bisulfite sequencing of Fmr1^hs341 littermates. C, Bisulfite sequencing of unrelated C57BL/6 mice. D, Bisulfite sequencing of artificially methylated C57BL/6 mice, which served as a positive control that our methodology could detect methylated cytosines. Sequence traces can be found in Extended Data Figure 2-1.

Figure 3.

FMR^hs341 exhibits premutation molecular pathologies. All animals are referenced by F<litter>.<pup>. A, qRT-PCR quantification. Fmr1 mRNA is consistently higher in Fmr1^hs341 male mice (red) compared with their wild-type (blue) male sibling counterparts. Values are normalized to Gapdh and Actin expression across each brain region and presented as the mean ± SEM. fCORTEX, Frontal cortex; pCORTEX, posterior cortex. B, Western blot quantification. FMRP is drastically reduced in Fmr1^hs341 (red) mice compared with wild-type (blue) siblings. Values are normalized to wild-type GAPDPH expression levels.