CGG Repeat Expansion, and Elevated Fmr1 Transcription and Mitochondrial Copy Number in a New Fragile X PM Mouse Embryonic Stem Cell Model.

Inbal Gazy,Geum-Yi Kim,Karen Usdin,Carson J Miller

doi:10.3389/fcell.2020.00482

Abstract

The Fragile-X related disorders (FXDs) are Repeat Expansion Diseases (REDs) that result from expansion of a CGG-repeat tract located at the 5′ end of the FMR1 gene. While expansion affects transmission risk and can also affect disease risk and severity, the underlying molecular mechanism responsible is unknown. Despite the fact that expanded alleles can be seen both in humans and mouse models in vivo, existing patient-derived cells do not show significant repeat expansions even after extended periods in culture. In order to develop a good tissue culture model for studying expansions we tested whether mouse embryonic stem cells (mESCs) carrying an expanded CGG repeat tract in the endogenous Fmr1 gene are permissive for expansion. We show here that these mESCs have a very high frequency of expansion that allows changes in the repeat number to be seen within a matter of days. CRISPR-Cas9 gene editing of these cells suggests that this may be due in part to the fact that non-homologous end-joining (NHEJ), which is able to protect against expansions in some cell types, is not effective in mESCs. CRISPR-Cas9 gene editing also shows that these expansions are MSH2-dependent, consistent with those seen in vivo. While comparable human Genome Wide Association (GWA) studies are not available for the FXDs, such studies have implicated MSH2 in expansion in other REDs. The shared unusual requirement for MSH2 for this type of microsatellite instability suggests that this new cell-based system is relevant for understanding the mechanism responsible for this peculiar type of mutation in humans. The high frequency of expansions and the ease of gene editing these cells should expedite the identification of factors that affect expansion risk. Additionally, we found that, as with cells from human premutation (PM) carriers, these cell lines have elevated mitochondrial copy numbers and Fmr1 hyperexpression, that we show here is O2-sensitive. Thus, this new stem cell model should facilitate studies of both repeat expansion and the consequences of expansion during early embryonic development.

Highlights

The Fragile X-related disorders (FXDs), are members of the Repeat Expansion Disorders (REDs), a group of 35+ human diseases that arise due to an expansion or increase in the length of a disease-specific microsatellite
We found that R-loops form on the mouse Fmr1 locus in both WT and PM mouse embryonic stem cells (mESCs) (Figure 6)
We report the establishment of FX KI mESC lines that show a high frequency of progressive CGG repeat expansions in tissue culture

Summary

Introduction

The Fragile X-related disorders (FXDs), are members of the Repeat Expansion Disorders (REDs), a group of 35+ human diseases that arise due to an expansion or increase in the length of a disease-specific microsatellite. In addition to the risk of FXTAS and FXPOI, women carrying a PM allele are at risk of transmitting a much larger full mutation (FM) allele to their children. Such alleles contain more than 200 repeats and give rise to a third disorder, Fragile X syndrome, the leading cause of inherited intellectual disability and autism spectrum disorder (ASD; Crawford et al, 2001). PM pathology results from the deleterious consequences of the PM transcripts (Renoux and Todd, 2012) that are likely to be exacerbated by the elevated levels of the expanded CGG-repeat containing FMR1 transcript. While expansions have important consequences for disease pathology in humans, the underlying mechanism responsible for the expansion mutation is still largely unclear, as is the timing of the expansion from a PM to a FM allele

Methods

Results

Conclusion