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.

与脆性X相关的疾病（FXD）是重复扩张疾病（REDs），是由位于小鼠5'端的CGG重复通道扩张引起的。 FMR1基因。尽管扩展会影响传播风险，还会影响疾病风险和严重程度，但潜在的潜在分子机制尚不清楚。尽管事实上在人类和小鼠模型中都可以看到扩展的等位基因体内，即使经过长时间的培养，现有的患者衍生细胞也不会显示出明显的重复扩增。为了开发用于研究扩展的良好组织培养模型，我们测试了小鼠胚胎干细胞（mESCs）是否在内源性携带扩展的CGG重复序列调频1基因允许扩展。我们在这里显示，这些mESC具有很高的扩展频率，可以在几天之内看到重复数的变化。这些细胞的CRISPR-Cas9基因编辑表明这部分可能是由于这样的事实，即能够防止某些细胞类型扩增的非同源末端连接（NHEJ）在mESC中无效。CRISPR-Cas9基因编辑也显示这些扩增是MSH2依赖性的，与所见的一致体内。尽管没有可比的人类全基因组协会（GWA）研究用于FXD，但此类研究暗示MSH2参与了其他RED的扩展。对于这种类型的微卫星不稳定性，MSH2共有不同寻常的要求，这表明这种新的基于细胞的系统与理解负责人类这种特殊类型突变的机制有关。这些细胞的高扩增频率和基因编辑的简便性应加快鉴定影响扩增风险的因素。此外，我们发现，与人类突变（PM）携带者的细胞一样，这些细胞系的线粒体拷贝数和调频1我们在这里显示的超表达对O ₂敏感。因此，这种新的干细胞模型应有助于研究重复扩增和早期胚胎发育期间扩增的后果。