Mosaicism in CGG repeat length is

Mosaicism in CGG repeat length is often observed in FXS patients, who carry both premutation and full mutation atp citrate lyase inhibitor and therefore differ in the proportion of cells with silenced FMR1, which contributes to the clinical spectrum of FXS phenotypes (Rousseau et al., 1994). Mosaicism in the methylation pattern of the expanded CGG repeats has also been described (Hagerman et al., 1994). So-called unmethylated full mutation (UFM) individuals represent an extreme case with all expanded alleles above 200 CGG repeats being unmethylated. These individuals display no signs of intellectual disability (Smeets et al., 1995; Tabolacci et al., 2008; Wohrle et al., 1998) and only a handful of cases have been identified worldwide. Molecular properties of the FMR1 promoter have been studied in lymphoblastoid cell lines and primary fibroblasts derived from UFM individuals (Pietrobono et al., 2005; Tabolacci et al., 2008). Normal or slightly elevated FMR1 transcription, with reduced FMRP level due to translational inefficiency, as well as euchromatic configuration of the FMR1 promoter have been demonstrated in these lines (Pietrobono et al., 2005; Tabolacci et al., 2008). However, it is not clear whether these cells have completely lost the ability to methylate FMR1. Human embryonic stem cells (ESCs) with more than 200 CGG repeats in the FMR1 locus as well as induced pluripotent stem cells (iPSCs) from FXS patients have been used to study the disease properties at a cellular level (Avitzour et al., 2014; Colak et al., 2014; Eiges et al., 2007; Sheridan et al., 2011; Telias et al., 2013; Urbach et al., 2010). These human ESCs serve as a model for developmental silencing of FMR1. In a fraction of ESC lines FMR1 is already repressed, whereas in some it is still active and becomes silenced during in vitro neuronal differentiation (Avitzour et al., 2014; Colak et al., 2014). In contrast, iPSCs derived from FXS patients do not reactivate FMR1, suggesting that the gene is locked in a silenced state that is resistant to epigenetic reprogramming (Sheridan et al., 2011; Urbach et al., 2010). Therefore, these cells are not used to study the mechanism of FMR1 silencing, but for modeling of neurological phenotypes of FXS (Sheridan et al., 2011; Telias et al., 2013). In a recent study, iPSCs have been also derived from one UFM individual (de Esch et al., 2014). It has been reported that the cells gained silencing of the FMR1 promoter upon reprogramming, hindering the use of these cells for further analyses of the UFM phenotype. In this study we used somatic reprogramming to dissect the relationship between repeat lengths and silencing status in iPSCs from two unrelated UFM individuals. We found that in the majority of iPSC clones FMR1 remained unmethylated and active. However, in a small proportion of clones which carried more than 400 CGGs FMR1 was silenced, suggesting that the CGG repeat number necessary to induce the silencing is ∼400 in UFM individuals and not ∼200 as described for FXS. Moreover, we demonstrate that upon selective pressure, unmethylated UFM clones gained methylation accompanied by an expansion of the CGG repeats above this higher threshold. Furthermore, the persistence of the UFM phenotype in iPSC-derived neurons allowed us to investigate whether cells carrying the expanded CGG repeat number and active FMR1 develop a neurodegenerative phenotype. Indeed, we found ubiquitin inclusion bodies in these cells, a phenotypic feature of FXTAS patients. We also observed that in UFM as well as in premutation, iPSC-derived neurons form FMRP inclusions that may contribute to the FXTAS pathology.
Results
Discussion FXS is caused by a CGG trinucleotide repeat expansion in the 5′ UTR of FMR1 that leads to its developmental silencing. There is a broadly accepted consensus that 200 CGGs represent the threshold above which the FMR1 promoter becomes methylated and its transcription is turned off (Biancalana et al., 2015; Willemsen et al., 2011). An exception to this rule has been identified in rare UFM individuals who do not silence FMR1 despite the expansion above this threshold (Smeets et al., 1995; Tabolacci et al., 2008; Wohrle et al., 1998). Whether these individuals have lost the capacity to silence FMR1 locus has so far remained elusive.