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    • Dioscin Recently iPSCs were derived from fibroblasts obtaine

    2018-10-24

    Recently, iPSCs were derived from fibroblasts obtained from another unrelated UFM subject (de Esch et al., 2014). The lymphoblastoid cell line from this subject has been previously characterized and showed the same epigenetic profile of the FMR1 promoter-like lymphoblastoid cells from UFM1 (Pietrobono et al., 2005; Tabolacci et al., 2008). However, the authors reported that upon iPSC derivation the FMR1 promoter gained methylation and the FMR1 expression was shut off in all derived clones (de Esch et al., 2014). Inter-individual heterogeneity of the UFM group may be the cause of different results of UFM reprogramming in our study. Alternatively, different sources of the material being fibroblasts and PBMCs in de Esch et al. (2014) in their two respective studies may have led to this discrepancy. However, we reproduced the results of Avitzour et al. (2014) obtaining a UFM-like clone from an independent FXS fibroblast line (GM09497, Figure 3), which suggest that different cell type origins alone would not result in clones with different silencing status. Furthermore, the results from de Esch et al. (2014) are in agreement with our observation that UFM subjects retain the ability to silence FMR1. In addition, the published data may well be interpreted in favor of an altered silencing threshold. The fibroblasts used by de Esch et al. (2014) for reprogramming carried the repeat sizes of 200–230 CGGs. The CGG size disclosed for two silent iPSC clones in de Esch et al. (2014) were 330 and 380 repeats. The difference between the original repeat size of the fibroblasts and the one reported in the iPSC clones with silent FMR1 is in line with our hypothesis of a shifted silencing threshold in UFM individuals. Our observation of the variability in the silencing threshold is not limited to the UFM group, as we also detected this phenomenon in FXS patients. From a UFM-related FXS subject F-N1 and from an unrelated patient, FX97 (derived from GM09497 fibroblasts), we derived iPSC clones (Figure 3). All clones from both individuals carried FMR1 Dioscin with more than 200 CGG repeats, most of them being silenced as previously described for FXS iPSCs (Sheridan et al., 2011; Urbach et al., 2010). However, some of the clones were active, hypomethylated, and had lower CGG repeat sizes than the silenced ones, consistent with a shift in the FMR1 silencing threshold in these cells. Interestingly, in a recent report iPSCs were derived from the same fibroblasts GM09497 (Avitzour et al., 2014). Among four clones one was carrying an active FMR1 allele with a fully expanded repeat, while the remaining three were silenced. The exact repeat size of the clones is not reported. Nevertheless, these data show the reproducibility of our results and indicate that the shift in the repeat threshold is an intrinsic property of the cells and not a random phenomenon. Our data support the hypothesis that the number of CGG repeats that triggers the epigenetic silencing of the FMR1 promoter may vary between individuals. In our model, the persistence of the increased silencing threshold in UFM iPSC-derived neurons prevents the development of the FXS phenotype. However, the expressed expanded CGG repeat is predicted to give rise to an additional phenotype in these neurons. Individuals that carry active FMR1 alleles with 50–200 CGG repeats (permutation) are at risk of developing the late-onset neurodegenerative disease FXTAS, and there have been reports of FXTAS diagnosis in UFM individuals (Basuta et al., 2015; Loesch et al., 2012). Key hallmarks of this disease are relatively large, single per cell, intra-nuclear ubiquitin-positive IBs found in postmortem brain samples from FXTAS patients as well as in mouse models (Greco et al., 2006; Wenzel et al., 2010). At the cellular level FXTAS phenotype has been explored in iPSCs from a premutation individual carrying an expansion of 94 CGG repeats (Liu et al., 2012). However, the authors did not report on the presence of ubiquitin IBs. In the FXTAS brain samples a correlation of the CGG repeat number and the percentage of neurons with IBs has been observed (Greco et al., 2006). Therefore, iPSCs that express FMR1 mRNA with more than 200 CGG repeats may potentially show enhanced or accelerated FXTAS phenotypes. Indeed, we find that neurons carrying more than 200 CGG repeats showed significantly increased numbers of ubiquitin-positive IBs compared with WT neurons (Figure 6B). This effect was not present in cells with silenced FMR1 as judged by FMRP staining, indicating that not the expansion per se but the expression of the expended repeat is necessary to trigger the effect.