Background: Expansion of intronic (CTG·CAG)n repeats in TCF4 is found in most Fuchs endothelial corneal dystrophy (FECD) patients.Results: RNA foci co-localizing with the splicing factor MBNL1 are found in FECD cells, and changes in mRNA splicing occur.Conclusion: Trinucleotide repeat expansion in FECD is associated with RNA focus formation and missplicing.Significance: RNA toxicity occurs in a disease affecting millions of patients.

Objective To investigate whether a histone deacetylase inhibitor (HDACi) would be effective in an in vitro model for the neurodegenerative disease Friedreich ataxia (FRDA) and to evaluate safety and surrogate markers of efficacy in a phase I clinical trial in patients. Methods We used a human FRDA neuronal cell model, derived from patient induced pluripotent stem cells, to determine the efficacy of a 2-aminobenzamide HDACi (109) as a modulator of FXN gene expression and chromatin histone modifications. FRDA patients were dosed in 4 cohorts, ranging from 30mg/day to 240mg/day of the formulated drug product of HDACi 109, RG2833. Patients were monitored for adverse effects as well as for increases in FXN mRNA, frataxin protein, and chromatin modification in blood cells. Results In the neuronal cell model, HDACi 109/RG2833 increases FXN mRNA levels and frataxin protein, with concomitant changes in the epigenetic state of the gene. Chromatin signatures indicate that histone H3 lysine 9 is a key residue for gene silencing through methylation and reactivation through acetylation, mediated by the HDACi. Drug treatment in FRDA patients demonstrated increased FXN mRNA and H3 lysine 9 acetylation in peripheral blood mononuclear cells. No safety issues were encountered. Interpretation Drug exposure inducing epigenetic changes in neurons in vitro is comparable to the exposure required in patients to see epigenetic changes in circulating lymphoid cells and increases in gene expression. These findings provide a proof of concept for the development of an epigenetic therapy for this fatal neurological disease.

Background: Friedreich ataxia is caused by a GAA⅐TTC triplet-repeat expansion in the first intron of the FXN gene. Results: Expansion of the repeats is observed in induced pluripotent stem cells (iPSCs) and can be blocked with either shRNAs to mismatch repair enzymes or small molecules targeting the repeats. Conclusion: MutS␣ and MutS␤ are involved in repeat expansion. Significance: iPSCs provide a model system for studying triplet-repeat expansion.

Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG·CAG triplet repeats in the 3' untranslated region of the DMPK1 gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG·CAG triplet-repeat instability is not fully understood. Herein, induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington's disease patient fibroblasts. We isolated 41 iPSC clones from DM1 fibroblasts, all showing different CTG·CAG repeat lengths, thus demonstrating somatic instability within the initial fibroblast population. During propagation of the iPSCs, the repeats expanded in a manner analogous to the expansion seen in somatic cells from DM1 patients. The correlation between repeat length and expansion rate identified the interval between 57 and 126 repeats as being an important length threshold where expansion rates dramatically increased. Moreover, longer repeats showed faster triplet-repeat expansion. However, the overall tendency of triplet repeats to expand ceased on differentiation into differentiated embryoid body or neurospheres. The mismatch repair components MSH2, MSH3 and MSH6 were highly expressed in iPSCs compared with fibroblasts, and only occupied the DMPK1 gene harboring longer CTG·CAG triplet repeats. In addition, shRNA silencing of MSH2 impeded CTG·CAG triplet-repeat expansion. The information gained from these studies provides new insight into a general mechanism of triplet-repeat expansion in iPSCs.