Year 3

Over the past year, our research efforts have focused on the generality of the results we found in human induced pluripotent stem cells derived from patients with the neurodegenerative disease Friedreich’s ataxia (FRDA). FRDA is one of the trinucleotide repeat (TNR) diseases, and our major previous finding was that the GAA•TCC trinucleotide repeats that cause FRDA expand during isolation and propagation of FRDA hiPSCs. This expansion was shown to be dependent on enzymes that are involved in the repair of mismatches in the human genome. To extend these studies, we have focused on hiPSCs from the related TNR diseases myotonic dystrophy type 1 (DM1), Huntington’s disease (HD), Fragile X syndrome (FXS), and Fuchs endothelial corneal dystrophy (FECD). DM1 is an inherited dominant muscular dystrophy caused by expanded CTG•CAG triplet repeats in the DMPK 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. hiPSCs were generated from DM1 and HD patient fibroblasts. Similar to our results in FRDA, DM1 hiPSCs show repeat instability, and repeat expansion is again dependent on the DNA mismatch repair system. We defined a threshold of repeat lengths where repeat expansion occurs. The relatively short repeats in the gene responsible for Huntington’s disease are below this threshold and hence do not expand in the iPSCs. We have also generated hiPSC lines from seven male subjects clinically diagnosed with fragile X syndrome. These hiPSCs have been thoroughly characterized with respect to pluripotency, DNA methylation status at the FMR1 gene, CGG repeat length, FMR1 expression and neuronal differentiation. In recent studies, we have turned our attention to the common eye disease FECD, where ~75% or so of Caucassian patients have a CTG•CAG triplet-repeat in an intron of the gene encoding the essential transcription factor TCF4. We find repeat instability in fibroblasts from FECD patient fibroblasts, and repeat expansion in the corresponding hiPSCs. Importantly, similar to DM1 with the same repeat sequence as in FECD, the pathological mechanism in both diseases appear to be similar, namely RNA toxicity caused by sequestering essential messenger RNA processing factors. We have also identified a potential small molecule therapeutic that binds CTG•CAG triplet-repeats and are currently testing this molecule in the relevant patient iPSC-derived cell types. The information gained from these studies provides new insight into a general mechanism of triplet repeat expansion in iPSCs and has revealed a new therapeutic approach for these diseases.