Year 1

Over twenty human genetic diseases are caused by expansion of simple trinucleotide repeat sequences within essential genes, resulting in toxic proteins (as in the polyglutamine expansion diseases, such as Huntington’s disease (HD)), toxic RNAs (as in Myotonic Dystrophy type 1), or gene repression (as in Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS)). Our laboratories have generated induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with Huntington’s disease (HD), Fragile X syndrome (FXS), Myotonic dystrophy type 1 (DM1) and Friedreich’s ataxia (FRDA). By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA and DM1 iPSCs, but not in HD iPSCs. During growth of the iPSCs in culture, the repeats continue to expand, suggesting that expansion might be linked to DNA replication in these cells. The expansion we observe in iPSCs does not occur in the fibroblast (skin cells) from which the iPSCs were derived. Similarly, on differentiation of the FRDA iPSCs into neurons (brain cells), repeat expansion stops. This observation suggests that some cellular factors necessary for expansion may be selectively expressed in iPSCs, but not in fibroblasts or neurons.

Over the past year, our studies have been aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. Previous studies have implicated the mismatch repair (MMR) enzymes in repeat expansion in mouse models for HD and DM1. We find that silencing of the MSH2 gene, encoding one of the subunits of the MMR enzymes, impedes repeat expansion in human FRDA iPSCs. We find that components of the human mismatch repair (MMR) system are associated with the disease alleles in the FRDA and DM1 iPSCs, and that silencing of these genes at the level of their messenger RNAs is sufficient to suppress repeat expansion. Moreover, we have monitored the levels of the MMR enzymes in fibroblasts, iPSCs and neurons, and as expected these enzymes are present at higher amounts in the iPSCs, suggesting that it is the availability of these enzymes in iPSCs that may be responsible for repeat expansion.

We wish to determine whether it is the DNA structure of triplet-repeats or protein recognition of the repeats that recruits the MMR enzymes to triplet repeats in iPSCs. To this end, we used a series of small molecule probes that can be designed to target particular DNA sequences in the human genome, and we find that a molecule that targets the GAA-TTC repeats in the FRDA frataxin gene displaces MMR enzymes and prevents repeat expansion. We are currently exploring the mechanism whereby this molecule displaces the MMR enzymes. A deeper understanding of the molecular events that lead to repeat expansion at the endogenous cellular genes responsible for these diseases will likely lead to discoveries of new therapeutic strategies for these currently untreatable disorders.