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.
Reporting Period:
Year 2
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 now focused on hiPSCs from the related TNR diseases myotonic dystrophy, Huntington's disease and Fragile X syndrome. Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG•CAG triplet repeats in the 3’ UTR 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. Human induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington’s disease (HD) 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 intergenerational expansion observed in DM1 patient families. 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. The relatively short repeats in the gene responsible for Huntington's disease are below this threshold and hence do not expand in the iPSCs. 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 to 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. 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. The information gained from these studies provides new insight into a general mechanism of triplet repeat expansion in iPSCs.
Reporting Period:
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.
Grant Application Details
Application Title:
Triplet Repeat Instability in Human iPSCs
Public Abstract:
Over twenty human genetic diseases are caused by expansion of simple DNA sequences composed of repeats of three nucleotides (such as CAG, CTG, CGG and GAA) within essential genes. These repeats can occur within the region of a gene that encodes the protein, generally resulting in proteins with large stretches of repeats of just one amino acid, such as runs of glutamine. These proteins are toxic, cause the death of specific types of brain cells and result in diseases such as Huntington’s disease (HD) and many of the spinocerebellar ataxias (a type of movement disorder). Other repeats can be in regions of genes that do not code for the protein itself, but are copied into messenger RNA, which is a copy of the gene that serves to generate the protein. These RNAs with expanded repeats are also toxic to cells, and sometimes these RNAs sequester essential cellular proteins. One example of this type of disease is Myotonic Dystrophy type 1, a form of muscular dystrophy. Lastly, there are two examples of repeat disorders where the repeats silence the genes harboring these mutations: these are Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS). One limitation in the development of drugs to treat these diseases is the lack of appropriate cell models that represent the types of cells that are affected in these human diseases. With the advent of the technology to produce induced pluripotent stem cells from patient skin cells, and our ability to turn iPSCs into any cell type, such as neurons (brain cells) that are affected in these triplet repeat diseases, such cellular models are now becoming available. Our laboratories have generated iPSCs from fibroblasts obtained from patients with HD, FXS and FRDA. By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA iPSCs, but not in HD iPSCs. This application is 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. While artificial systems with reporter gene constructs have reproduced triplet repeat expansion in bacteria, yeast and mammalian cells, no cellular models have previously been reported that recapitulate repeat expansions at the endogenous cellular genes involved in these diseases. Therefore, our observations that repeat expansion is found in FRDA iPSCs provides the first opportunity to dissect the mechanisms involved in expansion at the molecular level for the authentic cellular genes in their natural chromatin environment. Repeat expansion is the central basis for these diseases, no matter what the outcome of the expansion (toxic protein or RNA or gene silencing), and a fuller understanding of how repeats expand may lead to new drugs to treat these diseases.
Statement of Benefit to California:
A major obstacle in the development of new drugs for human diseases is our lack of cell models that represent the tissues or organs that are affected in these diseases. Examples of such diseases are the triplet-repeat neurodegenerative diseases, such as Huntington’s disease, the spinocerebellar ataxias, forms of muscular dystrophy, Fragile X syndrome and Friederich’s ataxia. These diseases, although relatively rare compared to cancer or heart disease, affect thousands of individuals in California. Recent advances in stem cell biology now make it possible to generate cells that reflect the cell types at risk in these diseases (such as brain, heart and muscle cells), starting from patient skin cells. Skin cells can be turned into stem cell-like cells (induced pluripotent stem cells or iPSCs), which can then give rise to just about any cell type in the human body. During the course of our studies, we found that iPSCs derived from Friedreich’s ataxia patient skin cells mimic the behavior of the genetic mutation in this disease. A simple repeat of the DNA sequence GAA is found in the gene encoding an essential protein called frataxin, and this repeat increases in length between generations in human families carrying this mutation. Over a certain threshold, the repeats silence this gene. It is also known that the repeats expand in brain cells in individuals with this disease. With the advent of patient derived iPSCs and neurons, we now have human model systems in which to study the mechanisms responsible for repeat expansion. We have already identified one set of proteins involved in repeat expansion and we now wish to delve more deeply into how the repeats expand. In this way, we may be able to identify new targets for drug development. We will extend our studies to Huntington’s disease and Fragile X syndrome. We have identified two possible therapeutic approaches for Friedreich’s ataxia, and identified molecules that either reactivate the silent gene or block repeat expansion. Our studies in related diseases may provide possible therapeutic strategies for these other disorders as well, which will be of benefit to patients suffering from these diseases, both in California and world-wide.
