SUMMARYFriedreich’s ataxia (FRDA) is a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy. FRDA is due to expanded GAA repeats within the first intron of the gene encoding frataxin, a conserved mitochondrial protein involved in iron-sulphur cluster biosynthesis. This mutation leads to partial gene silencing and substantial reduction of the frataxin level. To overcome limitations of current cellular models of FRDA, we derived induced pluripotent stem cells (iPSCs) from two FRDA patients and successfully differentiated them into neurons and cardiomyocytes, two affected cell types in FRDA. All FRDA iPSC lines displayed expanded GAA alleles prone to high instability and decreased levels of frataxin, but no biochemical phenotype was observed. Interestingly, both FRDA iPSC-derived neurons and cardiomyocytes exhibited signs of impaired mitochondrial function, with decreased mitochondrial membrane potential and progressive mitochondrial degeneration, respectively. Our data show for the first time that FRDA iPSCs and their neuronal and cardiac derivatives represent promising models for the study of mitochondrial damage and GAA expansion instability in FRDA.

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

Expansions of CTG/CAG trinucleotide repeats, thought to involve slipped DNAs at the repeats, cause numerous diseases including myotonic dystrophy and Huntington's disease. By unknown mechanisms, further repeat expansions in transgenic mice carrying expanded CTG/CAG tracts require the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSβ complex. Using an in vitro repair assay, we investigated the effect of slip-out size, with lengths of 1, 3, or 20 excess CTG repeats, as well as the effect of the number of slip-outs per molecule, on the requirement for human MMR. Long slip-outs escaped repair, whereas short slip-outs were repaired efficiently, much greater than a G-T mismatch, but required hMutSβ. Higher or lower levels of hMutSβ or its complete absence were detrimental to proper repair of short slip-outs. Surprisingly, clusters of as many as 62 short slip-outs (one to three repeat units each) along a single DNA molecule with (CTG)50•(CAG)50 repeats were refractory to repair, and repair efficiency was reduced further without MMR. Consistent with the MutSβ requirement for instability, hMutSβ is required to process isolated short slip-outs; however, multiple adjacent short slip-outs block each other's repair, possibly acting as roadblocks to progression of repair and allowing error-prone repair. Results suggest that expansions can arise by escaped repair of long slip-outs, tandem short slip-outs, or isolated short slip-outs; the latter two types are sensitive to hMutSβ. Poor repair of clustered DNA lesions has previously been associated only with ionizing radiation damage. Our results extend this interference in repair to neurodegenerative disease-causing mutations in which clustered slip-outs escape proper repair and lead to expansions.

Huntington's disease (HD) and myotonic dystrophy (DM1) are caused by trinucleotide repeat expansions. The repeats show different instability patterns according to the disorder, cell type and developmental stage. Here we studied the behavior of these repeats in DM1- and HD-derived human embryonic stem cells (hESCs) before and after differentiation, and its relationship to the DNA mismatch repair (MMR). The relatively small (CAG)44 HD expansion was stable in undifferentiated and differentiated HD hESCs. In contrast, the DM1 repeat showed instability from the earliest passages onwards in DM1 hESCs with (CTG)250 or (CTG)1800. Upon differentiation the DM1 repeat was stabilized. MMR genes, including hMSH2, hMSH3 and hMSH6 were assessed at the transcript and protein levels in differentiated cells. The coincidence of differentiation-induced down-regulated MMR expression with reduced instability of the long expanded repeats in hESCs is consistent with a known requirement of MMR proteins for repeat instability in transgenic mice. This is the first demonstration of a correlation between altered repeat instability of an endogenous DM1 locus and natural MMR down-regulation, in contrast to the commonly used murine knock-down systems.

Expansion of CAG/CTG trinucleotide repeats causes numerous inherited neurological disorders, including Huntington's disease (HD), several spinocerebellar ataxias and myotonic dystrophy type 1. Expanded repeats are genetically unstable with a propensity to further expand when transmitted from parents to offspring. For many alleles with expanded repeats, extensive somatic mosaicism has been documented. For CAG repeat diseases, dramatic instability has been documented in the striatum, with larger expansions noted with advancing age. In contrast, only modest instability occurs in the cerebellum. Using microarray expression analysis, we sought to identify the genetic basis of these regional instability differences by comparing gene expression in the striatum and cerebellum of aged wild-type C57BL/6J mice. We identified eight candidate genes enriched in cerebellum, and validated four--Pcna, Rpa1, Msh6 and Fen1--along with a highly associated interactor, Lig1. We also explored whether expression levels of mismatch repair (MMR) proteins are altered in a line of HD transgenic mice, R6/2, that is known to show pronounced regional repeat instability. Compared with wild-type littermates, MMR expression levels were not significantly altered in R6/2 mice regardless of age. Interestingly, expression levels of these candidates were significantly increased in the cerebellum of control and HD human samples in comparison to striatum. Together, our data suggest that elevated expression levels of DNA replication and repair proteins in cerebellum may act as a safeguard against repeat instability, and may account for the dramatically reduced somatic instability present in this brain region, compared with the marked instability observed in the striatum.