The National Institute of Diabetes & Digestive & Kidney Diseases

Repeat Expansion Diseases arise from an increase in the number of repeats in a specific tandem repeat tract. My group works on the diseases resulting from expansion at 2 different genomic loci. Friedreich ataxia is caused by the expansion of a GAA•TTC repeat in the first intron of the frataxin gene. This results in reduced levels of frataxin mRNA which leads to sensory motor neuron degeneration, diabetes and cardiomyopathy. Expansion of a CGG•CCG-repeat in the 5’ untranslated region of the FMR1 gene has different consequences depending on the number of repeats in the expanded allele. Carriers of so-called premutation alleles which have 55-200 repeats are at risk of a neurodegenerative disorder known as Fragile X associated tremor and ataxia syndrome (FXTAS). Female carriers are also at risk for Fragile X associated primary ovarian insufficiency. These individuals make more FMR1 mRNA than individuals with normal alleles. Female carriers are also at risk of transmission of an FMR1 allele with a greatly expanded repeat tract to their offspring. Alleles with more than 200 repeats are known as full mutation alleles. Carriers of such alleles are at very high risk of a developmental disorder known as Fragile X Syndrome in which the FMR1 gene is silenced. Symptoms of this disorder include moderate to severe intellectual disability, autistic behavior, connective tissue abnormalities, digestive difficulties, and occasionally hyperphagia and obesity. Expansion also results in the appearance of a folate-sensitive fragile site coincident with the expansion. Fragile sites are prone to breakage in vivo and these sites often coincide with deletion or translocation breakpoints in a number of malignancies.

These diseases are interesting not only because they provide a window into critical processes such as learning and memory, but also because there is evidence to suggest that some aspects of disease pathology may involve a variety of interesting and incompletely understood mechanisms including RNA toxicity and repeat-mediated chromatin remodeling. We are using a number of approaches to look at both the mechanism of expansion and the consequences of expansion in these two disorders. These include biochemical studies of the unusual nucleic acid structures formed by disease associated repeats, in situ hybridization and immunocytochemistry to examine the molecular basis of the chromosome fragility seen in individuals with Fragile X Syndrome, and the development of various in vitro, bacterial, tissue culture, and animal models for different aspects of these diseases.

Development of Fragile X premutation mice

The animal models we have developed include Fragile X premutation mice that contain 120 CGG•CGG-repeats in the endogenous murine Fmr1 gene (Entezam et. al., 2007). These mice recapitulate many aspects of disease pathology seen in human carriers of similar sized alleles including intranuclear neuronal inclusions and purkinje cell dropout. Like their human counterparts, these mice have elevated levels of Fmr1 mRNA. However, in spite of this, some parts of the brain like the inferior olive have a very severe deficiency of the Fmr1 gene product, FMRP. This deficiency probably results from difficulties in translating mRNA with large CGG-repeat tracts due to their ability to form very stable RNA hairpins (Handa, Saha and Usdin, 2003). A similar FMRP deficiency in human premutation carriers may explain some of the symptoms of Fragile X syndrome that are seen in this population group.

Expansion mechanisms

A small number of these mice have undergone large expansions to generate full mutation alleles (Entezam et. al., 2007). This is the first time such large expansions have been observed in mice. Thus contrary to what had previously been thought, mice may be good model animals for studying this unusual mutational process. We have shown that there are 2 mechanisms of expansion one that occurs in both males and females and one that occurs exclusively in females. We have shown that ATR normally protects the genome against this second sort of expansion that our data suggest is the result of some sort of aberrant DNA damage repair (Entezam and Usdin, 2008). Since these events can occur throughout gametogenesis, a process that lasts decades in human females, this could account for the female expansion bias of Fragile X mutations and why these events are much less common mice than they are in humans.

Repeat Mediated Epigenetic changes

We have also shown that the repeats responsible for Friedreich ataxia produce epigenetic changes in the region flanking the repeat (Greene et. al., 2007). These changes include DNA methylation and enrichment for histone modifications characteristic of transcriptionally silent chromatin. This suggests that Fragile X syndrome, Friedreich ataxia and another repeat expansion disease in which the repeat is transcribed but not translated, the congenital form of myotonic dystrophy type 1, may have a common molecular basis related to epigenetic effects of the expanded repeat tract on gene expression.

We have also recently shown that deacetylation of histone H4 at lysine 16 is a late step in the silencing of the FMR1 gene in Fragile X syndrome (Biacsi, Kumari and Usdin, 2008). We have shown that this step is carried out by SIRT1, a class III histone deacetylase and that inhibition of this enzyme reactivates the gene. As SIRT1 inhibitors do not require cell division to be effective, some reactivation of the FMR1 gene may be possible in neurons, where the effect of the gene silencing is most apparent but which no longer divide.

Kumari, D. and Usdin, K. Chromatin remodeling in the noncoding repeat expansion diseases. J Biol Chem. (284):7413-7417, 2009. [ Full Text / Abstract ]

Usdin, K. The biological effects of simple tandem repeats: lessons from the repeat expansion diseases. Genome Res . (18):1011-1019, 2008. [ Full Text / Abstract ]

Entezam, A. and Usdin, K. ATR protects the genome against CGG.CCG-repeat expansion in Fragile X premutation mice. Nucleic Acids Res (36): 1050-6, 2008. [ Full Text / Abstract ]

Biacsi, R., Kumari, D., and Usdin, K. SIRT1 inhibition alleviates gene silencing in Fragile X mental retardation syndrome. PLoS Genetics , 2008. (4):e1000017. [ Full Text / Abstract ]

Entezam, A., Biacsi, R., Orrison, B., Saha, T., Hoffman, G.E., Grabczyk, E., Nussbaum, R.L., Usdin, K. Regional FMRP deficits and large repeat expansions into the full mutation range in a new Fragile X premutation mouse model. Gene (395): 125-34, 2007. [ Full Text / Abstract ]

Greene, E., Mahishi, L., Entezam, A., Kumari, D., Usdin, K. Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia. Nucleic Acids Res (35): 3383-90, 2007. [ Full Text / Abstract ]

Mahishi, L. and Usdin, K. NF-Y, AP2, Nrf1 and Sp1 regulate the fragile X-related gene 2 (FXR2). Biochem J (400): 327-35, 2006. [ Full Text / Abstract ]

Greene, E., Entezam, A., Kumari, D. and Usdin, K. Ancient repeated DNA elements and the regulation of the human frataxin promoter. Genomics (85): 221-30, 2005. [ Full Text / Abstract ]

Handa, V., Goldwater, D., Stiles, D., Cam, M., Poy, G., Kumari, D. and Usdin, K. Long CGG-repeat tracts are toxic to human cells: implications for carriers of Fragile X premutation alleles. FEBS Lett (579): 2702-8, 2005. [ Full Text / Abstract ]

Handa, V., Yeh, H.J., McPhie, P. and Usdin, K. The AUUCU repeats responsible for spinocerebellar ataxia type 10 form unusual RNA hairpins. J Biol Chem (280): 29340-5, 2005. [ Full Text / Abstract ]

Handa, V., Saha, T. and Usdin, K. The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer. Nucleic Acids Res (31): 6243-8, 2003. [ Full Text / Abstract ]