The consequences of the unusual mutation responsible for the Repeat Expansion Diseases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Background: The Repeat Expansion Diseases (REDs) are a group of >46 severely life-limiting human genetic disorders that are caused by an expansion of a disease-specific tandem repeat or microsatellite. These diseases include 11 that result from the presence of a large CGG-repeat tract located in the transcriptional unit of the affected gene, but outside the canonical protein open reading frame. In those diseases involving >200 CGG-repeats, repeat-mediated gene silencing often occurs. This results in a deficiency of the protein product of the affected gene that is the proximal cause of disease pathology. In the case of Fragile X syndrome (FXS; OMIM #300624), the most common heritable cause of intellectual disability and autism, the silenced gene is the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene. As a result, the amount of the FMR1 gene product, FMRP, is severely reduced. FMRP is involved in, amongst many other things, the regulation of translation in the brain in response to synaptic activation. In contrast, repeats with 55-200 repeats, are often hyper-expressed, with pathology arising via some deleterious consequence of the expression of transcripts with larger than normal numbers of CGG-repeats, the nature of which is the subject of much debate. In many instances such alleles confer risk of neurodegeneration which in the case of the FMR1 gene is known as Fragile X associated tremor/ataxia syndrome (FXTAS; OMIM #300623). In the case of the FMR1 gene such alleles are also associated with form of ovarian dysfunction known as Fragile X associated primary ovarian insufficiency (FXPOI; OMIM #311360). In addition, the large, silent alleles in many of these diseases are associated with a folate-sensitive fragile site that colocalizes with the affected gene. Progress report: Our previous work has helped define the heterochromatin landscape associated with Fragile X alleles (Biacsi et. al., 2008; Kumari and Usdin, 2010; Kumari et. al., 2014; Zhou et. al., 2016; Kumari et. al., 2020), identified the likely timing of the silencing window (Zhou et. al., 2016) and implicated the FMR1 transcript in the silencing process (Kumari and Usdin, 2014). We were also amongst the first to show that repeat-mediated gene silencing also occurs in Friedreich’s ataxia (FRDA), an RED resulting from a GAA-repeat expansion in the first intron of the frataxin (FXN) gene (Greene et. al., 2007) and in a glutaminase deficiency disorder resulting from a CAG (GCA)-expansion in the 5 UTR of the Glutaminase 1 (GLS1) gene (OMIM #618412; van Kuilenberg et. al., 2019). More recently, we showed that the mechanisms involved in the maintenance of DNA methylation at different disease loci are different (Grant-Bier et. al., 2025). To better understand the mechanism responsible for the initiation of gene silencing and the maintenance of this silencing in FXS, we have developed an unbiased proximity labeling screen to identify proteins interacting with the FMR1 locus in patient cells. This screen has uncovered novel interactors that may contribute to FMR1 gene silencing. We had previously shown that the FX locus is prone to spontaneous DNA damage that is preferentially repaired by non-homologous end-joining and that gene silencing in FXS may occur, at least in part, to preserve genome integrity (Kumari et. al., 2024). We had also demonstrated that the repeats form structures that block the DNA polymerases necessary for their proper replication (Usdin and Woodford, 1995). Our data suggest a model in which transcription of the expanded CGG-repeat tract results in collisions between the transcription complex and the replication fork. This could result in the high frequency of repeat contractions associated with gene reactivation. We also demonstrated that there are at least two different mechanisms responsible for these repeat contractions, one that is MSH2-dependent and one that is not (Grant-Bier et. al., 2025). With respect to therapeutic approaches to treating these disorders, we have shown that CRISPR-mediated deletion of the repeats is not effective for restoring FMR1 expression in patient-derived neurons (manuscript in preparation). We have also extended our efforts to develop assays for the identification of new REDs and to help with the diagnosis of unusual cases of previously identified ones. Our efforts include helping define a new, severe early-onset neurodevelopmental disorder associated with a CGG-repeat expansion in DIP2B (Théberge et. al., 2025), contributing to the finding that Spinocerebellar ataxia type 27B involves a marked expansion bias in the cerebellum (Pellerin et. al., 2025) and helping to validate new bioinformatics approaches to the diagnosis of this group of diseases (Fazal et. al., 2025).