The pathology consequences of repeat expansion in the Repeat Expansion Diseases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Background: The Repeat Expansion Diseases are a group of 35+ human genetic disorders that are caused by expansions of a disease-specific tandem repeat. In a subset of these diseases, the expanded alleles become epigenetically silenced via a process we are still trying to understand. Silencing 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), the most common heritable cause of intellectual disability and autism, the missing protein FMRP, is involved in, amongst other things, the regulation of translation in the brain, as well as insulin signaling and glucose metabolism. In the case of FRDA, the protein is frataxin, a mitochondrial protein thought to be important in iron-cluster biogenesis. Progress report: In FXS, silencing occurs when the repeat number exceeds a certain critical threshold. Such alleles are referred to a Full mutation (FM) alleles. Alleles intermediate in length between FM and normal alleles are referred to as Premutation (PM) alleles. Using cells from FM carriers we have begun to dissect out the role of PRC2 and the FMR1 transcript in the silencing process. We also identified a new patient with FXS, who was very unusual in being mosaic for multiple, unmethylated FM alleles and 2 methylated PM alleles (Hayward et. al., 2019). The simplest interpretation of this finding is that this individual inherited a FM that was partially methylated during embryogenesis, with the methylated allele contracting to generate the 2 methylated PM alleles. This suggests that loss of the repeats may not be sufficient to remove methylation, a finding that has relevance for current attempts to use CRISPR-Cas9 deletion of the repeats to treat FXS. In this reporting period we were also involved in the description of a new Repeat Expansion Disorder, GLS Deficiency (van Kuilenburg et. al., 2019). We demonstrated in this work that the CAG-repeat expansion responsible for this disorder also results in epigenetic changes that are associated with a deficiency of the GLS gene product, glutaminase. The net result is that affected individuals do not produce enough glutamate, an important neurotransmitter. This likely accounts for most, if not all, of the early onset ataxia that is seen in this patient population. In the course of doing this work we also developed a reliable new assay for this disorder. This assay also showed that the expanded repeat tract is much larger than predicted, involving in some cases 1000s of repeats. Interestingly, while repeat-mediated gene silencing in this disorder involves the deposition of repressive chromatin modifications, in contrast to FXS, silencing occurs without any associated DNA methylation. This suggests that while gene silencing in FXS and GLS deficiency (and perhaps FRDA) share some common features, the mechanisms do not completely overlap. A comparative analysis of silencing in these disorders may help us understand the molecular events involved, and it is hoped, will ultimately allow the development of rational approaches to reducing pathology in these disorders.