The AAGGG repeat expansion in RFC1 associated with late-onset ataxia and sensory neuropathy: from genetic cause to defining the functional mechanism

Late-onset ataxia is a common reason for neurological consultation, but its cause often remains idiopathic. Ataxia primarily results from cerebellar dysfunction but can also be caused by disorders affecting the large-fibre sensory neurons (sensory neuronopathy) or the vestibular system. When in combination, this more severe late onset ataxia is termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We identified a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) as a common cause of CANVAS and late-onset ataxia. The AAGGG repeat expansion does not lead to overt loss of function of RFC1, which is unexpected given the recessive pattern of inheritance of the disease and suggests that novel disease-causing mechanisms could be involved. The main objective of this project is to
investigate the molecular mechanisms underlying neurodegeneration in the presence of biallelic AAGGG expansions in RFC1. We will use a combined approach by taking advantage of in vitro experiments, Drosophila model as well as patients'-derived cell lines and tissues in order to test the presence of a dose-dependent gain-of-function of toxic pentapeptide repeat proteins encoded by the transcribed intronic repeated sequence and/or unconventional loss-of-function of tissue specific RFC1 isoforms, non-coding transcripts, neighboring and distant genes and/or reorganization 3D chromatin structure.

Late-onset ataxia is a common reason for neurological consultation, but its cause often remains idiopathic. Ataxia primarily results from cerebellar dysfunction but can also be caused by disorders affecting the large-fibre sensory neurons (sensory neuronopathy) or the vestibular system. When in combination, this more severe late onset ataxia is termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We identified a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) as a common cause of CANVAS and late-onset ataxia. The AAGGG repeat expansion does not lead to overt loss of function of RFC1, which is unexpected given the recessive pattern of inheritance of the disease and suggests that novel disease-causing mechanisms could be involved. Main objective of this project is to
investigate the molecular mechanisms underlying neurodegeneration in the presence of biallelic AAGGG expansions in RFC1. We will use a combinational approach by taking advantage in vitro and Drosophila models as well as patients'-derived cell lines and tissues in order to test the presence of a dose-dependent gain-of-function of toxic pentapeptide repeat proteins encoded by the transcribed intronic repeated sequence and/or unconventional loss-of-function of tissue specific RFC1 isoforms, non-coding transcripts, neighbouring and distant genes and/or reorganization of topologically associated domains and 3D chromatin structure.

The frequency of the repeat expansion in 20% of undiagnosed ataxia cases and almost over 90% of typical CANVAS cases together with a carrier rate in the healthy population of ~1% makes the AAGGG repeat expansion in RFC1 as the most common cause of late-onset recessive ataxia. Therefore, we predict that the results of the study will be of great interest to the neurogenetics community and to the field of repeat expansion disorders, both at clinical and laboratory level.
Repeat expansions are still relatively hard to find and they may account for a relatively large proportion of missing heritability in neurological and non-neurological disorders. The majority of repeat expansions were discovered by positional cloning approaches and next generation sequencing technologies have so far had a limited impact on their identification. Our approach has unravelled that a significant part of undiagnosed ataxia and sensory neuronopathy cases is caused by a recessive repeat expansion, and we think that, if combined with novel tools for analysis of STR from WGS data (eg Expansion Hunter), it could inspire other researchers in academia or other research institutions, such as Genomic England, in the discovery of other recessive repeat expansion disorders.
Understandably, given the early days of this novel repeat expansion, little is known about the associated disease-causing mechanisms of this as well as other penta- and exanucleotide repeat disorders. However, this area has gained great importance as shown by the exponential growth of work following the discovery of GGGGCC expansion in c9orf72 in ALS/FTD and we now stand in the unique position to be the first ones to address the same mechanistic questions in this common type of recessive ataxia.
If confirmed, our hypothesis of a dose (and age) dependent toxic gain-of-function of AAGGG repeat expansion in RFC1 will represent a completely novel pathogenic mechanisms underlying a late-onset recessive Mendelian disorder, with broader implications to the general field of neurodegenerative diseases.
Moreover, the modified protocol we designed for recursive directional ligation, given the independence of restriction digestion and subsequent ligation from the repeated unit sequence, has the potential to enable the generation of increased repeat sizes of any nucleotide sequence and could represent a useful tool to basic scientists working on repeat expansion disorders and non-coding repetitive DNA elements.
Finally, one of the main aims of the project is to establish in vitro, cellular and Drosophila models of the disease which will be of use not only to the academic community but also to the pharmaceutical industry in order to test possible ways of future therapeutic intervention.