Genomic observation and perturbation: genetic modifiers to untangle disease mechanisms of RFC1 repeat expansion

Biallelic repeat expansion in RFC1, an essential gene involved in DNA replication and repair, are a leading cause of ataxia and neuropathy in the adult population. Nonetheless, the exact mechanism linking RFC1 repeat expansion and neurodegeneration remains elusive. The prevailing hypothesis is that the repeat may lead to a partial loss of function of RFC1, by affecting expression of neuron specific RFC1 isoforms or by impairing its fine regulation in terminally differentiated neurons, including sensory neurons and Purkinje cells.The objective of proposal is to leverage state-of the-art genomic medicine methods, including the observation of the naturally occurring genetic variation and controlled genome perturbation, to unravel disease relevant pathways. We will perform a genome-wide association (GWA) analysis complemented by long read sequencing to identify genetic modifiers of onset, phenotype and progression of RFC1 disease. We will take advantage of the Crispri technology in genetically engineered iSensoryNeurons to further investigate the effect of GWA loci and to identify additional factors which can modulate RFC1 expression and ameliorate or exacerbate the molecular phenotype. As modifiers are likely to act at cell specific and dynamic level, genetic data will be paralleled by single-cell approaches in post mortem cerebellum and dorsal root ganglia, as well as IPSC sensory neurons and cerebellar organoids. Finally, given a high carrier frequency of RFC1 expansion nearing one out of ten people, we will try to understand whether the repeat itself can act as a modifier of common health and disease traits at population level. The results will hopefully lead to a better understanding of disease causing mechanisms and identify potentially druggable targets. The study output may also have broader implications considering the central role of DNA repair in neuronal functioning and ageing.