Galassi Deforie, Valentina Irma; (2024) RFC1, KCNA6 and DIAPH1: Investigating the functional consequences. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The study of rare Mendelian disorders can provide insights into the complex genetics of neurological disorders. In my work, I have focused on four distinct genes, causing either a neurodevelopmental or neurodegenerative disorder. Firstly, I explored the ataxic disorder CANVAS and its genetic cause; a recessive AAGGG(exp) repeat expansion in RFC1. Using the pre-existing workflow of flanking PCR, RP-PCR and Southern blotting, and the in-depth optimisation of the Southern blotting technique have been imperative for the diagnosis, clinical understanding, and further genetic exploration of other pathogenic configurations in the RFC1 gene. Secondly, I functionally characterised variants found in the KCNA6 gene, which encodes for the voltage-gated potassium channel KV1.6, in individuals presenting with seizures, intellectual disability, and neurodevelopmental delay. By using two-electrode voltage clamp on injected Xenopus oocytes, I classified gain-of-function, loss-of-function, and both gain- and loss-of-function variants and explored the therapeutic potential of known potassium channel blockers on these mutated channels, thereby expanding the clinical and functional spectrum of voltage-gated potassium channels. Thirdly, I utilised CRISPR technology in two model organisms, Danio rerio and Xenopus tropicalis, to uncover the molecular and physiological implications of autosomal recessive truncating and frameshift variants identified in the DIAPH1 gene, involved in actin elongation, in patients presenting with seizures, cortical blindness, and microcephaly syndrome, SCBMS. Finally, I used the bioinformatics tool, ExpansionHunter, to understand the intergenerational instability of the CAG repeat expansion in the HTT gene, which causes Huntington’s Disease. By using whole genome sequencing data in the 100,000 Genome’s Project, I began to assess the impact that repeat configurations within this locus and CAG repeat size have on intergenerational instability. The diverse methods used here advance our understanding of a group of neurological disorders caused by an array of gene defects that include point and frameshift mutations, as well as repeat expansion defects.

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