CANVAS: a late onset ataxia due to biallelic intronic AAGGG expansions

Abstract

The ataxias are a group of disorders that manifest with balance, movement, speech and visual problems. They can arise due to dysfunction of the cerebellum, the vestibular system and/or the sensory neurons. Genetic defects are a common cause of chronic ataxia, particularly common are repeat expansions in this group of conditions. Co-occurrence of cerebellar ataxia with neuropathy and vestibular areflexia syndrome has been termed CANVAS. Although CANVAS is a rare syndrome, on discovery of biallelic expansions in the second intron of replication factor C subunit 1 (RFC1) gene, we and others have found the phenotype is broad and RFC1 expansions are a common cause of late-onset progressive ataxia.We aim to provide a review and update on recent developments in CANVAS and populations, where the disorder has been reported. We have also optimised a protocol for RFC1 expansion screening which is described herein and expanded phenotype after analysing late-onset ataxia patients from around the world.

Keywords: CANVAS; Late-onset ataxia; RFC1; Repeat expansion; Southern blot.

Fig. 1

Overall symptoms of CANVAS. Symptoms during the manifestation of the disease in 100 biallelic RFC1 expansion cases. Listed are the number of patients reporting specific symptoms and a combination of two or more symptoms (multiple symptoms) (Adapted from Cortese et al. 2020 [6])

Fig. 2

RP-PCR and Southern blotting. a RP-PCR with primers targeting the AAGGG pentanucleotide repeated unit. An ABI 3730 DNA Analyser was used to separate the products, and these were visualised using GeneMapper. The presence of a ‘sawtooth’ pattern is characteristic of a possible affected individual b Patients are characterised by either one overlapping band or two bands within the region 7–15 kb. Carriers are identified with one band residing between 7 and 15 kb and the other at 5–6.5 kb, equivalent to the non-expanded AAGGG sequence or a small AAGGG expansion. Non-affected individuals exhibit two bands in regions between 5 and 6.5 kb. Two ladders are needed for accurate measurements: DIG-labelled DNA Molecular Weight Marker II (Roche) (labelled as LADDER II) and DIG-labelled DNA Molecular Weight Marker III (Roche) (labelled as LADDER III). The left- and right-hand side of each panel documents the molecular weights represented by LADDER II and LADDER III, respectively. Overnight transfer with a 10 min exposure to the Fluorescent Detection Film

Fig. 3

Work flow diagram representing repeat expansion screening methodology. Flanking PCR and RP-PCR are used simultaneously on patient DNA to identify which are more likely to have two expanded alleles. If flanking PCR shows no amplifiable product and RP-PCR shows typical saw-tooth pattern, southern blotting is carried out on additional patient DNA if available