RFC1 in an Australasian neurological disease cohort: extending the genetic heterogeneity and implications for diagnostics

Abstract

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a progressive, generally late-onset, neurological disorder associated with biallelic pentanucleotide expansions in intron 2 of the RFC1 gene. The locus exhibits substantial genetic variability, with multiple pathogenic and benign pentanucleotide repeat alleles previously identified. To determine the contribution of pathogenic RFC1 expansions to neurological disease within an Australasian cohort and further investigate the heterogeneity exhibited at the locus, a combination of flanking and repeat-primed PCR was used to screen a cohort of 242 Australasian neurological disease patients. Patients whose data indicated large gaps within expanded alleles following repeat-primed PCR, underwent targeted long-read sequencing to identify novel repeat motifs at the locus. To increase diagnostic yield, additional probes at the RFC1 repeat region were incorporated into the PathWest diagnostic laboratory targeted neurological disease gene panel to enable first pass screening of the locus for all samples tested on the panel. Within the Australasian cohort, we detected known pathogenic biallelic expansions in 15.3% (n = 37) of cases. Thirty indicated biallelic AAGGG expansions, two had biallelic Māori alleles [(AAAGG)exp(AAGGG)exp], two samples were compound heterozygous for the Māori allele and a AAGGG expansion, two samples had biallelic ACAGG expansions and one sample was compound heterozygous for the ACAGG expansion and the AAGGG expansion. Forty-five samples tested indicated the presence of biallelic expansions not known to be pathogenic. A large proportion (84%) showed complex interrupted patterns following repeat-primed PCR, suggesting that these expansions are likely to be comprised of more than one repeat motif including previously unknown repeats. Using targeted long-read sequencing we identified three novel repeat motifs in expanded alleles. Here we also show that short read sequencing can be used to reliably screen for the presence or absence of biallelic RFC1 expansions in all samples tested using the PathWest targeted neurological disease gene panel. Our results show that RFC1 pathogenic expansions make a substantial contribution to neurological disease in the Australasian population and further extend the heterogeneity of the locus. To accommodate the increased complexity, we outline a multi-step workflow utilising both targeted short- and long-read sequencing to achieve a definitive genotype and provide accurate diagnoses for patients.

RFC1 Repeat Distribution in the Cypriot Population

“…We hereby report our current findings, including the diagnosis of 10 homozygous patients for the pathogenic AAGGG expansion and the high percentage of heterozygous AAGGG carriers both in the group of patients and controls. We also report the identification of 2 of the recently reported novel benign repeat configurations AAAGGG n and AAGAC n , 8 , 9 thus confirming their existence in another distinct population, and we highlight an increased frequency of the hexanucleotide AAAGGG n in the patient group.…”

“…This repeat has been reported only in compound heterozygosity with the AAGGG or other non-pathogenic expansions. 8 , 9 Homozygosity in a single patient presenting with ataxia and neuropathy is reported for the first time in the current study. Furthermore, compared with Dominik et al (2023), who found a similar frequency of the compound heterozygotes AAGGG/AAAGGG in patients (0.6%) and controls (0.4%), we identified 3 compound heterozygous AAGGG/AAAGGG patients (1.6%) and none in the controls (0%).…”

“…The currently reported diagnostic yield for the biallelic AAGGG repeat expansion in the group of CANVAS cases (57%) falls within the range reported by other studies (36%–92%). 3 , 4 , 9 , 13 , 26 , 31 Regarding our main cohort of undiagnosed cases presenting with pure or complex ataxia phenotypes, the calculated diagnostic yield for a homozygous expansion is much lower (2.7%). This percentage is close to the lower range of the other reported yields in different cohorts of mixed pure and complex ataxia cases rather than a full CANVAS phenotype (0.6% Canadian, 4.3% Brazilian, 2 1.0% French, 31 1.6% Dutch, 32 1.6% Greek, 33 7.6% German, 26 14% Caucasian, 13 and 15.9% British 3 ).…”

“…These have been specific for the distinct reported configurations ACAGG n , AGAGG n , and AAGAG n and the 2 novel AAAGGG n and AAGAC n that we had identified in our population before they were published by other groups. 8 , 9 The current study uncovered these 2 repeats using different approaches, as described below in the results section. All RP-PCR products were analyzed on an ABI 3500xL Genetic Analyzer [Applied Biosystems (ABI), CA, USA], and the results were visualized using the GeneMapper v5 software (ABI).…”

“… 8 Another recent Australian population study described 3 additional most probably non-pathogenic repeats (AGGGG n , AAAGGG n , and AAGAC n ), by using the targeted long-read sequencing approach. 9 …”