Comprehensive Characterisation of the RFC1 Repeat in an Australian Cohort

RFC1-related disease, which includes cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), is a late-onset neurodegenerative disorder primarily caused by biallelic AAGGG_(n) repeat expansions (RE) in RFC1. The RFC1 locus is highly polymorphic, with multiple pathogenic and non-pathogenic repeat motifs identified. This study aimed to characterise the structure of the RFC1 repeat and determine the pathogenic allele frequency in an Australian cohort. Using a combination of PCR and next generation sequencing techniques, we provide a comprehensive characterisation of the RFC1 repeat locus in an Australian cohort of 232 individuals with adult-onset ataxia and 269 healthy controls. Biallelic pathogenic RFC1 variants were identified in 34.1% of affected individuals. The overwhelming majority (93.7%) have biallelic AAGGG_(n) RE, although other pathogenic alleles, including ACAGG_(n), AAAGG_(>500) and the Māori AAAGG_(10-25)AAGGG_(n)AAAGG_(4-6) configuration were detected in some affected individuals. We also demonstrate the utility of targeted long-read sequencing in resolving complex alleles. The carrier frequency of the pathogenic AAGGG_(n) expansion was approximately 1 in 16 in controls, highlighting the potential for pseudodominant inheritance and the likelihood that RFC1-related disease is underdiagnosed. We further demonstrate the significant RFC1 repeat heterogeneity, identifying 16 distinct motifs, complex repeat structures, and at least six motifs with an allele frequency > 1%. The frequency of RFC1-related disease in individuals with adult-onset cerebellar ataxia and the high carrier frequency of pathogenic RFC1 alleles in the Australian population underscores the need for improved diagnostic strategies. Our findings indicate RFC1 RE are a major cause of late-onset cerebellar ataxia and sensory neuropathy in Australia and provide further insights into RFC1 repeat diversity.