Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis.

Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing (WGS) data from nearly 10,000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n=6 from 5 families), AAGGC (n=2 from 1 family), AGAGG (n=1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, here we show a pathogenic role for large AAAGG repeat configuration expansions (n=5). Long read sequencing was used to fully characterise the entire repeat sequence and revealed a pure AGGGC expansion in six patients, whereas the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs seem to have arisen from a common haplotype and are predicted to form highly stable G quadruplexes, which have been previously demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions, since the AAAGG motif when very large (>500 repeats) or in the presence of AAGGG interruptions. Accurate sizing and full sequencing of the satellite repeat with long read is recommended in clinically selected cases, in order to achieve an accurate molecular diagnosis and counsel patients and their families.