Background and ObjectiveCerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult-onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)_nrepeat expansions in the second intron of the replication factor complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expand the genotypic spectrum of CANVAS by identifying sequence variants inRFC1-coding region associated with this condition.MethodsFifteen individuals diagnosed with CANVAS and carrying only 1 heterozygous (AAGGG)_nexpansion inRFC1underwent whole-genome sequencing or whole-exome sequencing to test for the presence of a second variant inRFC1or other unrelated gene. To assess the effect of truncating variants onRFC1expression, we tested the level of RFC1 transcript and protein on patients' derived cell lines.ResultsWe identified 7 patients from 5 unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)_nexpansion together with a second truncating variantin transinRFC1, which included the following: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6), and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein.DiscussionOur report expands the genotype spectrum of RFC1 disease. FullRFC1sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)_nexpansions. In addition, it sheds further light on the pathogenesis of RFC1 CANVAS because it supports the existence of a loss-of-function mechanism underlying this complex neurodegenerative condition.