Whole-Genome and Long-Read Sequencing Identify a Novel Mechanism in <i>RFC1</i> Resulting in CANVAS Syndrome

<h4>Objectives</h4>Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) results from biallelic intronic pentanucleotide repeats in <i>RFC1.</i> We describe an adult male proband with progressive imbalance, cerebellar atrophy, somatosensory neuronopathy, and absence of peripheral vestibular function for whom clinical testing demonstrated a heterozygous <i>RFC1</i> expansion consistent with an unaffected carrier.<h4>Methods</h4>We performed whole-genome sequencing (WGS) on peripheral blood DNA samples from the proband and his unaffected mother. We performed DNA long-read sequencing and synthesized complementary DNA from RNA using peripheral blood from the proband.<h4>Results</h4>WGS confirmed the maternally inherited <i>RFC1</i> expansion and identified a rare, nonsense <i>RFC1</i> variant: c.C1147T; p.R383X in the proband but not the maternal DNA sample. <i>RFC1</i> variants were confirmed in <i>trans</i> with long-read sequencing. Functional studies demonstrated the absence of complementary DNA (cDNA) transcript from the c.C1147T; p.R383X variant supporting nonsense-mediated decay of this transcript.<h4>Discussion</h4>We report an adult with CANVAS due to compound heterozygous pathogenic <i>RFC1</i> variants: the pathogenic intronic pentanucleotide expansion confirmed in <i>trans</i> with a nonsense variant. This report represents a novel molecular mechanism for CANVAS. Sequencing for <i>RFC1</i> should be considered for adults meeting clinical criteria for the CANVAS phenotype if only a heterozygous pathogenic <i>RFC1</i> expansion is identified.