Streamlined two‐step fragment analysis PCR and exome sequencing of RFC1 for diagnostic testing of suspected CANVAS patients

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is caused by biallelic pathogenic expansions, or compound heterozygosity with other pathogenic variants in the RFC1 gene. CANVAS is estimated to be underdiagnosed, both because of the lack of formal diagnostic criteria and molecular challenges that translate to lesser access and high cost of routine testing. Our aim was to address the need for making CANVAS genetic testing routine, by designing a streamlined two‐step PCR consisting of a short‐allele screening PCR and a confirmatory PCR with fragment capillary electrophoresis detection. Exome sequencing of RFC1 was additionally foreseen to resolve potential compound heterozygosity cases. Specificity of our approach was evaluated using ataxia patients with known non‐CANVAS diagnoses, and optimized using Southern blot confirmed CANVAS patients. We evaluated our approach by testing patients consecutively referred for clinically suspected CANVAS using first the two‐step PCR, followed by exome sequencing. Our approach was able to accurately identify negative and confirm positive cases in prospectively collected suspected CANVAS patients presenting with at least three typical clinical signs. The proposed testing approach provides an alternative method able to clearly distinguish between CANVAS negative and positive cases and can be easily incorporated into the genetic diagnostic laboratory workflow. Genetic testing for CANVAS is currently complex. Our aim was to make routine genetic testing of CANVAS more available, by designing a specific two‐step PCR with fragment capillary electrophoresis detection, consisting of screening and confirmation steps. Finally, potential compound heterozygosity cases can be resolved using exome sequencing of RFC1.