20.500.12556/RUL-163127 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. CANVAS cerebellar ataxia neuronopathy vestibular areflexia syndrome clinical genetics fluorescent repeat primed PCR fragment length detection genetic testing molecular diagnostics true false true Angleški jezik Ni določen Članek v reviji 2024-10-02 10:59:17 2024-10-02 10:59:21 2024-10-07 03:37:28 0000-00-00 00:00:00 2024 0 0 Str. 632–637 iss. 5 Vol. 106 2024 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 61 1399-0004 10.1111/cge.14586 202723075 Clinical_Genetics_-_2024_-_Jaklic_-_Streamlined_two‐step_fragment_analysis_PCR_and_exome_sequencing_of_RFC1_for_diagnostic.pdf Clinical_Genetics_-_2024_-_Jaklic_-_Streamlined_two‐step_fragment_analysis_PCR_and_exome_sequencing_of_RFC1_for_diagnostic.pdf 1 34CA3ABEA40DF8E9F7843FEA932C5481 5039cd621085c099e4ef2daf863a21452bddb7f6bc1cce57ac5aad02f410ab6a a228e584-809c-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=192624 https://onlinelibrary.wiley.com/doi/10.1111/cge.14586 0 981dd93b-809c-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=192623 Medicinska fakulteta 0 0 0