RFC1 Repeat Expansion Analysis from Whole Genome Sequencing Data Simplifies Screening and Increases Diagnostic Rates

Biallelic expansions and a motif change in RFC1 are a common cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome. Molecular diagnosis relies on a complicated combination of repeat primed PCR and Southern blotting. We developed a whole genome sequencing based method for RFC1 repeat detection. The combination of sequence motifs and allele length analysis in 29,478 individuals showed that 92.5% of samples have no expanded allele or have expansions of known benign motifs on one allele. In total, 103 samples were classified as biallelic carriers of pathogenic expansions and the frequency of the most common pathogenic allele AAGGG was estimated as 3.9%. Our RFC1 classification method was validated by molecular diagnostic methods achieving sensitivity and specificity of 100% and 97.4%, respectively. Additionally, we catalogued 8 rare repeat motifs, further elucidating the high sequence complexity within the RFC1 locus. In this study we report a new method to identify patients carrying RFC1 pathogenic repeat expansions from WGS data, reducing the need for the inefficient molecular workflow currently used for clinical diagnoses. This method correctly identifies recessive pathogenic repeat expansions in RFC1 and allows screening of existing large scale WGS datasets of unsolved cases. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. In addition, RS is grateful to the Medical Research Council for funding this work (MRC UK MR/J004758/1, G0802760, G1001253). J.V. holds a fellowship from the Health Education England Genomics Education Programme. H.H. is grateful to the Medical Research Council (UK), the Wellcome Trust Synaptopathies Award, Ataxia UK, Rosetrees Trust, Brain Research UK, University College London Official Development Assistance and Low and Middle Income Country award, The Multiple System Atrophy Trust, Muscular Dystrophy UK, and Muscular Dystrophy Association. Andrea Cortese was supported by the Medical Research Council (MR/T001712/1), Fondazione Cariplo (grant n. 2019-1836), the Inherited Neuropathy Consortium, and Fondazione Regionale per la Ricerca Biomedica (Regione Lombardia, project ID 1751723). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics comittee of UCL Queen Square Institute of Neurology and the NHNN, London, UK gave ethical approval for this work. The National Genomic Research Library v5.1, Genomics England. approved this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Research on the de-identified patient data used in this publication can be carried out in the Genomics England Research Environment subject to a collaborative agreement that adheres to patient led governance. All interested readers will be able to access the data in the same manner that the authors accessed the data. For more information about accessing the data, interested readers may contact research-network{at}genomicsengland.co.uk or access the relevant information on the Genomics England website: .