Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis
Authors: Dominik Natalia , Magri Stefania , Currò Riccardo , Abati Elena , Facchini Stefano , Corbetta Marinella , Macpherson Hannah , Di Bella Daniela , Sarto Elisa , Stevanovski Igor , Chintalaphani Sanjog R , Akcimen Fulya , Manini Arianna , Vegezzi Elisa , Quartesan Ilaria , Montgomery Kylie-Ann , Pirota Valentina , Crespan Emmanuele , Perini Cecilia , Grupelli Glenda Paola , Tomaselli Pedro J , Marques Wilson ,   Genomics England Research Consortium, Ambrose J C , Arumugam P , Baple E L , Bleda M , Boardman-Pretty F , Boissiere J M , Boustred C R , Brittain H , Caulfield M J , Chan G C , Craig C E H , Daugherty L C , de Burca A , Devereau A , Elgar G , Foulger R E , Fowler T , Furió-Tarí P , Gustavsson E , Hackett J M , Halai D , Hamblin A , Henderson S , Holman J E , Hubbard T J P , Ibáñez K , Jackson R , Jones L J , Kasperaviciute D , Kayikci M , Lahnstein L , Lawson K , Leigh S E A , Leong I U S , Lopez F J , Maleady-Crowe F , Mason J , McDonagh E M , Moutsianas L , Mueller M , Murugaesu N , Need A C , Odhams C A , Patch C , Perez-Gil D , Polychronopoulos D , Pullinger J , Rahim T , Rendon A , Riesgo-Ferreiro P , Rogers T , Ryten M , Rugginini B , Savage K , Sawant K , Scott R H , Siddiq A , Sieghart A , Smedley D , Smith K R , Sosinsky A , Spooner W , Stevens H E , Stuckey A , Sultana R , Thomas E R A , Thompson S R , Tregidgo C , Tucci A , Walsh E , Watters S A , Welland M J , Williams E , Witkowska K , Wood S M , Zarowiecki M , Shaw Joseph , Polke James , Salsano Ettore , Fenu Silvia , Pareyson Davide , Pisciotta Chiara , Tofaris George K , Nemeth Andrea H , Ealing John , Radunovic Aleksandar , Kearney Seamus , Kumar Kishore R , Vucic Steve , Kennerson Marina , Reilly Mary M , Houlden Henry , Deveson Ira , Tucci Arianna , Taroni Franco , Cortese Andrea 
Published: July 2023

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions.

The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (&gt;500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families.