New Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome cases are caused by the presence of a nonsense variant in compound heterozygosity with the pathogenic repeat expansion in the <scp><i>RFC1</i></scp> gene

Arteche‐López, Ana; Ávila-Fernández, Almudena; Damián, Alejandra; Emma, Emma Soengas‐Gonda; Fuente, Rubén Pérez de la; Gómez, Patricia Ramos; Merlo, Jesús Gallego; Burgos, Laura Horcajada; Fernández, Carlos Cemillán; Rosales, Jose Miguel Lezana; Martínez, Juan Francisco González; Quesada‐Espinosa, Juan Francisco; Cortón, Marta; Guerrero-Molina, María Paz

doi:10.1111/cge.14249

Cited by 16 publications

(17 citation statements)

References 10 publications

(36 reference statements)

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“…CANVAS arises from a polymorphic set of biallelic RFC1 repeat expansion motifs comprising AAGGG, AAAGG, ACAGG, AAGGC, AGGGC, and AGAGG motifs in isolation or as heterozygous combinations 8,9,11,12 . Further, rare CANVAS patients harbor compound heterozygous monoallelic RFC1 expansions combined with loss-of-function mutations that result in RFC1 haploinsufficiency from the nonexpanded allele [17][18][19] . This, in combination with data indicating a recessive mode of inheritance, suggested to us and others that RFC1 loss-of-function is a pathogenic driver of CANVAS pathogenesis.…”

Section: Discussionmentioning

confidence: 99%

“…Initial studies suggested that RFC1 mRNA and protein expression are unaltered in the context of the repeat expansion, which is inconsistent with the classical mechanism by which recessively inherited repeat expansions elicit a loss of function for the genes in which they reside 8,10,15,16 . However, identification of rare compound heterozygotic CANVAS patients harboring RFC1 truncating and splicing loss-of-function mutations with single allele repeat-expansions suggests a role for RFC1 function in CANVAS [17][18][19] . Alternatively, the repeats could potentially elicit toxicity through dose-dependent gain of function mechanisms (such as repeat associated non-AUG initiated (RAN) translation or repeat RNA-protein complex formation) that only manifest in homozygosity or in combination with RFC1 haploinsufficiency 20,21 (Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

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AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS Neurons

Maltby,

Krans,

Grudzien

et al. 2023

Preprint

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Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a late onset, recessively inherited neurodegenerative disorder caused by biallelic, non-reference pentameric AAGGG(CCCTT) repeat expansions within the second intron of replication factor complex subunit 1 (RFC1). To investigate how these repeats cause disease, we generated CANVAS patient induced pluripotent stem cell (iPSC) derived neurons (iNeurons) and utilized calcium imaging and transcriptomic analysis to define repeat-elicited gain-of-function and loss-of-function contributions to neuronal toxicity. AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway functions. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins that selectively accumulate in CANVAS patient brains. However, neither these proteins nor repeat RNA foci were detected in iNeurons, and overexpression of these repeats in isolation did not induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded allele. These phenotypic deficits were not replicated by knockdown of RFC1 in control neurons and were not rescued by ectopic expression of RFC1. These findings support a repeat-dependent but RFC1-independent cause of neuronal dysfunction in CANVAS, with important implications for therapeutic development in this currently untreatable condition.SummaryHuman CANVAS neurons exhibit transcriptional and functional synaptic defects that are corrected by heterozygous repeat deletion but are independent of the gene within which they reside—RFC1.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS Neurons

Maltby,

Krans,

Grudzien

et al. 2023

Preprint

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“…Biallelic expansions of (A 2 G 3 ) n repeats cause a newly discovered repeat expansion disease (RED) named CANVAS ( c erebellar a taxia, n europathy, v estibular a reflexia s yndrome) ( 1 , 2 ). It is an autosomal recessive disease with a carrier frequency range from 0.7% to 4% in studied populations, resulting in a prevalence range from 1:20 000 to 1:625, respectively ( 1 , 3 ). This frequency establishes RFC1 -related ataxia as likely the most common cause of hereditary late-onset ataxia ( 1 , 2 , 4 ).…”

Section: Introductionmentioning

confidence: 99%

“…Given its essential role, it is not surprising that this is the first RFC1 mutation found to cause human disease. While the pathogenic mechanism of CANVAS remains uncertain, RFC1 loss of function is suspected due to (i) its recessive inheritance and (ii) the discovery of CANVAS-affected patients heterozygous for the repeat expansion and an RFC1 truncating mutation ( 3 , 8–11 ).…”

Section: Introductionmentioning

confidence: 99%

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures

Hisey,

Radchenko,

Mandel

et al. 2024

Nucleic Acids Research

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CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

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“…Therefore, the current data indicate that RFC1 expansions are likely the most frequent cause of recessive ataxias, and many patients remain undiagnosed. 10 , 16 More recently, the genotype spectrum of RFC1 -related disease has been expanded by identifying truncating, frameshift, and nonsense variants in compound heterozygosity with an AAGGG expansion. 14 , 16 , 17 Regarding the disease pathophysiologic mechanism in the case of homozygous expansions, it proved to be puzzling and remains undetermined.…”

Section: Introductionmentioning

confidence: 99%

RFC1 Repeat Distribution in the Cypriot Population

Votsi,

Tomazou,

Nicolaou

et al. 2024

Neurol Genet

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Background and Objectives The intronic biallelic AAGGG expansion in the replication factor C subunit 1 ( RFC1 ) gene was recently associated with a phenotype combining cerebellar ataxia, neuropathy, and vestibular areflexia syndrome, as well as with late-onset ataxia. Following this discovery, studies in multiple populations extended the phenotypic and genotypic spectrum of this locus. Multiple benign and additional pathogenic configurations are currently known. Our main objectives were to study the prevalence of the pathogenic AAGGG expansion in the Cypriot population, to further characterize the RFC1 repeat locus allele distribution, and to search for possible novel repeat configurations. Methods Cypriot undiagnosed patients, in the majority presenting at least with cerebellar ataxia and non-neurologic disease controls, were included in this study. A combination of conventional methods was used, including standard PCR flanking the repeat region, repeat-primed PCR, long-range PCR, and Sanger sequencing. Bioinformatics analysis of already available in-house short-read whole-genome sequencing data was also performed. Results A large group of undiagnosed patients (n = 194), mainly presenting with pure ataxia or with ataxia accompanied by neuropathy or additional symptoms, as well as a group of non-disease controls (n = 100), were investigated in the current study. Our findings include the diagnosis of 10 patients homozygous for the pathogenic AAGGG expansion and a high percentage of heterozygous AAGGG carriers in both groups. The benign AAAAG n , AAAGG n , and AAGAG n configurations were also identified in our cohorts. We also report and discuss the identification of 2 recently reported novel and possibly benign repeat configurations, AAAGGG n and AAGAC n , thus confirming their existence in another distinct population, and we highlight an increased frequency of the AAAGGG n in the patient group, including a single case of homozygosity. Discussion Our findings indicate the existence of genetic heterogeneity regarding the RFC1 repeat configurations and that the AAGGG pathogenic expansion is a frequent cause of ataxia in the Cypriot population.

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New Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome cases are caused by the presence of a nonsense variant in compound heterozygosity with the pathogenic repeat expansion in the RFC1 gene

Cited by 16 publications

References 10 publications

AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS Neurons

AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS Neurons

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures

RFC1 Repeat Distribution in the Cypriot Population

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