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. 2023 Jan 31;100(5):e543-e554.
doi: 10.1212/WNL.0000000000201486. Epub 2022 Oct 26.

Truncating Variants in RFC1 in Cerebellar Ataxia, Neuropathy, and Vestibular Areflexia Syndrome

Riccardo Ronco  1 , Cecilia Perini  1 , Riccardo Currò  1 , Natalia Dominik  1 , Stefano Facchini  1 , Alice Gennari  1 , Roberto Simone  1 , Skye Stuart  1 , Sara Nagy  1 , Elisa Vegezzi  1 , Ilaria Quartesan  1 , Amar El-Saddig  1 , Timothy Lavin  1 , Arianna Tucci  1 , Agnieszka Szymura  1 , Luiz Eduardo Novis De Farias  1 , Alexander Gary  1 , Megan Delfeld  1 , Priscilla Kandikatla  1 , Nifang Niu  1 , Sanjukta Tawde  1 , Joseph Shaw  1 , James Polke  1 , Mary M Reilly  1 , Nick W Wood  1 , Emmanuele Crespan  1 , Christopher Gomez  1 , Jin Yun Helen Chen  1 , Jeremy Dan Schmahmann  1 , David Gosal  1 , Henry Houlden  1 , Soma Das  1 , Andrea Cortese  2
Affiliations

Truncating Variants in RFC1 in Cerebellar Ataxia, Neuropathy, and Vestibular Areflexia Syndrome

Riccardo Ronco et al. Neurology. .

Abstract

Background and objective: Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult-onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)n repeat expansions in the second intron of the replication factor complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expand the genotypic spectrum of CANVAS by identifying sequence variants in RFC1-coding region associated with this condition.

Methods: Fifteen individuals diagnosed with CANVAS and carrying only 1 heterozygous (AAGGG)n expansion in RFC1 underwent whole-genome sequencing or whole-exome sequencing to test for the presence of a second variant in RFC1 or other unrelated gene. To assess the effect of truncating variants on RFC1 expression, we tested the level of RFC1 transcript and protein on patients' derived cell lines.

Results: We identified 7 patients from 5 unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)n expansion together with a second truncating variant in trans in RFC1, which included the following: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6), and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein.

Discussion: Our report expands the genotype spectrum of RFC1 disease. Full RFC1 sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)n expansions. In addition, it sheds further light on the pathogenesis of RFC1 CANVAS because it supports the existence of a loss-of-function mechanism underlying this complex neurodegenerative condition.

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Figures

Figure 1
Figure 1. Truncating Variants in RFC1 in Compound Heterozygous State With Pathogenic (AAGGG)n Expansions Cause CANVAS
(A) Family pedigrees. (B) Schematic diagram showing the location of mutations identified in this study on RFC1 gene (NM_002913.5). Abbreviation: CANVAS = cerebellar ataxia, neuropathy, and vestibular areflexia syndrome; RFC1 = replication factor complex subunit 1.
Figure 2
Figure 2. Nonsense-Mediated mRNA Decay and Haploinsufficiency of RFC1
(A) Real-Time qPCR showed a 40% reduction of RFC1 mRNA in 2 CANVAS cases carrying c.1267C> T/(AAGGG)exp (n = 2) or c.2876del/(AAGGG)exp (n = 1) mutations compared with healthy controls (n = 4, p value = 0.0305) and with biallelic (AAGGG)exp/(AAGGG)exp CANVAS cases (n = 4, p value <0.0001). No significant reduction was seen in control vs biallelic CANVAS cases (p = 0.154) (B) RNAseq confirmed a significant reduction in RFC1 transcript level in patients I-1 and I-2 compared with healthy controls (n = 6, p = 0.0086). (C) Binary Alignment Map (BAM) data from RNAseq and WGS performed in individuals from family 1 showed in I-1 and I-2 a lower number of reads containing the c.1267C>T mutation (green) compared reads mapping to the transcript derived from the second (brown), as opposed to an equal representation of the 2 alleles on WGS, supporting the presence of nonsense-mediated Decay (NMD) of the c.1267C>T mutant transcript. Schematic representation of the percentage of cytosine and thymine called at c.1267 of RFC1 in WGS and RNAseq for family 1. (D) Case II RFC1 gDNA and mRNA sequencing. The top left electropherogram show the presence on gDNA of the c.2876del frameshift variant, which is not evident on mRNA sequencing (bottom left) due to nonsense-mediated decay. Conversely, the cytosine allele at base 2,511, which is heterozygously expressed in gDNA and is part of the (AAGGG)n expansion–containing haplotype, seems as “homozygous” in the mRNA sequencing due to nonsense-mediated decay of the second allele, which contains the c.2876del truncating variant. Together, the findings support the location in trans of (AAGG)n repeat expansion and c.2876del variant in this case. (E) Western blotting revealed a 50% reduction of full-length RFC1 protein expression (red arrow) in CANVAS patients (I-1 and I-2) vs healthy control (control) and their unaffected sibling (I-3) (p = 0.0263) along with a reduction of RFC1 in HEK293 cells on siRFC1 transfection. Nontargeting siRNA-transfected cells were used as a control. RFC1 densitometric values were normalized to GAPDH. No significant reduction in RFC1 protein expression was seen between biallelic (AAGGG)exp/(AAGGG)exp CANVAS patient–derived fibroblasts (n = 5) and healthy control–derived fibroblasts (n = 5) (p = 0.94). Abbreviations: CANVAS = cerebellar ataxia, neuropathy, and vestibular areflexia syndrome; RFC1 = replication factor complex subunit 1.
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References

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