Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis

Abstract

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 (>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.

Keywords: RFC1; CANVAS; ataxia; long-read sequencing; neuropathy; repeat expansions.

Figure 1

Long-read sequencing defines the precise sequence of the novel pathogenic RFC1 motifs. (A) Pedigrees. P = proband. (B) RP-PCR plots and, where available, Southern blot images and optical genome mapping plots. (C) Long-read sequencing results of representative patients with AAGGC, AGGGC, AGAGG and AAAGG expansions (Cases I-1, III-1, VII-1 and XII-1). In Case III-1, only partial reads, which did not span the entire RFC1 repeat locus, could be obtained from the AAGGG allele.

Figure 2

RFC1 repeat expansion size. (A) Comparison of repeat sizes of alleles carrying AAGGG, AAAGG, AAGGC, AGGGC and AGAGG expansions from this and previous studies.^,, The dotted lines refer to the smallest pathogenic expansion of 250 AAGGG repeats identified so far. (B) Comparison of the AAAGG repeat sizes in the compound heterozygous state with the AAGGG expansion in patients with CANVAS/spectrum disorder versus controls. CANVAS = cerebellar ataxia, neuropathy and vestibular areflexia syndrome.

Figure 3

A shared ancestral haplotype in patients with pathogenic RFC1 motifs. Graphical representation of the haplotypes associated with AAGGG, ACAGG and novel pathogenic repeat motifs identified in this study. For each single nucleotide polymorphism, the reference allele is represented in blue, while the alternative allele is represented in yellow. The repeat expansion locus is marked with a red line (R). There is a shared region (B–C, -rs2066782-rs6851075, chr4:39302305–39366034, hg38) of 66 kb for all novel configurations. A larger region of 207 kb (A–C, rs148316325- rs6851075, chr4:39158847–39366034, hg38), which is flanked by two recombination hotspots (arrows), is also shared among all but one allele for Case III-1, suggesting a recombination event at B (rs2066782) in this family. The shared haplotype lies in a region of low recombination rate (HapMap data) and is delimited by small peaks at A and C. A smaller increase in the recombination rate is also visible at B. hom = homozygous.

Figure 4

Normal and pathogenic significance of repeat expansion motifs at the RFC1 locus.