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Biallelic expansion of an intronic repeat in RFC1 is a common cause of late-onset ataxia

An Author Correction to this article was published on 26 April 2019

This article has been updated


Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG)11 allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia.

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Fig. 1: Clinical spectrum and pedigrees of late-onset ataxia.
Fig. 2: Identification of CANVAS locus.
Fig. 3: Recessive expansion of a mutated AAGGG repeated unit in intron 2 of RFC1 causes CANVAS and late-onset ataxia in familial and sporadic cases.
Fig. 4: Polymorphic configurations of the repeat expansion locus and allelic distribution in healthy controls.
Fig. 5: Pathology of cerebellar degeneration in a patient with CANVAS carrying the recessive AAGGG repeat expansion.
Fig. 6: RFC1 expression is not affected by the AAGGG repeat expansion.

Data availability

The genotyping microarray and sequence data obtained by WGS and RNA-seq are available upon request from the corresponding authors (A.C., H.H.). They are not publicly available because some of the study participants did not give their full consent to release data publicly. Since WGS data are protected by the Personal Information Protection Law, availability of these data is under the regulation of the institutional review board. The data obtained from RNA-seq have been deposited on the NCBI Sequence Read Archive under accession no. SRP186868.

Change history

  • 26 April 2019

    In the version of this article initially published, the name of author Wai Yan Yau was misspelled. The error has been corrected in the HTML and PDF versions of the article.


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A.C. is funded by the Inherited Neuropathies Consortium (INC), which is part of the National Institutes of Health Rare Diseases Clinical Research Network (RDCRN) (grant no. U54NS065712) and the Wellcome Trust (grant no. 204841/Z/16/Z). A.M.R. is funded by a Wellcome Trust Postdoctoral Fellowship for Clinicians (no. 110043/Z/15/Z). H.H. is supported by the Rosetree Trust, Ataxia UK, MSA Trust, Brain Research UK, Muscular Dystrophy UK, Muscular Dystrophy Association, Higher Education Commission of Pakistan and Wellcome Trust (Synaptopathies Strategic Award, 165908). M.M.R. is grateful to the Medical Research Council (MRC), MRC Centre grant (G0601943) and to the National Institutes of Neurological Diseases and Stroke and Office of Rare Diseases (U54NS065712) for their support. The INC (grant no. U54NS065712) is part of the National Center for Advancing Translational Sciences (NCATS) RDCRN. The RDCRN is an initiative of the NCATS Office of Rare Diseases Research and is funded through a collaboration between NCATS and the National Institute of Neurological Disorders and Stroke. S.Z. thanks the National Institutes of Health (NIH; grant no. 4R01NS075764) for its support. This research was also supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre (176718). Neuromuscular and brain tissue samples were obtained from University College London Hospitals NHS Foundation Trust as part of the UK Brain Archive Information Network (BRAIN UK), which is funded by the Medical Research Council and Brain Tumour Research and the NIH-funded NeuroBioBank. We also thank F. Launchbury from the UCL IQPath laboratory for her technical assistance in histology slide preparation. We would also like to thank Muscular Dystrophy UK and Muscular Dystrophy Association USA (award 171011). P.F. is funded by an MRC/MNDA CSF (MR/M008606/1).

Author information




A.C. designed the study, collected the clinical data, performed the genetic analysis that led to the discovery of the AAGGG repeat expansions, analyzed the data, and drafted the manuscript together with contributions from J.V., R.Simone, R.Sullivan, and J.H. R.Simone, N.S.A., E.T., E.B., A.R., W.Y.Y., and M.I. performed the investigation on RFC1 expression. J.V. performed the computational genetic analysis. R.Sullivan and H.T. collected and analyzed the genetic data in healthy controls. P.J.T., W.J.M., A.B., G.D., I.C., M.V., D.K., V.S., S.E., N.W.W., and A.M.R. contributed with the collection of clinical data and patient samples. J.H., P.S., and P.F. performed the RNA-seq analysis. Z.J. performed the pathological investigation. R.Simone, A.M.R., P.F., and J.P. contributed to the design of the study. S.Z. contributed to the design of the study and analyzed the data. H.H. and M.M.R. designed the study, collected the patient clinical data and biological samples and analyzed the data. All authors revised the manuscript.

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Correspondence to Andrea Cortese or Henry Houlden.

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Supplementary Figure 1 Homozygosity mapping in a consanguineous CANVAS family.

(a,b) Homozygosity mapping plot (a) and genomic positions (b) defining the homozygous regions shared by Fam7-1 and Fam7-2. The blue arrow in a points to a ~ 12-Mb homozygous region on chromosome 4 (bold highlighted in b), which encompass the AAGGG repeat expansion locus.

Supplementary Figure 2 Southern blots.

Southern blotting of genomic DNA from 18 patients from 11 families and 16 sporadic cases. Patients show two discrete or overlapping bands of 7 to 15 kb. Unaffected siblings, indicated by “ * ”, carry an expanded allele and one allele in the normal range. In controls (CTRL), one 5-kb band corresponding to the expected size for reference allele (AAAAG)11 is usually observed, but bands of increased size can also be seen. In some blots, an unspecific band at 2 kb is also observed. Ladder used are DIG-labelled DNA Molecular Weight Marker II (Roche) (LADDER II, left) containing 8 fragments with the following base pair lengths: 125 (not shown), 564, 2,027, 2,322, 4,361, 6,557, 9,416, and 23,130 bp and DIG-labelled DNA Molecular Weight Marker III (Roche) (LADDER III, right) containing 13 fragments with the following base pair lengths: 125 (not shown), 564, 831, 947, 1,375, 1,584, 1,904, 2,027, 3,530, 4,268, 4,973, 5,148, and 21,226 bp. N.I., sample not included in this study; rep, repeated sample.

Supplementary Figure 3 Morphological findings in the brain from patient with CANVAS.

(a) Histological examination reveals some age-related changes in the medial temporal lobe with frequent diffuse parenchymal amyloid-β deposits (a) in the neocortex, subiculum, CA1 and caudate nucleus (Thal phase 3). Rare leptomeningeal amyloid angiopathy (not shown) is also seen in the cerebral hemisphere. Neurofibrillary tangle tau pathology (d) is restricted to the medial temporal lobe with the extent corresponding to Braak and Braak stage IV. There are no α-synuclein (g) or TDP43 (h) positive pathological inclusions in the medial temporal lobe. (b) In the brainstem, the locus coeruleus (b) and pontine base nuclei, and white matter tracts show no apparent neuronal loss. In the midbrain, the substantia nigra shows no significant reduction of pigmented neurones (e). In the inferior olivary nucleus, however, there is widespread depletion of neurones and marked chronic gliosis (i). (c) In the cerebellar hemisphere and cerebellar vermis, haematoxylin and eosin stained section shows prominent cortical atrophy with more prominent atrophy seen in the superior cerebellar vermis (c and f, blue square) when compared with the inferior cerebellar vermis (c and j, red square). There are no p62 positive pathological inclusions (f and j) in the cerebellum. Scale bar: 3 mm in a and d; 200 µm in g and e; 40 µm in h; 100 µm in b, i, f and j; 35 mm in c. Stainings were carried out once on patients’ samples with appropriate controls according to standard practice and histopathology procedures in an ISO15189 accredited laboratory.

Supplementary Figure 4 Morphological findings in the nerve and muscle biopsy from a representative patient with CANVAS.

Sural nerve biopsy (a, c, and e). a and c, semi-thin resin section stained with methylene blue azure-basic fuchsin; e, immunostaining for neurofilament with SMI31. Quadriceps skeletal muscle biopsy (b, d and f). b, stained with haematoxylin and eosin; d and f, histochemical staining with ATP 4.2 (d) and ATP9.4 (f). The nerve biopsy demonstrates marked loss of large and small myelinated fibres (a, c, and e), blue arrowheads) with no active axonal degeneration or any signs of regeneration. The unmyelinated fibres (e) are comparably better preserved. The skeletal muscle biopsy reveals angulated fibres and particularly small atrophic fibres (b, yellow arrowhead). There is grouping of type 1 and type 2 fibres (d red arrows and f), in keeping with chronic denervation with reinnervation. Scale bar: 100 µm in a, d and f; 50 µm in c, e and b. Stainings were carried out once on patients’ samples with appropriate controls according to standard practice and histopathology procedures in an ISO15189 accredited laboratory.

Supplementary Figure 5 RNA fluorescence in situ hybridization (FISH).

(a-e) No endogenous RNA foci were detected using oligonucleotides (AAGGG)5 (a, b, c, d, e) or (TTCCC)5 (f, g, h, I, j) probes targeting transcripts containing sense (TTCCC) or antisense (GGGAA) repeated units in unaffected individuals (a, f), pathological controls (b, g), or CANVAS patient (c, h). As a technical control, SH-SY5Y cells were transfected with plasmids expressing mutant sense (TTCCC)94 (d), mutant anti-sense (AAGGG)54 (i), wild-type sense (TTTTC)11 (e) or wild-type anti-sense (AAAAG)11 (j) and analyzed 24 h after transfection by RNA FISH. RNA foci were detected in d and i (red). Nuclei were stained with DAPI (blue). Scale bar: 20 µm in a, b, c, f, g, h; 10 µm in d, e, i, j. Experiments were repeated independently twice with similar results.

Supplementary Figure 6 RFC1 intron 2 retention in multiple tissue types in CANVAS.

Expression levels of intron 2 in RFC1 pre-mRNA as measured by qRT-PCR using primers cF1-iR1 in control (n = 3) and CANVAS (n = 2) lymphoblasts; control (n = 3), Friedreich’s ataxia (n = 3) and CANVAS (n = 1) cerebellum and frontal cortex; control (n = 5) and CANVAS muscles (n = 7). Bar graphs show mean ± s.d. and data distribution (black dots). Two-tailed t-test was performed to compare the expression level of intron 2 in RFC1 pre-mRNA in patients versus healthy or disease controls. All experiments were repeated independently twice with similar results. CANVAS, cerebellar ataxia, neuropathy, vestibular areflexia syndrome; CBM, cerebellum; Ctrl, control; FCX, frontal cortex; FRDA, Friedreich’s ataxia; IR, intron 2 retention; LBLs, lymphoblasts.

Supplementary Figure 7 Western blots for RFC1-encoded protein expression.

(a,b) Uncropped gels showing RFC1-encoded protein levels as measured by Western blotting using the polyclonal antibody (GTX129291) and normalized to β-actin in (a) control (n = 5, lanes 1-5) and CANVAS (n = 5, lanes 6-10) fibroblasts, control (n = 3, lanes 12-14) and CANVAS (n = 4, lanes 15-18) lymphoblasts and (b) CANVAS (n = 1, lane 2), control (n = 3, lanes 3-5), Friedreich’s ataxia (n = 3, lanes 6-8) vermis and CANVAS (n = 1, lane 9), control (n = 3, lanes 10-12) and Friedreich’s ataxia (n = 3, lanes 13-15) frontal cortex. Green arrow indicates the expected 128 kDa band corresponding to RFC1-encoded protein, and the red arrow a 45 kDa band of β-actin. Ladder in lanes 11 (a) and 1 (b) is SeeBlue Plus2 (Thermofisher).

Supplementary Figure 8 Response to DNA damage in CANVAS.

(a) Cell viability after DNA damage inducing treatments of CANVAS (n = 5) and control (n = 5) fibroblasts. (b) Quantification of γH2AX protein expression levels normalized to actin and plotted relative to controls. (c) Representative western blot of cells treated with different DNA damage inducing agents. Bar graphs show mean ± s.d. and data distribution (dots). All experiments were repeated independently twice with similar results.

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Cortese, A., Simone, R., Sullivan, R. et al. Biallelic expansion of an intronic repeat in RFC1 is a common cause of late-onset ataxia. Nat Genet 51, 649–658 (2019).

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