ABSTRACT Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) has recently been attributed to biallelic repeat expansions in _RFC1_. More recently, the disease entity

has expanded to atypical phenotypes, including chronic neuropathy without cerebellar ataxia or vestibular areflexia. Very recently, _RFC1_ expansions were found in patients with Sjögren

syndrome who had neuropathy that did not respond to immunotherapy. In this study _RFC1_ was examined in 240 patients with acute or chronic neuropathies, including 105 with Guillain-Barré

syndrome or Miller Fisher syndrome, 76 with chronic inflammatory demyelinating polyneuropathy, and 59 with other types of chronic neuropathy. Biallelic _RFC1_ mutations were found in three

CANVAS-related _RFC1_ mutations in patients with treatable immune-mediated neuropathy or demyelinating neuropathy. SIMILAR CONTENT BEING VIEWED BY OTHERS AUTOREACTIVE T CELLS TARGET

PERIPHERAL NERVES IN GUILLAIN–BARRÉ SYNDROME Article Open access 17 January 2024 RECESSIVE CEREBELLAR AND AFFERENT ATAXIAS — CLINICAL CHALLENGES AND FUTURE DIRECTIONS Article 24 March 2022

CLINICAL AND MOLECULAR SPECTRUM OF TK2-DEFICIENCY: A LARGE BRAZILIAN COHORT Article Open access 15 March 2025 INTRODUCTION Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome

(CANVAS) has been recently found to be caused by biallelic repeat expansions in the intron of _RFC1_1. More recently, the disease entity has expanded to chronic neuropathy without cerebellar

ataxia or vestibular areflexia2. Repeat configuration includes AAGGG, ACAGG, AGGGC, or combinations thereof without clear genotype and phenotype relationship2,3,4. Immune-mediated

neuropathy can be categorized into acute and chronic. Guillain-Barré syndrome (GBS) or Miller Fisher syndrome (MFS) is representative of acute immune-mediated neuropathy, whereas chronic

inflammatory demyelinating polyneuropathy (CIDP) is representative of chronic immune-mediated neuropathy. Myelin-associated glycoprotein (MAG) neuropathy, originally derived from CIDP, is

characterized by sensory and autonomic neuropathy with positive serum monoclonal IgM antibody against MAG5 and is at least partly treatable by immunotherapy including rituximab6. Notably,

neuropathies cannot be solely attributed to the presence of antibodies. In fact, our previous study showed that among two siblings who simultaneously suffered from _Campylobacter

jejuni_-associated diarrhea and subsequently tested positive for serum anti-ganglioside antibodies, only one developed GBS, suggesting the involvement of some unknown host factors7. Our

previous study showed that CAG repeat expansions in _ATXN2_, which causes spinocerebellar ataxia type 2 (SCA2), were found in patients with CIDP or immune-mediated neuropathy8. A recent

review also suggested the association between repeat expansions and various diseases, including immune-mediated diseases9. Very recently, _RFC1_ expansions were found in patients with

Sjögren syndrome who had neuropathy that did not respond to immunotherapy, suggesting that neuropathy could be attributed to _RFC1_ expansions10. Thus, a previous study suggested that

unnecessary immunotherapy should be avoided in patients who have immune-mediated diseases with _RFC1_ mutations10. However, we speculate that immunotherapy might be effective in some

patients with certain unknown conditions. We therefore hypothesized that repeat expansion may be a background genetic factor for immune-mediated neuropathy. Accordingly, the current study

examined _RFC1_ in 240 patients with immune-mediated neuropathy, such as GBS, CIDP, and MAG neuropathy, as well as other types of neuropathies. PATIENTS AND METHODS PATIENTS This study

enrolled Japanese patients and controls from the Kinki region of Japan between 2005 and 2023. _RFC1_ was analyzed in 240 Japanese patients with neuropathy (146 men and 94 women; mean age, 54

 ± 18 years), including 105 with GBS or MFS (62 men and 43 women, mean age, 50 ± 19 years), 76 patients with CIDP (44 men and 32 women, mean age, 56 ± 17 years), and 59 with other types of

chronic neuropathy (40 men and 19 women, mean age, 61 ± 17 years), which included eight patients with MAG neuropathy, four with paraneoplastic syndrome (without MAG antibody), three with

Sjögren syndrome, and three with multifocal neuropathy. Patients with known genetic causes were excluded. The clinical diagnosis was established after the first admission for diagnostic

workup when DNA samples were collected. Chronic neuropathy was herein defined as weakness or sensory disturbance in two or more limbs over at least 2 months with other signs or symptoms

being minimal and corresponding abnormalities in nerve conduction studies (NCS). A control group consisting of 160 apparently healthy participants (90 men and 70 women; mean age, 64 ± 17 

years) was also included. The nerve biopsy specimen was analyzed as described previously11. GENETIC ANALYSES DNA was extracted from peripheral blood using the Qiagen Kit (Qiagen, Hilden

Germany). Primers used for the amplification of the short range of the repeat region in _RFC1_ were as described. When no normal size band was detected, the sample was subjected to

repeat-primed polymerase chain reaction (PCR) for AAGGG (pathogenic), ACAGG (pathogenic), AGGGC (pathogenic), AGAGG (possibly pathogenic), AAGGC (possibly pathogenic), AAAGG (variable

penetrance), AAAAG (likely non-pathogenic), AAAGGG (likely non-pathogenic), and AAGAG (likely non-pathogenic) repeat configurations1,2,4. Primers used for repeat-primed PCR are described in

supplemental material. All patients provided written informed consent for genetic analyses. All methods in this study were performed in accordance with the Declaration of Helsinki. The study

protocol was approved by the Institutional Review Boards of Kagoshima University and Kindai University. AUTOANTIBODY MEASUREMENT Anti-ganglioside antibodies were examined in patients with

GBS, MFS, CIDP, and IgM paraproteinemic neuropathy using GM1, GM2, GM3, GD1a, GD1b, GD3, GT1b, and GQ1b as reported previously12,13. Anti-GalNAc-GD1a antibody was additionally examined in

patients with GBS and MFS, and anti-GT1a antibody was examined when anti-GQb1 antibody was detected12,13. Anti-MAG antibody and glycolipid sulfoglucuronyl paragloboside (SGPG) antibody,

cross-reactive to MAG, were examined in patients with IgM paraproteinemic neuropathy as reported previously5. All methods in this study were performed in accordance with the Declaration of

Helsinki. The study protocol was approved by the Institutional Review Boards of Kindai University. RESULTS _RFC1_ ANALYSES _RFC1_ mutations were found in three patients with immune-mediated

neuropathy, such as GBS, MAG neuropathy, and idiopathic sensory ataxic neuropathy and one patient with sensory autonomic neuropathy (Fig. 1). No expansion of AGGGC, AGAGG, AAGGC, AAAGG,

AAAAG, AAAGGG, or AAGAG was found in the four patients. No mutations were found in the control group. CLINICAL INFORMATION OF PATIENTS WITH RFC1 MUTATIONS Clinical and genetic information of

patients with _RFC1_ mutations is summarized in Table 1. Detailed clinical information is provided upon request. Results of NCS are summarized in Supplementary Table 1. NERVE BIOPSY

FINDINGS OF PATIENT 4 Sural nerve biopsy revealed loss of myelinated and unmyelinated nerves (Fig. 2A and B), a typical finding for CANVAS-related neuropathy1. Amyloidosis was excluded by

Congo red staining (not shown). An electron microscopic image revealed many collagen pockets (Fig. 2C). Notably, electron microscopic analysis of Schwann cells revealed cytoplasmic inclusion

bodies, dense material, or accumulated membranous material (Fig. 2D–G). No apparent abnormality was found in the vascular systems (Fig. 2H). DISCUSSION We found biallelic _RFC1_ mutations

in three patients with immune-mediated neuropathy and one with non-immune mediated neuropathy. This has been the first study to show an association between repeat expansions in _RFC1_ and

treatable immune-mediated neuropathy. Patient 1 had typical monophasic GBS with anti-ganglioside antibodies wherein weakness was completely resolved after IVIG, but dysesthesia in the upper

limbs remained 3 years after disease onset. Patient 2, who suffered from idiopathic sensory ataxic neuropathy with mild motor deficit, had a recurrent and remission clinical course with some

improvement in motor and sensory conditions after repeated IVIG treatment. Patient 3 had MAG demyelinating neuropathy with stable symptoms and decreased IgM levels after rituximab

treatment. All three patients tested positive for autoantibodies. Although positivity for rheumatoid factor, IgM anti-IgG, found in patient 2 might not directly cause neuropathy, a previous

study demonstrated that rheumatic patients with rheumatoid factor had significantly more frequent neuropathies than those without rheumatoid factor (83% vs. 44%)14. Common symptoms in the

patients included sensory disturbances, similar to those in Patient 4 with chronic sensory autonomic neuropathy with mild motor deficit and in previous studies on CANVAS2. Pathological

studies for CANVAS have been limited, with electron microscopic analyses being reported in only one study15. While the reported electron microscopic findings were limited to descriptions

regarding Schwann cells associated with unmyelinated axons, we found several rare findings concerning myelinating Schwann cells, with a cytoplasmic inclusion body, dense materials, or

accumulated membranous materials. Although Patient 4 did not have electrophysiological evidence of demyelinating neuropathy, subclinical changes in Schwann cells may have occurred. In

Patient 2, demyelination was suggested to have occurred in the sural nerve on biopsy at the early stage and in the tibial nerves on NCS at the late stage. In addition, Patient 3 with MAG

neuropathy had demyelination in the median nerve on NCS. These findings may indicate that patients with _RFC1_ mutations occasionally develop demyelination neuropathy or Schwann cell damage.

A possible mechanism by which _RFC1_ mutations are associated with immune-mediated neuropathy includes the vulnerability of the nerves themselves or that of Schwann cells to autoantibodies.

In fact, we showed subclinical abnormal findings in Schwann cells producing myelin as mentioned above. Another possible mechanism is the abnormal function of blood–nerve barrier, given that

the blood–nerve barrier may protect the nervous system from toxic materials, including autoantibodies16. However, we found no abnormalities around blood vessels, the site of the blood–nerve

barrier, though the absence of morphological changes cannot rule out its functional change. Alternatively, abnormal immune-response or production of autoantibodies may be promoted in such

patients. For example, neuronal or myelin antigens, or an expanded repeat RNA, might be abnormally exposed as immunogens17. Despite these fascinating mechanisms, the association between

_RFC1_ mutations and immune-mediated neuropathy remains inconclusive, because the relatively low incidence of mutations among a large cohort of studied patients may raise the possibility

that the association is coincidental. Biallelic mutations may suggest loss of function in RFC1, which is involved in DNA repair18. A recent report describing expansion mutations in an allele

and truncation mutations in another supports this mechanism19. Loss of DNA repair protein function in neuropathy and cerebellar ataxia is reminiscent of Aprataxin-related disorders20.

However, a previous study did not find any evidence suggesting loss of DNA repair function of RFC1 in fibroblasts1. Our electron microscopic findings suggested cytoplasmic inclusions in

Schwann cells, a situation similar to that of SCA2-related neuropathy, a gain of function disease8. Further accumulation of evidence is needed to clarify the pathomechanism using neuronal or

glial cell models or animal models. One limitation of this study is the small number of patients with immune-mediated neuropathy who were positive for _RFC1_ mutations. In addition,

detailed pathological studies in patients with immune-mediated neuropathy were lacking, as mentioned above. In summary, we found CANVAS-related _RFC1_ mutations in patients with treatable

immune-mediated neuropathy or demyelinating neuropathy. Thus, immunotherapy should not be terminated solely based on the identification of _RFC1_ mutations; however, repeated immunotherapy

to unresponsive patients should be avoided10,21. Nonetheless, future large-scale studies are needed before definitive conclusions can be established. DATA AVAILABILITY Detailed clinical

information of patients with _RFC1_ mutations were available in the supplemental material. REFERENCES * Cortese, A. _et al._ Biallelic expansion of an intronic repeat in RFC1 is a common

cause of late-onset ataxia. _Nat. Genet._ 51, 649–658 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ando, M. _et al._ Genetic and clinical features of cerebellar ataxia

with RFC1 biallelic repeat expansions in Japan. _Front. Neurol._ 13, 952493 (2022). Article  PubMed  PubMed Central  Google Scholar  * Yuan, J. H. _et al._ Multi-type RFC1 repeat expansions

as the most common cause of hereditary sensory and autonomic neuropathy. _Front. Neurol._ 13, 986504 (2022). Article  PubMed  PubMed Central  Google Scholar  * Dominik, N. _et al._ Normal

and pathogenic variation of RFC1 repeat expansions: Implications for clinical diagnosis. _Brain_ https://doi.org/10.1093/brain/awad240 (2023). Article  PubMed  Google Scholar  * Hamada, Y.

_et al._ Binding specificity of anti-HNK-1 IgM M-protein in anti-MAG neuropathy: Possible clinical relevance. _Neurosci. Res._ 91, 63–68 (2015). Article  CAS  PubMed  Google Scholar  *

Parisi, M. _et al._ Efficacy of rituximab in anti-myelin-associated glycoprotein demyelinating polyneuropathy: Clinical, hematological and neurophysiological correlations during 2 years of

follow-up. _Eur. J. Neurol._ 29, 3611–3622 (2022). Article  PubMed  PubMed Central  Google Scholar  * Hirano, M. _et al._ A family with Campylobacter enteritis: Anti-GD1a antibody

with/without Guillain-Barre syndrome. _Neurology_ 60, 1719–1720 (2003). Article  CAS  PubMed  Google Scholar  * Inada, R. _et al._ Phenotypic and molecular diversities of spinocerebellar

ataxia type 2 in Japan. _J. Neurol._ 268, 2933–2942 (2021). Article  CAS  PubMed  Google Scholar  * Fotsing, S. F. _et al._ The impact of short tandem repeat variation on gene expression.

_Nat. Genet._ 51, 1652–1659 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fernandez-Eulate, G. _et al._ Sjogren syndrome and RFC1-CANVAS sensory ganglionopathy:

Co-occurrence or misdiagnosis?. _J. Neurol._ 270, 460–465 (2023). Article  PubMed  Google Scholar  * Oka, N., Kawasaki, T., Unuma, T., Shigematsu, K. & Sugiyama, H. Different profiles of

onion bulb in CIDP and CMT1A in relation to extracellular matrix. _Clin. Neuropathol._ 32, 406–412 (2013). Article  PubMed  Google Scholar  * Kusunoki, S. _et al._ N-acetylgalactosaminyl

GD1a is a target molecule for serum antibody in Guillain-Barre syndrome. _Ann. Neurol._ 35, 570–576 (1994). Article  CAS  PubMed  Google Scholar  * Yamagishi, Y. _et al._ Markers for

Guillain-Barre syndrome with poor prognosis: A multi-center study. _J. Peripher. Nerv. Syst._ 22, 433–439 (2017). Article  CAS  PubMed  Google Scholar  * Biswas, M. _et al._ Prevalence,

types, clinical associations, and determinants of peripheral neuropathy in rheumatoid patients. _Ann. Indian Acad. Neurol._ 14, 194–197 (2011). Article  PubMed  PubMed Central  Google

Scholar  * Magy, L. _et al._ Early diagnosis in cerebellar ataxia, neuropathy, vestibular Areflexia syndrome (CANVAS) by focusing on major clinical clues: Beyond ataxia and vestibular

impairment. _Biomedicines_ 10, 2046 (2022). Article  PubMed  PubMed Central  Google Scholar  * Kanda, T. Biology of the blood-nerve barrier and its alteration in immune mediated

neuropathies. _J. Neurol. Neurosurg. Psychiatry_ 84, 208–212 (2013). Article  PubMed  Google Scholar  * Wang, E. T. _et al._ What repeat expansion disorders can teach us about the central

Dogma. _Mol. Cell_ 83, 324–329 (2023). Article  CAS  PubMed  Google Scholar  * Dilley, R. L. _et al._ Break-induced telomere synthesis underlies alternative telomere maintenance. _Nature_

539, 54–58 (2016). Article  CAS  PubMed  PubMed Central  ADS  Google Scholar  * Ronco, R. _et al._ Truncating variants in RFC1 in cerebellar ataxia, neuropathy, and vestibular Areflexia

syndrome. _Neurology_ 100, e543–e554 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hirano, M. _et al._ DNA single-strand break repair is impaired in aprataxin-related

ataxia. _Ann. Neurol._ 61, 162–174 (2007). Article  CAS  PubMed  Google Scholar  * Curro, R. _et al._ RFC1 expansions are a common cause of idiopathic sensory neuropathy. _Brain_ 144,

1542–1550 (2021). Article  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This study was partly supported by KAKEN (22K07510 to MH), 2023 Kindai University

Research Enchancement Grant (KD2306 to MH), KAKEN (20K07894 to MK), KAKEN (21K15702 and 23K06931 to MA), Transformative Research Areas (A) (Multifaceted Proteins) (20H05927 to YN) from

Ministry of Education, Culture, Sports, Science and Technology (MEXT), Scientific Research (B) (21H02840 to YN) from Japan Society for the Promotion of Science (JSPS), Practical Research

Projects for Rare/Intractable Diseases (JP21ek0109532 to YN) from Japan Agency for Medical Research and Development (AMED), and Research on Measures for Intractable Diseases (20FC1041 to YN)

from the Ministry of Health, Labour and Welfare (MHLW). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Neurology, Kindai University, Faculty of Medicine, Ohno-Higashi,

Osakasayama, Osaka, 589-8511, Japan Makito Hirano, Motoi Kuwahara, Yuko Yamagishi, Makoto Samukawa, Kanako Fujii, Shoko Yamashita, Toshihide Takeuchi, Kazumasa Saigoh, Susumu Kusunoki & 

Yoshitaka Nagai * Department of Neurology and Geriatrics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan Masahiro Ando & Hiroshi Takashima *

Department of Neurology, NHO Minami-Kyoto Hospital, Joyo, Japan Nobuyuki Oka * Department of Anatomy, Kindai University, Faculty of Medicine, Osakasayama, Japan Mamoru Nagano * Division of

Neurology, Anti-Aging, and Vascular Medicine, Department of Internal Medicine, National Defense Medical College, Tokorozawa, Japan Taro Matsui Authors * Makito Hirano View author

publications You can also search for this author inPubMed Google Scholar * Motoi Kuwahara View author publications You can also search for this author inPubMed Google Scholar * Yuko

Yamagishi View author publications You can also search for this author inPubMed Google Scholar * Makoto Samukawa View author publications You can also search for this author inPubMed Google

Scholar * Kanako Fujii View author publications You can also search for this author inPubMed Google Scholar * Shoko Yamashita View author publications You can also search for this author

inPubMed Google Scholar * Masahiro Ando View author publications You can also search for this author inPubMed Google Scholar * Nobuyuki Oka View author publications You can also search for

this author inPubMed Google Scholar * Mamoru Nagano View author publications You can also search for this author inPubMed Google Scholar * Taro Matsui View author publications You can also

search for this author inPubMed Google Scholar * Toshihide Takeuchi View author publications You can also search for this author inPubMed Google Scholar * Kazumasa Saigoh View author

publications You can also search for this author inPubMed Google Scholar * Susumu Kusunoki View author publications You can also search for this author inPubMed Google Scholar * Hiroshi

Takashima View author publications You can also search for this author inPubMed Google Scholar * Yoshitaka Nagai View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS M.H., M.K., KS., H.T., S.K. and Y.N. contributed to the conception and design of the study; Y.Y., M.S., K.F., S.Y., M.A., N.O., M.N., T.M., T.T. and K.S. contributed to

the acquisition and analysis of data; M.H. and N.Y. contributed to drafting the text or preparing the figures. CORRESPONDING AUTHOR Correspondence to Makito Hirano. ETHICS DECLARATIONS

COMPETING INTERESTS MH, MS, HT, and YN have received honoraria from Takeda. MH has received a research grant unrelated to this study from Takeda. HT and SK received honoraria from Japan

Blood Product Organization. HT and SK have received honoraria from CSL Behring. All other authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE Springer Nature

remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE 1. SUPPLEMENTARY INFORMATION. RIGHTS AND

PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any

medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The

images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is

not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission

directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hirano,

M., Kuwahara, M., Yamagishi, Y. _et al._ CANVAS-related _RFC1_ mutations in patients with immune-mediated neuropathy. _Sci Rep_ 13, 17801 (2023). https://doi.org/10.1038/s41598-023-45011-8

Download citation * Received: 01 September 2023 * Accepted: 14 October 2023 * Published: 18 October 2023 * DOI: https://doi.org/10.1038/s41598-023-45011-8 SHARE THIS ARTICLE Anyone you share

the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative