Serologic Anti-GD3 IgM-positive CANVAS Syndrome with RFC1 rare mutation type: A Case Report

In 1998, Rinne et al. reported cerebellar degeneration in 7 out of 51 patients (13%) with bilateral vestibular failure, which included 4 patients with mild peripheral neuropathy [4]. Vera C. Zingler et al. analyzed the etiology and epidemiology of 255 patients with bilateral vestibular lesions, finding that a significant subgroup exhibited cerebellar dysfunction and peripheral polyneuropathy [5]. Szmulewicz et al. retrospectively analyzed 18 subjects meeting the criteria for bilateral vestibulopathy with cerebellar ataxia, finding that all had sensory neuropathy. MRI scans revealed cerebellar atrophy in 16 patients. This led to the proposal of a new syndrome termed cerebellar ataxia with neuropathy and bilateral vestibular reflex syndrome (CANVAS) [6]. MRI in patients with CANVAS demonstrated a pattern of anterior and dorsal cerebellar atrophy, specifically in lobules VI, VIIa, and VIIb, while lateral atrophy predominantly affected the cerebellar hemispheres, corresponding to lobule VII [7]. Microscopically, diffuse loss of Purkinje cells is evident, primarily affecting the vermis region but also significantly impacting the lateral cerebellum [8]. Recently, Cortese et al. identified a double allelic intron (AAGGG)n repeat amplification in the poly-A tail at the 3' end of the Alu element of replication factor C1 (RFC1) as the causative mutation in CANVAS [9]. Notably, no correlation was observed between the age of onset of neuropathy in CANVAS syndrome and the size of the RFC1 repeat expansion [3].

In this paper, we report a unique case of CANVAS in a patient with a heterozygous mutation in exon 11 of the RFC1 gene, as well as multimodal mutations of (AAAAGG)exp and (AAAGG)exp in the bi-allelic introns, combined with serological positivity for anti-GD3 IgM. This finding is relatively rare and offers valuable clinical insights. In clinical practice, CANVAS syndrome is often misdiagnosed or underdiagnosed, particularly when it overlaps with other conditions. Upon reviewing this case, we noted that the patient exhibited abnormal limb sensations, weakened tendon reflexes upon examination, and positive findings for anti-GD3 IgM. These indicators facilitated the diagnosis of Guillain-Barre syndrome (GBS). Following treatment with hormone and gammaglobulin shock therapy, the patient's symptoms improved, although he continued to experience unsteady gait. Concurrently, the patient exhibited prolonged sensory abnormalities in the extremities, a positive bilateral bedside head-shaking test, signs of cerebellar atrophy on imaging, and vestibular function tests suggesting bilateral vestibular hypoplasia, which could not be solely attributed to GBS. Consequently, we suspected that the patient might be comorbid with other ataxic disorders. To investigate this, we conducted genetic testing for various ataxic types (SCA1/2/3/6/7/8/10/12/17/36/DRPLA and FRDA), ruling out SCA-related disorders and Friedreich's ataxia, ultimately diagnosing CANVAS syndrome [10]. Additionally, we identified a rare mutation locus in the RFC1 gene in this patient with CANVAS syndrome. Whole-exon sequencing revealed a heterozygous deletion mutation at the locus of exon 11 of the RFC1 gene. Most literature indicates that the causative mutation in RFC1 for CANVAS patients is the amplification of the AAGGG sequence in intron 2 [3]. Consequently, we designed amplification primers targeting the repeated sequences of the RFC1 gene in the poly-A tail, specifically (AAAAG)n or (AAGGG)n within the Alu element, to amplify blood nucleic acids from patients. Concurrently, six randomly selected blood samples from healthy individuals were amplified using the same PCR system as controls. The amplification results for the patient samples revealed a single amplified band close to 1000 bp, indicating a pure synonymous mutation. Comparisons with healthy controls suggest that the patient harbors a mutation in the (AAAAG)n or (AAAGG)n repeats of the RFC1 gene. Subsequent sequencing identified multiple mutation types at the (AAAAG)n position of the Alu element in the patient's RFC1 gene, specifically (AAAAGG)exp and (AAAGG)exp. The former represents a 6-nucleotide repeat sequence amplification, which is less commonly reported. Given the unclear pathogenesis of CANVAS, we speculate that this mutation may represent a rarer mutation type associated with the disease.

In recent years, there have been several reports concerning the RFC1 non-AAGGG mutations in CANVAS syndrome. Natalia Dominik et al. expanded the genetic heterogeneity of RFC1-associated CANVAS disorders, identifying three additional pathogenic repeat motifs: AAGGC, AGGGC, and AGAGG [11]. Carolin K. Scriba et al. screened an Asia-Pacific CANVAS cohort and identified a novel RFC1 repeat motif, (ACAGG), amplified in three affected individuals. This motif was associated with additional clinical features, including muscle fasciculations and elevated serum creatine kinase levels [12]. Sacha Weber et al. reported genetic and clinical data from two families with heterozygous mutations in the RFC1 gene, specifically in a splice variant located in exon 19 and another in exon 20, respectively [13]. RFC1 encodes the large subunit of replication factor C, a five-subunit DNA polymerase accessory protein. It facilitates the loading of proliferating cell nuclear antigen onto DNA, activates DNA polymerases δ and ε, and promotes the coordinated synthesis of both DNA strands during DNA replication or DNA damage. The 3' end of the Alu element in RFC1 contains a long A-rich region that is crucial for its amplification mechanism [14]. The cerebellum and peripheral nerves are particularly vulnerable to reduced DNA repair function [15]. Therefore, when the 3' end of the Alu element is mutated, it may undergo abnormal over-amplification, leading to pathogenic mRNA accumulation and resulting in CANVAS [3].

In summary, we present a case of CANVAS syndrome involving mutations in both the intron and exon of RFC1 in a patient with a recent history of GBS. This case provides valuable clinical insights for diagnosing similar combined disorders and expands the genetic heterogeneity of CANVAS syndrome. The specific role of this RFC1 mutation warrants further investigation.

Author contributions: Lu Gengxin and Qi Weiwei are responsible for data collection, analysis and paper writing; Li Minping is responsible for data collection and paper revision; Huang Haiwei is responsible for research guidance, paper revision and financial support.

Funding source: This study was supported by grants from National Natural Science Foundation of China(82271410) .

Data Availability: Anonymized data can be requested from any qualified researcher.