In one of the largest studied ataxia cohorts consisted of 2523 patients from Poland in whom the unequivocal genetic cause was not determined, the biallelic pathogenic (AAGGG) repeat expansions in the RFC1 were observed in 4.6% of patients. These results were based on PCR analysis – RP-PCR, a method which cannot estimate the size of the expansions, however, which can indicate the presence or absence of known pathogenic configurations and flanking PCR which can indicate a presence or absence of normal and to some extend -pathogenic alleles. All patients included in the study showed clinical picture of cerebellar ataxia, but they were not divided into subgroups of presented clinical symptoms due to insufficient medical data and not fully determined clinical phenomenology, considering particular sensory neuropathy and vestibulopathy.

Southern blotting was performed on selected patients DNA to confirm RP-PCR results and gain insight into the size of the expanded alleles. The OGM was used in 3 clinically suspected CANVAS patients. The highly restricted procedure of DNA extraction and staining yielded reliable results. The overall bench working time is comparable to the Southern blotting, with the assurance of consistently good-quality results. Despite its expenses, it is worthwhile to apply the procedure for sizing expansions in particular cases. Unfortunately, none of both methods gives the detailed structure of the motif. OGM provides very accurate sizing and SB only approximate size of the expansion. Recent advances in long-read sequencing (LRS) technologies, such as those offered by Oxford Nanopore and PacBio platforms, have introduced highly accurate methods for identifying and characterizing repetitive sequences, including intronic AAGGG expansions in the RFC1 gene (Nakamura et al. 2020). These technologies enable direct measurement of repeat number and structure across long genomic regions, overcoming many of the limitations inherent to traditional PCR-based approaches and SB. Although our current study relied on RP-PCR combined with confirmatory SB and OGM, in selected cases, we acknowledge that LRS represents a comprehensive analysis of pathogenic repeat expansions. Future studies incorporating LRS are likely to enhance diagnostic precision and may uncover additional variability in repeat structures that remain undetectable by conventional methods.

It is increasingly evident that CANVAS is a heterogeneous disorder with different configuration motifs and sizes contributing to the phenotypic spectrum. This is a very important issue in the context of diagnostics and genetic counseling for people from families affected by CANVAS and disease spectrum. The biallelic expansions of the non-pathogenic AAAAG/AAAAG motif is also quite frequent in our studied group, observed in 253 patients (10%), and the frequency of such genotype seems to be too high to have a serious effect on neurogenerative mechanisms (patient 3B—929 AAGGG repeats/443 repeats AAAAG, and patients 3 C AAGGG 825 repeats and 547 AAAAG repeats). However, we cannot exclude that such expansions > 500 repeats may reveal other functional effects within the nervous system. Current knowledge does not allow for precise determination of the pathogenicity of these expansions. Despite suggestions that expansions of other motifs are significant, their role has not been confirmed. Outcomes of our research corresponds with other European populations where the frequency of pathogenic biallelic AAGGG expansion estimates between 0.7% up to 22% of undiagnosed ataxia patients which shows that CANVAS syndrome has been underestimated in clinical diagnosis so far (Cortese et al. 2019; Dominik et al. 2021; Rafehi et al. 2019). Patients were genetically diagnosed – PCR, RP-PCR, to determine the motif, and selected samples were used in SB and OGM to determine their size. Table 3 presents studies on the genetics of CANVAS syndrome conducted across various populations (Cortese et al. 2019, 2020; Akçimen et al. 2019; Aboud Syriani et al. 2020; Fan et al. 2020; Tsuchiya et al. 2020; Beecroft et al. 2020; Kontogeorgiou et al. 2021; Tagliapietra et al. 2021; Montaut et al. 2021; Ghorbani et al. 2022).

To date, CANVAS patients are mainly of European origins with a few studies of other populations with different expanded motifs (Tsuchiya et al. 2020; Tyagi et al. 2023). Analysis of the AAGGG core haplotype suggests that the pathogenic repeat expansion arose in Europe, approximately 50,000 years ago (Dominik et al. 2023; Rafehi et al. 2019). The study of Rafehi et al.(Rafehi et al. 2019), it was indicated that in a North American/Australian cohort, the majority of individuals carrying AAGGG expansion were of European ancestry. However, it was also present in non-European individuals i.e., East Asians with admixed Native American ancestry, despite the disorder being highly overrepresented in European populations (Rafehi et al. 2019; Tsuchiya et al. 2020). Also, the pathogenic AAGGG expansion carrier frequency ranges from ~ 1 to 5% in the healthy population as it was previously reported (Cortese et al. 2019; Akçimen et al. 2019; Sułek-Piątkowska et al. 2010). Compared to the previously reported control cohort by Cortese et al.(Cortese et al. 2019), our data suggest a higher prevalence of pathogenic AAGGG motifs in the general population. While Cortese’s study, which included 304 healthy individuals, identified four pathogenic-length expansions (0.7% of 608 chromosomes), our cohort of 537 individuals yielded 48 AAGGG-positive chromosomes via RP-PCR (4.47%). Although we could not directly confirm expansion size in our control group, prior SB analysis in patients showed that 3 out of 8 RP-PCR-positive individuals carried truly pathogenic-length expansions. Extrapolating this proportion to our control group suggests that around 1.67% of alleles might be pathogenic in length (over twice the frequency reported by Cortese et al. (Cortese et al. 2019)). It remains unclear whether the Cortese cohort included heterozygous or homozygous carriers, and whether shorter, non-pathogenic expansions were present, which limits direct comparability but further emphasizes the variability in population data. Nevertheless, it might be beneficial for patients with ataxic disorders but no clear genetic diagnosis to be tested for the RFC1 repeat expansions, particularly if the patients present with sensory neuropathy or/and unexplained cough.

In our study, we have found that some patients with cerebellar ataxia may have (1) pathogenic AAGGG motif only in one allele, (2) expansion of non-pathogenic genotypes AAAGG or AAAAG, (3) heterogenous arrangements of both alleles with pathogenic expansion of AAGGG on one allele and non-pathogenic AAAGG or AAAAG expansion on the other allele; (4) pathogenic genotype AAGGG with normal nucleotide repeats < 250. Our findings showed that the biallelic AAGGG expansion in the RFC1 gene was found in 4.6% of patients with late-onset cerebellar ataxia of unknown cause. Whitmore, if we hypothesize that the presence of only one AAGGGexp allele (AAGGGexp/AAAAGexp or AAAGGexp) within genotype is sufficient to cause CANVAS, the prevalence of this disorder among undiagnosed ataxias would be even higher (6.7%). However, the significance of such findings in relation to CANVAS phenomenology is unknown. We have only few patients with precise neurological diagnostic work-up so far and one patient with severe clinical symptomatology of CANVAS had AAGGG expansion with 825 repeats in only one allele and the expansion of 547 AAAAG repeats on the other allele, as measured by OGM. We cannot exclude that the abnormalities pointed 1 to 4 are genetic risk factors for the developing of the disease, are the only cause of the disease or are the cause of other, non-CANVAS ataxia. So, it is possible that minority of the cases that not fully blown symptomatology of CANVAS (e.g., ataxia with the absence of vestibulopathy and sensory neuropathy) may be caused by expansion of other than AAGGG allele or even normal repeat length of pathogenic AAGGG motif on both alleles. They may also have other genetic cause of the disease. It is worth mentioning that two patients, who show to be a carrier of the pathogenic motif, were initially suspected of CANVAS syndrome based on clinical observation. These patients underwent reexamination by professionals, confirming clinical presentation of CANVAS syndrome. This gives a further notice to conclude that this is very fragile matter. Determination of genotypes and presented symptoms are not that obvious.

Following other issues of CANVAS syndrome, that had been reported lately (Dominik et al. 2023; Akçimen et al. 2019; Nakamura et al. 2020; Tyagi et al. 2023; Scriba et al. 2020). Tyagi et al. have investigated the RFC1 locus in diverse neurodegenerative outcomes in an India cohort (Tyagi et al. 2023). They have confirmed the presence of the pathogenic motif AAGGG and reported cases with new pathogenic expansion of (AAAGG)exp/(AAGGG)exp in the same locus (for different repeat motifs see Table 3.). Another group has identified novel conformations AAGAG and AGAGG (Akçimen et al. 2019). Perhaps, as in the case of other recessive diseases caused by dynamic mutations such as Friedreich's ataxia or Unverricht-Lundborg myoclonic epilepsy, a complex mutation including a repeat expansion on one allele and a point mutation on the other may cause CANVAS syndrome (Benkirane et al. 2022; Ronco et al. 2023). Several other (Cortese et al. 2019, 2020; Akçimen et al. 2019; Aboud Syriani et al. 2020; Fan et al. 2020; Tsuchiya et al. 2020; Beecroft et al. 2020; Kontogeorgiou et al. 2021; Tagliapietra et al. 2021; Montaut et al. 2021; Ghorbani et al. 2022; Tyagi et al. 2023) researchers have been reporting pathogenic motifs in their cohorts and assumptions that different may also appear and that further investigation is required. These findings could correspond to our study, where some of the patients demonstrated no product in flanking and RP-PCR, suggesting the possible presence of different motifs. This raises a question on the exact method of testing CANVAS syndrome patients. In our study, we have limited the examination of the conformation of repeat motifs to RP-PCR. The SB and OGM was used to see whether sizing are appropriate as other groups have reported (Cortese et al. 2019, 2020; Akçimen et al. 2019; Aboud Syriani et al. 2020; Fan et al. 2020; Tsuchiya et al. 2020; Beecroft et al. 2020; Kontogeorgiou et al. 2021; Tagliapietra et al. 2021; Montaut et al. 2021; Ghorbani et al. 2022; Tyagi et al. 2023). Our study confirms accurate sizing is crucial for characterizing patients’ genotypes. Dominik et al. had given implications for genetic testing and clinical diagnosis of variants of the RFC1 repeat expansions and agreed on sizing the microsatellite repeats in clinical diagnosis (Dominik et al. 2023).

From the research conducted so far, the molecular mechanism of pathogenic expansion is not fully elucidated to assess the role of currently known as non-pathogenic or smaller pathogenic expansions. Perhaps, they also influence the development of the disease, posing an open question that needs to be addressed in the future to characterized the genetic content of CANVAS syndrome. Moreover, detection of the pathogenic expansion in patients with other neurological conditions such as MSA or parkinsonism may expand the spectrum of disorders related to mutations in the RFC1 gene.