Myotonic dystrophy type 2 (DM2; MIM#602668) is an autosomal dominant multisystem disorder characterized by progressive proximal muscle weakness, myotonia, myalgia, calf hypertrophy, and multiorgan involvement with cataract, cardiac conduction defects, and endocrine disorders (Meola and Cardani, 2015; Montagnese et al., 2017). The disease is caused by a (CCTG)_n repeat expansion in intron 1 of the CNBP gene (previously ZNF9; MIM*116955) on chromosome 3q21.3 (Liquori et al., 2001). The CCTG repeat tract is part of a complex (TG)_v (TCTG)_w (CCTG)_x motif that, in healthy-range alleles, is generally interrupted by one or more GCTG, TCTG, or ACTG (NCTG) motifs that confer repeat stability (Radvanszky et al., 2013; Mahyera et al., 2018; Guo and Lam, 2016). Nonpathogenic alleles contain up to 26 CCTG repeat units, whereas premutations are composed of <75 ‘pure’ CCTG blocks whose clinical significance remains unclear (Mahyera et al., 2018; Botta et al., 2021). In DM2 patients, the number of repeats is between ~75 and >11,000 units, among the largest reported so far in repeat-expansion disorders (Depienne and Mandel, 2021). The DM2 mutation shows marked somatic instability and tends to increase in length over time within the same individual, but it does not show a strong bias toward intergenerational expansion, and genetic anticipation is rarely seen in DM2 families (Liquori et al., 2001; Kamsteeg et al., 2012; Mahyera et al., 2018; Thornton, 2014; Udd et al., 2003).

The few genotype–phenotype studies reported so far in DM2 patients did not reveal any significant associations between the severity of the disease, including the age at onset, and the number of CCTG repeats (Day et al., 2003; Udd et al., 2003). The identification of such correlations is hindered by heterogeneity across tissues, somatic instability, and the technical challenges that must be overcome to measure repeat lengths accurately in such large microsatellite expansions. This has prevented the discovery of additional in cis genetic modifiers that may ameliorate or exacerbate DM2 disease symptoms. The genetic features of the CNBP microsatellite locus, as well as its extreme length and high CG content, have frustrated attempts to size and sequence expanded alleles in DM2 patients. Even the investigators in the original gene-discovery study were unable to sequence the entire CCTG array because of its length and the high level of somatic mosaicism (Liquori et al., 2001; Bachinski et al., 2003; Day et al., 2003).

According to international guidelines, current best practice for DM2 genetic testing relies mainly on PCR-based approaches (Botta et al., 2006; Kamsteeg et al., 2012). These include an initial short-range PCR (SR-PCR) to exclude a DM2 diagnosis when two normal-range alleles are detected. When only one allele is visible, (CCTG)_n expansions can be identified by long-range PCR (LR-PCR) or quadruplet-repeat primed PCR (QP-PCR), leading to a ~99% detection rate (Kamsteeg et al., 2012). However, neither method can define the exact length of large DM2 expansions, which requires the Southern blot analysis of digested genomic DNA and has a sensitivity of ~80% (Day et al., 2003). The latter method is time consuming, requires large amounts of DNA, and is not included in the routine workflow of most diagnostic centers. Moreover, none of the methods described above can resolve the expansion to the single-nucleotide level and they have a limited ability to detect minor alleles and the degree of somatic mosaicism.

Third-generation long-read sequencing technologies provide an unprecedented opportunity to fully characterize DM2 expansions in terms of repeat size, allele configuration, and base composition. Whereas most second-generation sequencing methods produce short reads, third-generation methods such as Oxford Nanopore Technologies (ONT) and PacBio SMRT sequencing facilitate the analysis of DNA fragments multiple kilobases in length, including large repetitive elements and their flanking regions. Furthermore, third-generation methods are generally based on PCR-free workflows, thus avoiding amplification-related biases (Hommelsheim et al., 2014) and challenges caused by regions with a high CG content (such as the CNBP locus). However, these technologies are more expensive and error-prone than first- and second-generation methods. Targeted approaches that maximize data production on a selected region of interest can compensate for such errors and provide cost-effective alternatives to whole-genome sequencing.

Targeted enrichment approaches coupled to long-read sequencing have already been used for the in-depth characterization of repeat expansions in fragile X syndrome (FMR1), Huntington’s disease (HTT), and neuronal intranuclear inclusion disease (NOTCH2NLC), although these expansions are significantly shorter than the (CCTG)_n repeats in DM2 patients (DeJesus-Hernandez et al., 2021; Ebbert et al., 2018; Giesselmann et al., 2019; Grosso et al., 2021; Hafford-Tear et al., 2019; Höijer et al., 2018; Mizuguchi et al., 2021; Sone et al., 2019; Tsai et al., 2017; Wallace et al., 2021; Wieben et al., 2019; Mitsuhashi and Matsumoto, 2020). The longest allele thus far characterized by third-generation sequencing is a 21-kbp allele of the C9orf72 gene that causes amyotrophic lateral sclerosis and frontotemporal dementia (DeJesus-Hernandez et al., 2021). In addition, many of the works cited above combined PacBio SMRT sequencing with LR-PCR (Mangin et al., 2021; Ciosi et al., 2021; Cumming et al., 2018), which is unsuitable for the analysis of DM2 expansions due to their extreme length and high GC content. Importantly, PacBio reads do not exceed 20 kbp in a PCR-free enrichment (DeJesus-Hernandez et al., 2021; Ebbert et al., 2018; Hafford-Tear et al., 2019; Höijer et al., 2018; Tsai et al., 2017; Wieben et al., 2019), and would not completely span DM2 pathogenic expansions (20 kbp on average, but up to 50 kbp).

To address these issues, we assessed the analysis of CNBP expansions using a combination of CRISPR/Cas9-based enrichment (Cas9-enrichment) and ONT sequencing. The latter can generate reads >100 kbp in length (Payne et al., 2019; Iyer et al., 2022) and recently demonstrated valuable for the analysis of very long repetitive elements, like telomeric and centromeric regions in the completion of human genome (Sergey et al., 2022; Consortium and The Telomere-to-telomere T T, 2022) and microsatellite expansions in the pathogenic range (Stevanovski et al., 2022). In this manner, we sequenced full-length (CCTG)_n expansions in nine DM2 patients including one mutated allele 47 kbp in length. Because this approach achieves single-nucleotide resolution, we were able to detect a previously unreported (TCTG)_n motif at the 3′ end of the CNBP expansion in seven of the DM2 patients. Our pilot study demonstrated that Cas9-mediated enrichment and long-read sequencing improves the DM2 diagnostic workflow, facilitating the in-depth characterization of CNBP expansions by accurately reporting the repeat length, structure/motif, and degree of somatic mosaicism in a single analysis. In the future, this approach may enable more precise genotype–phenotype correlations and thus improve patient stratifications in clinical trials for personalized therapies.

The analysis of extremely long microsatellite expansions is challenging, preventing the in-depth characterization of CNBP mutations underlying DM2 and its relationship with the clinical phenotype. To date, the genotype–phenotype correlation issue in DM2 is still unsolved and relies on a single study from Day et al., 2003 in which Southern blot analysis was used to determine the length of DM2 mutation. Because of the extremely large size of the CCTG expansions and somatic instability of the repeat, Southern blot fails to detect the DM2 mutation in about 20% of known carriers, whose expansion length remains undeterminable. Moreover, currently no diagnostic method allows to sequence through fully expanded CNBP microsatellites. Here, we have demonstrated for the first time the use of Cas9-mediated enrichment and ONT long-read sequencing to analyze the full (CCTG)_n expansion in DM2 patients at single-nucleotide resolution. We were able to characterize normal and expanded CNBP alleles, simultaneously revealing the repeat length, structure/motif, and extent of somatic mosaicism, which is not possible with traditional methods, even if several are used in combination.

The paucity of genotype–phenotype data for DM2 reflects the historical inability to determine the size of CCTG repeats, especially in the largest expansions, which traditionally was based on the Southern blot analysis of genomic DNA. This labor-intensive and time-consuming technique is becoming obsolete because it requires large amounts of high-molecular-weight DNA. LR-PCR can be used instead, but the performance of this technique is poor in regions with a high CG content, and it cannot accommodate the very large expanded alleles (>15 kbp) often found in DM2. Therefore, although LR-PCR achieves the sensitive detection of DM2 mutations, the full length of the (CCTG)_n expansion cannot be determined in all patients. Our Cas9-targeted sequencing protocol overcomes these limits by focusing long-read sequencing data on the CNBP microsatellite, with reads spanning the entire expansion. The length of normal and expanded alleles in DM2 patients determined using this new approach closely matched the values obtained with the traditional reference methods. The mean size of normal CNBP alleles was 134 bp whereas expanded alleles ranged from 344 to 46,685 bp, in agreement with previous reports (Meola and Cardani, 2015; Montagnese et al., 2017; Botta et al., 2021). A single incongruence was observed for one expanded allele in patient B, where ONT sequencing underestimated the size determined by Southern blotting (~20 vs. ~40 kbp). A possible explanation is the presence of damaged DNA in the sample, reflecting its long-term storage in a biobank. Southern blotting involves the fractionation of double-stranded DNA, which would be unaffected by the presence of nicked strands, whereas ONT sequencing involves the analysis of single DNA strands, so the presence of nicks would have a profound effect (Oxford Nanopore Community, 2021). Even so, Cas9-mediated enrichment allowed us to sequence DM2 alleles up to ~50 kbp in length at single-nucleotide resolution, which has not been reported previously for DM2 or any other repeat-expansion disorder using this approach (Giesselmann et al., 2019; Mizuguchi et al., 2021; Sone et al., 2019; Wallace et al., 2021; Gilpatrick et al., 2020; Iyer et al., 2020). The analysis of such long and repetitive alleles required the coupling of a PCR-free enrichment protocol to ONT sequencing because even other long-read sequencing technologies cannot accommodate this read length in targeted sequencing experiments. For example, PacBio long-read sequencing was previously used to sequence repeat expansions in DM1, which is also characterized by long alleles of 4–6 kbp, but the microsatellites were first amplified by PCR (Mangin et al., 2021; Cumming et al., 2018). Even when coupled to PCR-free enrichment approach based on Cas9, the length of PacBio sequencing reads could not exceed 20 kbp (DeJesus-Hernandez et al., 2021; Ebbert et al., 2018; Hafford-Tear et al., 2019; Höijer et al., 2018; Tsai et al., 2017; Wieben et al., 2019).

Although ONT sequencing had been already utilized for the analysis of the microsatellite within the CNBP gene, this was confined to CNBP alleles in the normal range only (Stevanovski et al., 2022; Mohammad et al., 2022). Moreover, the work of Mitsuhashi et al. exploited ONT whole-genome sequencing, that is not applicable in the routine due to the very high costs (Mohammad et al., 2022). The group of Stevanovski utilized the recently introduced ‘Read Until’ feature of ONT sequencing for the analysis of microsatellites in 37 disease-associated loci. This allows the selective sequencing of predefined genomic regions, thus enabling a targeted sequencing with similar advantages of the Cas9-mediated sequencing presented hereby. However, enrichment levels achieved by ‘Read Until’ (5×) are consistently lower than those obtained with the Cas9 approach (500×), due to higher background (Stevanovski et al., 2022). This may constitute an important issue when dealing with extremely long CNBP alleles that can be disadvantaged in sequencing as compared to shorter contaminating fragments (Holgersen et al., 2021).

From a technical perspective, we achieved >500× enrichment on CNBP using the Cas9 protocol, which is robust and comparable to similar assays for the assessment of microsatellite length (Giesselmann et al., 2019; Mizuguchi et al., 2021; Sone et al., 2019; Wallace et al., 2021). We also compared Cas9-mediated singleplex versus multiplex enrichment protocols for the first time on clinical samples and observed a consistently lower performance (10-fold lower enrichment, with 70% unclassified reads) in the multiplex environment, as reported by other ONT users for other type of samples (Oxford Nanopore Community, 2022). Further improvements are therefore required before the multiplexing protocol is suitable, for example by combining the Cas9 protocol with a second enrichment step using the ‘Read Until’ feature of ONT sequencing to exclude the background noise. Alternatively, costs could be optimized by analyzing single samples using Flongles, which produce less sequencing data than regular ONT flow cells but reduce costs by 90%.

The bioinformatic pipeline we used to analyze the CNBP microsatellite sequences also allowed us to recognize the repeat pattern and precisely count the repeats, including the short interrupted and uninterrupted alleles typifying the Italian population (Botta et al., 2021). Consensus sequences generated from the normal allele shared a high degree of identity (99.5%) and significant correlation with state-of-the-art Sanger sequences, aside from a few nucleotide positions. Similar small discrepancies have also been reported in characterization of the (TG)_v motif upstream of the (CCTG)_n repeated array in familial cases A1–A4. These inconsistences probably reflect ONT sequencing errors and could be addressed by using the most recent base-calling algorithm and eventually the more accurate Q20+ chemistry.

In the expanded alleles of seven DM2 patients, the anticipated (CCTG)_n repeat was accompanied by a previously unreported (TCTG)_n repeat located at the 3′ end of the (CCTG)_n array. When present, the atypical motif varied in length between donors and within each sample (40–8000 bp). Repeat interruptions within the expanded array have been reported in 3%–8% of DM1 patients (Tomé et al., 2018; Braida et al., 2010; Santoro et al., 2017; Santoro et al., 2013; Ballester-Lopez et al., 2020; Pešović et al., 2018; Miller et al., 2020; Radvansky et al., 2011; Siena et al., 2018; Botta et al., 2017; Santoro et al., 2015; Addis et al., 2012; Fontana et al., 2020; Lian et al., 2016; Leeflang and Arnheim, 1995; Musova et al., 2009; Cumming et al., 2018), but have not been described in DM2 before. This may reflect the challenge of sequencing complete CNBP expanded alleles and/or the use of a primer containing ‘pure’ (CCTG)_n repeats for diagnostic QP-PCR. Given that (TCTG)_n repeats were present in a highly variable proportion of expanded alleles (11%–86%), always in the presence of the typical (CCTG)_n repeats, technical bias may have revealed only the ‘pure’ (CCTG)_n repeats. Indeed, a modified QP-PCR protocol using a primer containing five TCTG units – (TCTG)₅T – was able to confirm the ONT sequencing data. From a biological perspective, the (TCTG)_n motif may have arisen through DNA duplication/repair errors or spontaneous DNA damage in the somatic cells of DM2 patients. Although the presence of this motif may be biologically relevant in the context of DM2, our data must be interpreted with caution. First, the motif was discovered in a small set of patients, most belonging to the same family, so confirmation requires a larger prospective DM2 cohort enrolled in multicenter studies, in which DNA samples are collected in order to ensure the optimal quality for ONT sequencing. Second, considering the known limitations of ONT sequencing when presented with low-complexity regions such as homopolymers, the length and recurrence of such motifs should be investigated using other long-read methods when they are sufficiently advanced to sequence the expanded alleles completely.

The Cas9-targeted sequencing approach also allowed us to estimate the degree of somatic mosaicism for the mutated alleles, either ‘pure’ or ‘interrupted’. On average, the allele length within each patient varied by 65%. Mosaicism plays an important role in the development of disease symptoms, so establishing the relative proportion of expanded alleles in the lower and upper mutation range could add prognostic value, significantly improving genetic counseling for DM2. Extreme mosaicism (more than expected based on previous studies) has also been detected when long-read sequencing is applied to other repeat-expansion disorders, suggesting that such techniques achieve higher resolution (Loomis et al., 2013; Mizuguchi et al., 2021; Mangin et al., 2021). As already demonstrated for DM1 (Cumming et al., 2019; Monckton et al., 1995), the progenitor allele length (i.e., the length of the CCTG repeat transmitted by the affected parent) is one genetic determinant that influences the age at onset of DM2 symptoms, and that age is further modified by individual-specific differences in the level of somatic instability. Notably, our method accurately distinguished between the shortest expanded allele and the normal allele.

Another advantage of the approach demonstrated hereby is that PCR-free analysis potentially allows the direct assessment of DNA methylation, as already reported for other repeat-linked diseases (Fukuda et al., 2021; Giesselmann et al., 2019). This can provide additional information concerning the impact of expansions on the functionality of the CNBP gene. The methylation of the CNBP gene has been analyzed using a pyrosequencing method, revealing hypomethylation of CpG sites upstream and hypermethylation of CpG sites downstream of the (CCTG)_n expansion in DM2 patients and healthy individuals, with no significant differences between these groups (Santoro et al., 2018). However, it remains possible that the DM2 mutation could have epigenetic effects in other regulatory regions of the CNBP gene and/or in different tissues.

Given the ability of our method to simultaneously determine the size, single-nucleotide composition and degree of somatic mosaicism of DM2 repeat expansions, ONT sequencing could be included in the DM2 diagnostic workflow to improve the information content available for genetic counseling. To date, the cost for Cas9-mediated sequencing of a single patient is relatively high and not comparable with the PCR-based approaches used in the routine of the DM2 molecular diagnostics. Nevertheless, targeted long-read sequencing might help to solve unusual large and complex (CCTG)_n expansions not detectable with conventional methods and identifies noncanonical repetitive motif conformations and sequence interruptions. Taken together, this information will allow more precise correlation between the length and composition of DM2 expansion and the clinical phenotype. In a next future, further evolution of ONT chemistry and the optimization of multiplexing strategies are expected to drastically decrease the costs of the analysis, making the Cas9-mediated sequencing more easily accessible in the clinical practice.

Taken together, our pilot study has demonstrated the potential of PCR-free long-read sequencing for the genetic assessment of DM2, allowing us to investigate both the length and genetic features of normal and expanded alleles in a single round of analysis. The use of such an approach in larger cohorts will increase the accuracy of genotype–phenotype correlations and enhance the information content available for DM2 genetic counseling.

The sequencing data generated in this study have been submitted to the NCBI BioProject database under accession number PRJNA818354 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA818354).

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   ID PRJNA818354. Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing.

   https://www.ncbi.nlm.nih.gov/bioproject/PRJNA818354

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Author details

1. Massimiliano Alfano

   Department of Biotechnology, University of Verona, Verona, Italy

   Contribution

   Investigation, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Luca De Antoni

   Competing interests

   No competing interests declared

2. Luca De Antoni

   Department of Biotechnology, University of Verona, Verona, Italy

   Contribution

   Investigation, Writing - original draft

   Contributed equally with

   Massimiliano Alfano

   Competing interests

   No competing interests declared

3. Federica Centofanti

   Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata, Rome, Italy

   Contribution

   Validation

   Competing interests

   No competing interests declared

4. Virginia Veronica Visconti

   Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata, Rome, Italy

   Contribution

   Validation

   Competing interests

   No competing interests declared

5. Simone Maestri

   Department of Biotechnology, University of Verona, Verona, Italy

   Contribution

   Software, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1192-0684

6. Chiara Degli Esposti

   Department of Biotechnology, University of Verona, Verona, Italy

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5974-4915

7. Roberto Massa

   Department of Systems Medicine (Neurology), University of Rome Tor Vergata, Rome, Italy

   Contribution

   Resources

   Competing interests

   No competing interests declared

8. Maria Rosaria D'Apice

   Laboratory of Medical Genetics, Tor Vergata Hospital, Rome, Italy

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

9. Giuseppe Novelli

   1. Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata, Rome, Italy
   2. IRCCS Neuromed, Via Atinense, Molise, Italy
   3. Department of Pharmacology, School of Medicine, University of Nevada Reno, Reno, United States

   Contribution

   Supervision

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7781-602X

10. Massimo Delledonne

    1. Department of Biotechnology, University of Verona, Verona, Italy
    2. Genartis s.r.l., Via P. Mascagni, Castel D'azzano, Italy

    Contribution

    Conceptualization, Supervision, Funding acquisition, Methodology

    Competing interests

    is a partner of Genartis srl

11. Annalisa Botta

    Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata, Rome, Italy

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - review and editing

    For correspondence

    botta@med.uniroma2.it

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4031-5624

12. Marzia Rossato

    1. Department of Biotechnology, University of Verona, Verona, Italy
    2. Genartis s.r.l., Via P. Mascagni, Castel D'azzano, Italy

    Contribution

    Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Writing - review and editing

    For correspondence

    marzia.rossato@univr.it

    Competing interests

    is a partner of Genartis srl, Verona

    "This ORCID iD identifies the author of this article:" 0000-0002-6101-1550

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