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

Abstract

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully-expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3′ end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype-phenotype correlations for the better stratification of DM2 patients in clinical trials.

Data availability

The sequencing data generated in this study have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA818354. For revision purposes, reviewers can download data from the following link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA818354?reviewer=1g4lp9ijkgv5s07is6imnot6ch

The following data sets were generated

Article and author information

Author details

  1. Massimiliano Alfano

    Department of Biotechnology, University of Verona, Verona, Italy
    Competing interests
    No competing interests declared.
  2. Luca De Antoni

    Department of Biotechnology, University of Verona, Verona, Italy
    Competing interests
    No competing interests declared.
  3. Federica Centofanti

    Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
    Competing interests
    No competing interests declared.
  4. Virginia Veronica Visconti

    Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
    Competing interests
    No competing interests declared.
  5. Simone Maestri

    Department of Biotechnology, University of Verona, Verona, Italy
    Competing interests
    No competing interests declared.
    ORCID icon "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
    Competing interests
    No competing interests declared.
    ORCID icon "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
    Competing interests
    No competing interests declared.
  8. Maria Rosaria D'Apice

    Laboratory of Medical Genetics, University of Rome Tor Vergata, Rome, Italy
    Competing interests
    No competing interests declared.
  9. Giuseppe Novelli

    Laboratory of Medical Genetics, University of Rome Tor Vergata, Rome, Italy
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7781-602X
  10. Massimo Delledonne

    Department of Biotechnology, University of Verona, Verona, Italy
    Competing interests
    Massimo Delledonne, is a partner of Genartis srl.
  11. Annalisa Botta

    Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
    For correspondence
    botta@med.uniroma2.it
    Competing interests
    No competing interests declared.
  12. Marzia Rossato

    Department of Biotechnology, University of Verona, Verona, Italy
    For correspondence
    marzia.rossato@univr.it
    Competing interests
    Marzia Rossato, is a partner of Genartis srl, Verona.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6101-1550

Funding

Muscular Dystrophy Association (MDA 876149)

  • Massimo Delledonne

Italian DiMio onlus association (DM0000010)

  • Annalisa Botta

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Murim Choi, Seoul National University, Republic of Korea

Ethics

Human subjects: The study was approved by the institutional review board of Policlinico Tor Vergata (document no. 232/19). All experimental procedures were carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consent was obtained from all nine participants and all samples and clinical information were anonymized immediately after collection using a unique alphanumeric identification code.

Version history

  1. Preprint posted: May 12, 2022 (view preprint)
  2. Received: May 12, 2022
  3. Accepted: August 25, 2022
  4. Accepted Manuscript published: August 26, 2022 (version 1)
  5. Version of Record published: September 9, 2022 (version 2)

Copyright

© 2022, Alfano et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Massimiliano Alfano
  2. Luca De Antoni
  3. Federica Centofanti
  4. Virginia Veronica Visconti
  5. Simone Maestri
  6. Chiara Degli Esposti
  7. Roberto Massa
  8. Maria Rosaria D'Apice
  9. Giuseppe Novelli
  10. Massimo Delledonne
  11. Annalisa Botta
  12. Marzia Rossato
(2022)
Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing
eLife 11:e80229.
https://doi.org/10.7554/eLife.80229

Share this article

https://doi.org/10.7554/eLife.80229

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