Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing
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- Massimiliano Alfano
- Department of Biotechnology, University of Verona
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- Luca De Antoni
- Department of Biotechnology, University of Verona
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- Federica Centofanti
- Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata
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- Virginia Veronica Visconti
- Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata
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- Simone Maestri
- Department of Biotechnology, University of Verona
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- Chiara Degli Esposti
- Department of Biotechnology, University of Verona
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- Roberto Massa
- Department of Systems Medicine (Neurology), University of Rome Tor Vergata
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- Maria Rosaria D'Apice
- Laboratory of Medical Genetics, Tor Vergata Hospital
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- Giuseppe Novelli
- Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata
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- Massimo Delledonne
- Department of Biotechnology, University of Verona
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- Annalisa Botta
- Department of Biomedicine and Prevention, Medical Genetics Section, University of Rome Tor Vergata
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- Marzia Rossato
- Department of Biotechnology, University of Verona
抄録
<jats:p>Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the <jats:italic>CNBP</jats:italic> 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 <jats:italic>CNBP</jats:italic> 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.</jats:p>
収録刊行物
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- eLife
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eLife 11 2022-08-26
eLife Sciences Publications, Ltd