Characterization of full-length CNBP expanded alleles in DM2 patients by Cas9-mediated enrichment and nanopore sequencing

Expansion of a CCTG microsatellite in the CNBP gene is causative of the Myotonic dystrophy type 2 (DM2). The extreme length of the repeat (from 75 to >11.000 units) and its strong mosaicism deeply challenge the molecular diagnosis of DM2 patients and hampered the sequencing of fully expanded alleles so far. To overcome such limitations, we have setup a PCR-free Cas9-mediated nanopore sequencing for the characterization, at single-nucleotide resolution, of CNBP repeat expansions in DM2 patients. The approach was applied in a pilot group of DM2 patients (n=9) where it allowed to properly assess the length of both normal and expanded alleles, in good agreement with traditional methods, and to recognize the presence of repeat mosaicism. Of note, nanopore allowed to sequence through a full expansion of 50Kbp, a dimension never achieved before, nor for DM2 or other repeat-expansion disorders. The approach properly identified and counted the repeat pattern characterizing the CNBP gene, for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only 2 DM2 samples showed the expected pure CCTG pattern, while the other 7 DM2 samples presented at the 3 prime end a mixed pattern consisting of TCTG and CCTG blocks, never described so far in DM2 patients, but confirmed hereby with orthogonal methods. Allowing the determination of expanded repeat length, structure/motif and level of somatic mosaicism in a single analysis, the approach has the potential to improve DM2 molecular diagnosis, thus leading to more accurate genotype-phenotype correlations and better stratification of DM2 patient in clinical trials.