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.

CNBP基因（CNBP gene）中CCTG微卫星（CCTG microsatellite）的扩增是肌强直性营养不良2型（Myotonic dystrophy type 2, DM2）的致病诱因。该重复序列的长度跨度极大（75至>11000个重复单元）且嵌合性极强，长期以来给DM2患者的分子诊断带来严峻挑战，也阻碍了完全扩增等位基因的测序工作。为突破上述局限，我们建立了无PCR的Cas9介导纳米孔测序（PCR-free Cas9-mediated nanopore sequencing）技术，可实现单核苷酸分辨率（single-nucleotide resolution）下DM2患者CNBP基因重复扩增的精准表征。该方法应用于由9例DM2患者组成的试点队列（n=9），成功准确测定了正常等位基因与扩增等位基因的长度，结果与传统检测方法吻合度良好，同时可识别重复序列嵌合性的存在。值得关注的是，纳米孔测序成功完成了50Kbp全长扩增序列的测序——这一长度此前从未在DM2或其他重复扩增疾病中实现。该方法可准确识别并计数CNBP基因的重复序列模式，涵盖短的间断型与连续型等位基因。有趣的是，在扩增等位基因中，仅2例DM2样本呈现预期的纯CCTG模式，其余7例DM2样本的3'端呈现由TCTG与CCTG模块组成的混合模式——这一现象此前从未在DM2患者中被报道，但通过正交验证方法（orthogonal methods）得到了证实。该方法可通过单次分析同时完成扩增重复序列的长度、结构/基序以及体细胞嵌合水平的测定，有望改善DM2的分子诊断，从而实现更精准的基因型-表型关联（genotype-phenotype correlations），并为临床试验中的DM2患者分层提供更可靠的依据。