Cas9-enriched nanopore sequencing identifies repeat expansions in undiagnosed cerebellar ataxia
Investigators developed an amplification-free Cas9-targeted nanopore sequencing platform paired with a dedicated analysis algorithm to detect short tandem repeat expansions in patients with cerebellar ataxia. Testing genomic DNA from 37 genetically undiagnosed individuals revealed pathogenic repeat expansions in 12 patients (32.4%) across multiple loci including FGF14, RFC1, and NOP56, with cascade screening confirming additional affected relatives. The workflow also captured CpG methylation patterns, suggesting that methylation status may modulate disease severity. This integrated nCATS-STRiker approach provides molecular laboratories with a robust alternative to conventional PCR and short-read sequencing for resolving complex repeat expansion disorders.
The original study
Optimized Cas9-Enriched Nanopore Sequencing and Analysis Workflow for Clinical Diagnosis of Repeat Expansion Disorders.
- Authors
- Lee S, Jung C, Kim M, Kim N, Hwang GH, Lee ST, et al.
- Journal
- Advanced science (Weinheim, Baden-Wurttemberg, Germany)
- Type
- Journal Article
- PMID
- 42667167
Original abstract
Short tandem repeat (STR) expansion is a major genetic mechanism underlying numerous neurogenetic disorders. However, traditional PCR amplification and short-read next-generation sequencing-based methods often fail to detect large-scale, complex expansions and to capture methylation information. Thus, this study aimed to modify an amplification-free nanopore Cas9-targeted sequencing (nCATS) platform to achieve uniform coverage across 56 currently defined STR loci using a single test with genomic DNA from patient-derived blood cells and to develop a dedicated analysis algorithm, STRiker, capable of identifying internal motif contexts and de novo repeat structures. Ultimately, this study identified pathogenic repeat expansions in 12 of 37 patients (32.4%) with cerebellar ataxia who remained genetically undiagnosed despite extensive prior genetic testing, in FGF14 (n = 4), ATXN8OS, NOP56, RFC1 (n = 2 each), and PRNP and NOTCH2NLC (n = 1 each). Additionally, family-based cascade screening revealed six relatives with repeat expansions in five families. These results demonstrate a broader diversity of pathogenic repeat structures, particularly in FGF14, and illustrate that CpG methylation can mitigate the pathogenic effects of repeat expansions. This nCATS-STRiker workflow offers a powerful strategy for improving the diagnosis of STR-related neurogenetic diseases, such as cerebellar ataxia and other diseases.