Optimized Cas9‐Enriched Nanopore Sequencing and Analysis Workflow for Clinical Diagnosis of Repeat Expansion Disorders

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.