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

Abstract

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.