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

Amplification-free nanopore Cas9-targeted sequencing across 56 short tandem repeat loci, with a dedicated algorithm identifying internal motif contexts, de novo repeat structures and CpG methylation.

Short tandem repeat expansions frequently escape PCR amplification and short-read sequencing, which detect neither large or complex expansions nor methylation information. The authors adapted an amplification-free nanopore Cas9-targeted sequencing platform to achieve uniform coverage across the 56 currently defined loci in a single test from blood-derived genomic DNA, and developed a dedicated algorithm, STRiker, able to identify internal motif contexts and de novo repeat structures. Among 37 patients with cerebellar ataxia who remained genetically undiagnosed despite extensive prior testing, pathogenic expansions were identified in 12 (32.4%): FGF14 (n = 4), ATXN8OS, NOP56 and RFC1 (n = 2 each), and PRNP and NOTCH2NLC (n = 1 each). Family-based cascade screening revealed six relatives with repeat expansions across five families, and the results suggest that CpG methylation can mitigate the pathogenic effects of expansions.

Synthesis written by Geno'X. For the full original abstract, please refer to the source publication.

A third of diagnoses among already extensively investigated patients is the kind of figure that justifies placing targeted long-read early in the ataxia pathway rather than as a last resort after conventional testing is exhausted. The most instructive point is the diversity of FGF14 repeat structures: a test that only measures expansion size loses part of the information, and that is exactly where the algorithm contributes. Caveat: a small, selected cohort, with no prospective comparison against the standard strategy and no specificity figures.

Analysis by Dr Thibaut Benquey