Scholarlink

<p>Abnormal expansion of nucleotide repeats was first identified 34 years ago as a unique mutational mechanism. It is now linked to numerous neurogenetic disorders, several of which discovered only recently. The identification of these expansions has led to various classifications based on clinical presentation, repeat nature and genomic location (coding or non-coding regions). Precise diagnosis of these conditions relies on molecular testing, currently performed on a gene-by-gene basis. Their analysis remains challenging, especially for long expansions. We evaluated CRISPR-Cas9-mediated target enrichment coupled to Oxford Nanopore Technologies (ONT) long read sequencing, to accelerate and improve the time-consuming molecular diagnosis of repeat expansion disorders. We simultaneously targeted nine loci involved in 10 repeat expansion disorders in a single capture panel, including <i>FMR1</i>, <i>HTT</i>, <i>DMPK</i>,<i> CNBP/ZNF9</i>,<i> ATXN2</i>,<i> JPH3</i>,<i> FXN</i>,<i> C9ORF72</i> and <i>RFC1</i>, covering a broad range of repeat types, sizes and diagnostic needs. Results were compared with standard routine testing methods. ONT sequencing using Flongle flow cells yielded results consistent with standard techniques for most loci, particularly for non-complex repeats. However, limitations were observed for structurally complex regions such as <i>RFC1</i>, and inter-run variability required the aggregation of multiple Flongle runs per sample to achieve robust genotyping. These findings highlight both the potential and current limitations of CRISPR-Cas9-enriched ONT sequencing for multiplex diagnosis of repeat expansion disorders in a clinical setting. The approach deserves further development, particularly optimisation of protocols, inclusion of larger sample sizes, and comparison with alternative technologies.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Towards routine genetic testing of repeat expansions in neurogenetic diseases using multiplex CRISPR-Cas9-targeted long read sequencing

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年8月3日

查看全文
  • Patricia Fergelot,
  • Christophe Boury,
  • Benjamin Penaud,
  • Anne-Gaelle Giguet-Valard,
  • Marie-Claire Vincent,
  • Kevin Mouzat,
  • Virginie Raclet,
  • Marie-Pierre Baudier,
  • Caroline Rooryck,
  • Benoit Arveiler,
  • Rémi Bellance,
  • Claire Guissart,
  • Cécilia Marelli,
  • Olivier Lepais,
  • Cyril Goizet,
  • Giovanni Stevanin

摘要

Abnormal expansion of nucleotide repeats was first identified 34 years ago as a unique mutational mechanism. It is now linked to numerous neurogenetic disorders, several of which discovered only recently. The identification of these expansions has led to various classifications based on clinical presentation, repeat nature and genomic location (coding or non-coding regions). Precise diagnosis of these conditions relies on molecular testing, currently performed on a gene-by-gene basis. Their analysis remains challenging, especially for long expansions. We evaluated CRISPR-Cas9-mediated target enrichment coupled to Oxford Nanopore Technologies (ONT) long read sequencing, to accelerate and improve the time-consuming molecular diagnosis of repeat expansion disorders. We simultaneously targeted nine loci involved in 10 repeat expansion disorders in a single capture panel, including FMR1, HTT, DMPK, CNBP/ZNF9, ATXN2, JPH3, FXN, C9ORF72 and RFC1, covering a broad range of repeat types, sizes and diagnostic needs. Results were compared with standard routine testing methods. ONT sequencing using Flongle flow cells yielded results consistent with standard techniques for most loci, particularly for non-complex repeats. However, limitations were observed for structurally complex regions such as RFC1, and inter-run variability required the aggregation of multiple Flongle runs per sample to achieve robust genotyping. These findings highlight both the potential and current limitations of CRISPR-Cas9-enriched ONT sequencing for multiplex diagnosis of repeat expansion disorders in a clinical setting. The approach deserves further development, particularly optimisation of protocols, inclusion of larger sample sizes, and comparison with alternative technologies.