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在患者 iPSC 中C9orf72重复扩张的单分子测序

Yu-Chih Tsai¹, Katherine A Brown^2,3, Mylinh T Bernardi⁴

¹Pacific Biosciences, Menlo Park, CA, USA.

|September 16, 2024

概括

一种新的PacBio测序方法准确地检测和大小重复扩张在C9orf72基因中,这是ALS和FTD的关键原因,没有放大. 这一突破改善了这些神经退行性疾病的基因测试.

科学领域:

遗传学是一个遗传学.
神经科学是一个神经科学.
分子生物学分子生物学

背景情况:

在C9orf72基因中GGGGCC重复扩张是肌缩侧面硬化症 (ALS) 和前性痴呆症 (FTD) 的主要遗传原因.
目前的诊断方法,如远程PCR和南方斑,不准确,缺乏检测这些扩张的灵敏度.
C9orf72位点的重复性和GC丰富性阻碍了基于放大的测序方法.

研究的目的:

开发一种新的,无放大方法,用于准确检测和测量C9orf72重复扩张.
克服C9orf72相关ALS和FTD诊断现有技术的局限性.

主要方法:

使用PacBio单分子测序与CRISPR/Cas9向丰富策略相结合.
从患者衍生的诱导多能干细胞 (iPSC) 中分离出高分子量基因组DNA.
使用CRISPR/Cas9切除了C9orf72重复区域,并使用绑定适配器对目标区域进行了测序,而无需PCR放大.

主要成果:

开发的方法准确和一致地大小C9orf72重复扩展,包括那些有数千次重复.
在患者衍生的iPSC中成功应用,并适用于来自各种细胞类型,血液或组织的DNA.
在没有放大的情况下实现了C9orf72重复区域的丰富,克服了以前的测序障碍.

结论:

使用CRISPR/Cas9丰富的PacBio单分子测序为大小C9orf72重复扩张提供了一种敏感和准确的方法.
这种非放大方法为C9orf72相关的ALS和FTD的临床和研究诊断提供了重大进展.
该协议可适应各种样本类型,并与特定的PacBio Sequel平台兼容.

关键词:

C9orf7272 是一个很好的方法.没有放大,没有放大.重复扩张的扩张.单分子测序的测序方法对于 iPSCs 来说,这是一个很好的选择.

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