检测和验证神经退行性病变患者中的 MAPT 突变，并分析外显子剪接的后果。

Detecting and Validating MAPT Mutations in Neurodegeneration Patients and Analysis of Exon Splicing Consequences.

机构信息

Brain and Mind Centre and School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Camperdown, NSW, Australia.

New South Wales Brain Tissue Resource Centre, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Camperdown, NSW, Australia.

出版信息

Methods Mol Biol. 2024;2754:411-433. doi: 10.1007/978-1-0716-3629-9_22.

DOI:10.1007/978-1-0716-3629-9_22

PMID:38512679

Abstract

Mutation of MAPT has been observed in patients with parkinsonism, progressive supranuclear palsy, and corticobasal degeneration and is a significant cause of frontotemporal dementia. In this chapter, we discuss considerations for next-generation sequencing analysis to identify MAPT mutations in patient genomic DNA and describe the validation of these mutations by Sanger sequencing. One of the most common effects of MAPT mutations is differential splicing of exon 10, which leads to an imbalance in the proportion of 3-repeat and 4-repeat tau isoforms. We describe how to investigate the effect of novel DNA variants on the splicing efficiency of this exon in vitro using the exon-trapping technique, also known as the splicing reporter minigene assay.

摘要

MAPT 突变已在帕金森病、进行性核上性麻痹和皮质基底节变性患者中被观察到，是额颞叶痴呆的重要病因。在本章中，我们讨论了用于鉴定患者基因组 DNA 中 MAPT 突变的下一代测序分析的注意事项，并描述了通过 Sanger 测序对这些突变进行验证的方法。MAPT 突变最常见的影响之一是外显子 10 的差异剪接，导致 3 重复和 4 重复 tau 异构体比例失衡。我们描述了如何使用外显子捕获技术（也称为剪接报告基因 minigene 测定）在体外研究新型 DNA 变异对该外显子剪接效率的影响。