Nguyen Khue Vu, Nyhan William L
Department of Medicine, Biochemical Genetics and Metabolism, The Mitochondrial and Metabolic Disease Center, School of Medicine, University of California, San Diego, Building CTF, Room C-103, 214 Dickinson Street, San Diego, CA, 92103-8467, USA; Department of Pediatrics, University of California, San Diego, School of Medicine, San Diego, La Jolla, CA, 92093, USA.
Department of Pediatrics, University of California, San Diego, School of Medicine, San Diego, La Jolla, CA, 92093, USA.
Neurosci Lett. 2017 Mar 16;643:52-58. doi: 10.1016/j.neulet.2017.02.016. Epub 2017 Feb 10.
The present work is the development of a simple and specific kinetic method based on RT-PCR technique coupled with direct sequencing for quantification of various amyloid precursor protein-mRNA isoforms (APP-mRNA isoforms) in biological samples, especially for identifying the most abundant one that may decisive for the normal status or disease risk. Application of this kinetic method to the Lesch-Nyhan disease (LND) was performed and results indicated an epistasis between mutated hypoxanthine phosphoribosyltransferase1 (HPRT1) and APP genes. APP-mRNA isoform of 624bp, with a deletion starting after 49bp of the 5' end of exon 3 followed by a complete deletion of exons 4-15, mutations in exon 1: c.22C>T, p.L18F, and exon 3: c.269A>G, p.Q90R encoding APP isoform, was the most abundant one in most of the LND patients and would be responsible for the neurobehavioral syndrome in these patients. The method is useful for identifying the defective APP-mRNA isoform in LND patients, and in neurodevelopmental and neurodegenerative disorders in which the APP gene is involved in the pathogenesis of diseases such as autism, fragile X syndrome, amyotrophic lateral sclerosis, and Alzheimer's disease, and may pave the way for new strategies applicable to rational antisense drugs design.
本研究基于逆转录聚合酶链反应（RT-PCR）技术结合直接测序法，开发了一种简单且特异的动力学方法，用于定量生物样品中各种淀粉样前体蛋白信使核糖核酸（APP-mRNA）异构体，特别是用于鉴定对正常状态或疾病风险可能起决定性作用的最丰富的异构体。将这种动力学方法应用于莱施-奈恩病（LND），结果表明突变的次黄嘌呤磷酸核糖转移酶1（HPRT1）和APP基因之间存在上位性。624bp的APP-mRNA异构体，在第3外显子5'端49bp后开始缺失，随后第4-15外显子完全缺失，第1外显子中的突变：c.22C>T，p.L18F，以及第3外显子中的突变：c.269A>G，p.Q90R编码APP异构体，是大多数LND患者中最丰富的异构体，并且是这些患者神经行为综合征的原因。该方法有助于鉴定LND患者中缺陷的APP-mRNA异构体，以及鉴定APP基因参与疾病发病机制的神经发育和神经退行性疾病，如自闭症、脆性X综合征、肌萎缩侧索硬化症和阿尔茨海默病，并且可能为适用于合理反义药物设计的新策略铺平道路。
