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首页> 外文期刊>Journal of Neuroscience Methods >High capacity and low cost detection of prion protein gene variant alleles by denaturing HPLC.
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High capacity and low cost detection of prion protein gene variant alleles by denaturing HPLC.

机译：通过变性HPLC对病毒蛋白基因变异等位基因进行高容量和低成本检测。

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摘要

Mutations in the human prion protein gene (PRNP) are responsible for hereditary diseases called transmissible spongiform encephalopathies (TSE) and a polymorphic site at codon 129 determines sensitivity to infectious forms of these maladies. More recently, codon 129 has been related to cognition performance in the elderly, in Alzheimer disease (AD) and in Down syndrome. Furthermore, a rare polymorphism at codon 171 was described in 23% of patients with mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS), the most common form of surgically remediable epileptic syndrome. Thus, a method that permits fast and efficient screening of PRNP mutations and polymorphisms in patients, in high risk populations, and in family members is desirable. In the present study, we established the conditions for analysis of the PRNP open reading frame using denaturing high-performance liquid chromatography (DHPLC), whereby unpurified PCR products were subjected to denaturing and reannealing steps leading to heteroduplex formation. We described specific profiles for the PRNP polymorphisms at codons 129 (M/V), 117 (A/A silent), 219 (E/K), 171 (N/S), and the octarepeat deletion using amplified DNA from 562 samples. The chromatograms for TSE-associated mutations at codons 102 (P/L), 183 (T/A), and 210 (V/I) were also determined. Specificity of the DHPLC profile for each PRNP variant allele was confirmed in 100% of the samples by direct and cloned DNA sequencing in addition to endonuclease digestion when applicable. Therefore, the present study shows that DHPLC is a rapid, highly accurate and efficient technique for the detection of PRNP genetic variants.
机译：人类病毒蛋白基因（PRNP）中的突变与遗传性疾病有关，称为遗传性传染性海绵状脑病（TSE），第129位密码子的多态性位点决定了这些疾病的传染性敏感性。最近，密码子129与老年人，阿尔茨海默氏病（AD）和唐氏综合症的认知能力有关。此外，在23％的与海马硬化症（MTLE-HS）有关的中颞叶癫痫患者中发现了一种罕见的171位密码子多态性，海马硬化症是外科可治的癫痫综合症的最常见形式。因此，需要一种允许快速，有效地筛查患者，高危人群和家庭成员中PRNP突变和多态性的方法。在本研究中，我们建立了使用变性高效液相色谱（DHPLC）分析PRNP开放阅读框的条件，未纯化的PCR产物经过变性和再退火步骤导致异源双链体形成。我们描述了使用来自562个样本的扩增DNA在129个密码子（M / V），117个（A / A沉默），219个（E / K），171个（N / S）和八边形缺失处PRNP多态性的具体概况。还确定了密码子102（P / L），183（T / A）和210（V / I）处与TSE相关的突变的色谱图。除适用时，除核酸内切酶消化外，还通过直接和克隆的DNA测序方法在100％的样品中证实了每种PRNP变异等位基因的DHPLC图谱的特异性。因此，本研究表明DHPLC是检测PRNP遗传变异的快速，高度准确和高效的技术。

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