Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, CIBERNED, Instituto de Salud Carlos III, Madrid, Spain.
Prion. 2009 Jul-Sep;3(3):146-50. doi: 10.4161/pri.3.3.9339. Epub 2009 Jul 23.
The complete molecular characterization of human genetic prion diseases from different backgrounds is important for clinical diagnosis and epidemiological classification. The characterization of the PRNP gene should always include the description of the pathogenic mutation, as well as the status at each allele of the polymorphic codon 129 (M129V), a well-established susceptibility marker and phenotypic variability factor for different types of human prion diseases. Indeed, the phenotypical expression of two of the most common mutations in the human PRNP gene associated with genetic prion diseases, D178N and E200K, is clearly modulated by the codon 129 polymorphism. Here, we describe two simple, fast, cost-effective and suited for high-throughput protocols to resolve cis-trans ambiguities between these mutations respect the M129V polymorphism. This methodology is based on differential amplification by allele-specific primers using Real-time PCR monitored by SYBR Green dye. The main advantages of these protocols are their relative simplicity and the reduced cost compared to other methods such as cloning protocols, and that it may be readily applicable to the characterization of other mutations with codon 129-dependent expression, e.g., P102L.
从不同背景下对人类遗传朊病毒病进行完整的分子特征分析，对于临床诊断和流行病学分类非常重要。PRNP 基因的特征分析应始终包括致病性突变的描述，以及多态性密码子 129 （M129V）每个等位基因的状态，该密码子是不同类型人类朊病毒病的一个既定的易感性标志物和表型变异性因素。事实上，与遗传性朊病毒病相关的人类 PRNP 基因中两种最常见突变（D178N 和 E200K）的表型表达明显受到 M129V 多态性的调节。在这里，我们描述了两种简单、快速、具有成本效益且适合高通量的方案，可以解决这些突变与 M129V 多态性之间的顺式-反式歧义。该方法基于等位基因特异性引物的实时 PCR 监测 SYBR Green 染料的差异扩增。这些方案的主要优点是相对简单，与克隆方案等其他方法相比成本降低，并且易于应用于其他依赖 M129 表达的密码子的突变的特征分析，例如 P102L。
