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Allelic discrimination of genetic human prion diseases by real-time PCR genotyping
Olga Calero1, Rafael Hortigüela, Carmen Albo
1Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, CIBERNED, Instituto de Salud Carlos III, Madrid, Spain.
Abstract:
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.
Insights
Accurately characterizing human prion diseases requires analyzing PRNP gene mutations and the M129V polymorphism. New, rapid, and cost-effective real-time PCR methods simplify this genetic analysis for better diagnosis and classification.
Area of Science:
- Neurogenetics
- Molecular Biology
- Medical Diagnostics
Background:
- Human genetic prion diseases necessitate comprehensive molecular characterization for accurate clinical diagnosis and epidemiological classification.
- The PRNP gene's pathogenic mutations and the polymorphic codon 129 (M129V) status are crucial, as M129V influences susceptibility and disease phenotype.
- The phenotypic expression of common PRNP mutations like D178N and E200K is significantly modulated by the M129V polymorphism.
Purpose of the Study:
- To develop simple, fast, and cost-effective protocols for resolving cis-trans ambiguities between PRNP mutations and the M129V polymorphism.
- To provide high-throughput methods suitable for routine genetic analysis in clinical and research settings.
Main Methods:
- Development of two distinct protocols utilizing allele-specific primers and Real-time PCR.
- SYBR Green dye was employed for monitoring amplification in real-time.
- Differential amplification strategies were designed to distinguish the co-occurrence of specific mutations with M129V alleles.
Main Results:
- Successful implementation of two protocols for differentiating mutations (D178N, E200K) in relation to the M129V polymorphism.
- Demonstrated simplicity, speed, and cost-effectiveness compared to traditional methods like cloning.
- Validated applicability for other codon 129-dependent mutations, such as P102L.
Conclusions:
- The developed real-time PCR protocols offer an efficient and accessible method for the molecular characterization of genetic prion diseases.
- These methods facilitate precise genetic analysis, aiding in improved diagnosis and understanding of genotype-phenotype correlations.
- The protocols are adaptable for broader application in characterizing other PRNP mutations influenced by the M129V polymorphism.
