Allelic discrimination of genetic human prion diseases by real-time PCR genotyping

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.

Figures and Tables

Figure 1 PRNP genotypes vs Ct (M129-178/200 wildtype, circles; M129-178/200 mutant, squares; V129-178/200 wildtype, triangles and V129-178/200 mutant, rhombus and negative controls, NC).

Figure 2 Amplification plots (cycle vs ΔRn) in a StepOne Real-Time PCR System of the different PRNP genotypes (M129-178/200 wildtype in red, M129-178/200 mutant in green, V129-178/200 wildtype in blue and V129-178/200 mutant in magenta). ΔRn is an indicator of the magnitude of the signal generated by the PCR reaction.

Figure 3 Performance of the allelic discrimination protocol for the M129V-D178N genetic background as a function of the initial DNA amount vs Ct (M129-178 wildtype, circles; M129-178 mutant, squares; V129-178 wildtype, triangles; V129-178 mutant, rhombus).

Figure 4 Performance of the allelic discrimination protocol for the M129V-E200K genetic background as a function of the initial DNA amount vs Ct (M129-200 wildtype, circles; M129-200 mutant, squares; V129-200 wildtype, triangles; V129-200 mutant, rhombus).

Table 1 Nucleotide sequence and characteristics of the primers used for PRNP genotyping

Table 2 Reaction mixtures and lengths of the amplification products expected for PRNP genotyping