title: 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.
authors.affiliation: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, CIBERNED, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: Calero
authors.forename: Rafael
authors.initials: R
authors.lastname: Hortigüela
authors.forename: Carmen
authors.initials: C
authors.lastname: Albo
authors.forename: Jesés
authors.initials: J
authors.lastname: de Pedro-Cuesta
authors.forename: Miguel
authors.initials: M
authors.lastname: Calero
mesh_terms: D000483:Alleles; D001483:Base Sequence; D003062:Codon; D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011110:Polymorphism, Genetic; D017096:Prion Diseases / Q000235:genetics*; D011328:Prions / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D013696:Temperature
chemical_list: D003062:Codon; D017931:DNA Primers; D011328:Prions
references.citation: J Neurol Neurosurg Psychiatry. 2005 Jul;76(7):906-19
references.pmid: 15965195
references.citation: Ann Neurol. 1999 Jun;45(6):812-6
references.pmid: 10360778
references.citation: J Gen Virol. 2009 Mar;90(Pt 3):546-558
references.pmid: 19218199
references.citation: Ann Neurol. 2000 Aug;48(2):269-70
references.pmid: 10939584
references.citation: Curr Opin Genet Dev. 1992 Jun;2(3):448-54
references.pmid: 1504620
references.citation: Science. 1991 Jun 14;252(5012):1515-22
references.pmid: 1675487
references.citation: Ann Neurol. 1992 Mar;31(3):274-81
references.pmid: 1353341
references.citation: Brain Res Mol Brain Res. 1997 Feb;44(1):147-50
references.pmid: 9030710
references.citation: Cell. 1986 Aug 1;46(3):417-28
references.pmid: 2873895
references.citation: Hum Genet. 2005 Nov;118(2):166-74
references.pmid: 16187142
references.citation: Lancet. 1990 Aug 25;336(8713):514-5
references.pmid: 1975028
references.citation: Annu Rev Neurosci. 2008;31:439-77
references.pmid: 18558863
references.citation: Eur J Hum Genet. 2006 Mar;14(3):273-81
references.pmid: 16391566
references.citation: J Neurol Neurosurg Psychiatry. 2005 Nov;76(11):1491-6
references.pmid: 16227536