Rapid genotyping of the M129V polymorphism of prion protein using real-time fluorescent PCR.
The human prion diseases are fatal neurodegenerative disorders that may present as sporadic, hereditary, or infectious processes. The most common is the sporadic form associated with a typical Creutzfeldt-Jakob disease (CJD) phenotype; heritable forms may have, in addition to a CJD phenotype, Gerstmann-Straussler-Scheinker or fatal familiar insomnia phenotypes. The infectious forms may occur as CJD or kuru. It has been proposed that the codon 129 polymorphism in the prion protein gene (PRNP) is largely correlated with phenotype heterogeneity of these diseases (1). This correlation has suggested the use of the polymorphism in the clinicopathologic classification of the sporadic CJD (2).
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Recently, a novel form of human prion disease (nvCJD) that is thought to be linked with bovine spongiform encephalopathy has been described. Almost all cases of nvCJD with available genetic analysis were methionine homozygotes at codon 129 of the PRNP gene and died at an early age (mean, 29 years). This finding suggests that Met/Met homozygosity at codon 129 of the PRNP gene is a risk factor for nvCJD.
The majority of available methods for the study of single-nucleotide polymorphisms and point mutations are time-consuming and require multiple manual steps. To overcome these problems, new and single-tube genotyping methods are being proposed. We describe a rapid and accurate method for the genotyping of the PRNP codon 129 polymorphism based on real-time PCR mutation detection by melting-point analysis with fluorescent hybridization probes with fluorescence resonance energy transfer (3,4).
DNA was extracted from peripheral blood by a phenol-chloroform procedure. The primers and hybridization probes were designed and synthesized by TIB MOLBIOL (Berlin, Germany). The primers used for the amplification were as follows: PRNP Forward (5'-CCAAAAACCAACATGAAGCAC-3') and PRNP Reverse (5'-GTGGTTGTGGTGACCGTGT-3'). The sequence of the sensor 3'-fluorescein-labeled probe was 5'-TTCCCAGCACGTAGCCGCC-3', and that of the anchor 5'-LC-Red 640-labeled probe was 5'-A000CCCCCACCACTGCCCCA-3'.
LightCycler (Roche, Mannheim, Germany) amplification was performed in a final volume of 15 [micro]L containing 1.5 [micro]L of DNA solution of a concentration ~100 ng/[micro]L, 0.75 [micro]L of each primer (10 [micro]mol/L), 0.3 [micro]L of each probe (4 [micro]mol/L), 1.8 [micro]L of Mg[Cl.sub.2] (25 mmol/L), 8.1 [micro]L of distilled water, and 1.5 [micro]L of the DNA Master hybridization probes (Roche). A negative control without DNA was included in all assays. Reaction mixtures were loaded into glass capillaries (Roche), centrifuged, and placed in the LightCycler carousel. The reaction mixture was denatured at 95[degrees]C for 20 s, followed by 35 cycles of denaturation at 95[degrees]C for 0 s, annealing at 55[degrees]C for 5 s, and extension at 72[degrees]C for 10 s. After amplification, the melting was performed by denaturation at 95[degrees]C for 5 s, annealing at 55[degrees]C for 10 s, and increasing the temperature to 90[degrees]C with a ramp rate of 0.2[degrees]C/s. The fluorescence emitted was measured during this process, and the melting curves were generated by plotting fluorescence (F) vs temperature (T) and automatically converted to melting peaks (-dF/dT). The resulting melting peaks allowed the discrimination among different genotypes: the homozygous 129A/A (Met/Met) showed a melting temperature of 61.7[degrees]C, the homozygous 129G/G (Val/Val) presented a peak at 70.5[degrees]C, and the heterozygous genotype 129G/A (Met/Val) showed two peaks at 61.7 and 70.5[degrees]C (Fig. 1). Among 30 local volunteers, 46% were heterozygous Met/Val, 40% were homozygous Met/Met, and 4% were homozygous Val/Val. These genotypes were confirmed in each case by direct sequencing.
We conclude that real-time fluorescence PCR and melting curve analysis are ideal methods to study the PRNP M129V polymorphism. The genotype at this codon is important for the clinicopathologic classification of sporadic CJD, and it also defines risk populations for nvCJD. The method we present here permits in a rapid and accurate way, large population studies.
We thank Olfert Landt (TIB MOLBIOL, Berlin, Germany) for designing the LightCycler hybridization probes. This work was supported by a Project of the Xunta de Galicia, Conselleria de Sanidade (Project 6040.XA44.64200)
(1.) Goldfarb LG, Petersen RB, Tabaton M, Brown P, LeBlanc AC, Montagna P, et al. Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism. Science 1992;258:806-8.
(2.) Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl 0, et al. Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol 1999;46:224-33.
(3.) Wittwer CT, Ririe KM, Andrew RV, David DA, Goundry RA, Balis UJ. The LightCycler [TM]; a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 1997;22: 130-8.
(4.) Lay MJ, Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem 1997;43:2262-7.
Ana Vega *
Unidad de Medicina Molecular-INGO
Universidad de Santiago de Compostela
Hospital de Conxo
15706 Santiago de Compostela, Spain
* Address correspondence to this author at: Unidad de Medicina Molecular, Hospital de Conxo, Rua Ramon Baltar s/n, 15706 Santiago de Compostela, Spain. Fax 34-981-951679; e-mail firstname.lastname@example.org.
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|Author:||Vega, Ana; Ruiz-Ponte, Clara; Carracedo, Angel; Barros, Francisco|
|Article Type:||Letter to the editor|
|Date:||Oct 1, 2001|
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