RESUMO
Pathogenesis and transmission of the prion disorders (transmissible spongiform encephalopathies, TSEs) are mediated by a modified isoform of the prion protein (PrP). Prion protein gene (PRNP) alleles associated with relative susceptibility to TSE have been identified in sheep, humans and possibly elk. Comparable data have not been derived for mule deer, a species susceptible to the TSE chronic wasting disease (CWD). Initial analysis of the open reading frame (ORF) in exon 3 of the mule deer PRNP gene revealed polymorphisms in all 145 samples analyzed, with 10 potential polymorphic sites. Because 144/145 (99.3%) of the samples were heterozygous for a coding change (N/S) at codon 138 (bp 412) and a non-coding polymorphism at bp 418, and individual deer with three or four different alleles were identified a possible gene duplication was indicated. Analysis of BAC clones containing mule deer PRNP genes revealed a full length functional gene and a processed pseudogene. The pseudogene was characteristic of previously described retroelements, in that it lacks introns and is flanked by repeat sequences. Three alleles of the functional gene were identified, with coding changes only at codons 20 (D/G) and 225 (S/F). Determination of PRNP functional gene alleles from 47 CWD-positive mule deer showed the predominant allele encoded 20D225S (frequency 0.85). When alleles were grouped by coding changes in the functional gene, four of the six possible peptide combinations were identified in infected deer. Three pseudogene alleles with coding changes in exon 3 were identified in the mule deer samples examined. Because the TSEs appear to be "protein only" disorders, the presence of an untranslated pseudogene is not expected to affect disease resistance. Therefore, selection of a genotyping method specific for the functional gene is critical for large-scale studies to identify the role of the PRNP gene in susceptibility to CWD in mule deer.
Assuntos
Cervos/genética , Príons/genética , Pseudogenes/genética , Alelos , Animais , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Frequência do Gene , Genes/genética , Heterogeneidade Genética , Íntrons , Dados de Sequência Molecular , Polimorfismo Genético , Análise de Sequência de DNA , Doença de Emaciação Crônica/genéticaRESUMO
A monoclonal antibody dot-blot assay was used to evaluate detergent lysates of tonsil tissue from mule deer to detect PrP(CWD), the marker for the cervid transmissible spongiform encephalopathy chronic wasting disease (CWD). Samples of formalin-fixed brain and tonsil tissues from mule deer were examined for PrP(CWD) using immunohistochemistry (IHC) with Mab F99/97.6.1, the gold standard for diagnosis of preclinical CWD. The contralateral tonsil from each of the 143 deer was prepared for confirmatory IHC and as a 10% (wt/vol) detergent lysate without purification or enrichment steps for monoclonal antibody dot-blot assay. PrP(CWD) was detected by dot-blot assay in 49 of 50 samples considered positive by IHC. Forty-eight of the positive samples were evaluated with a quantitative dot-blot assay calibrated with recombinant PrP. Tonsillar PrP(CWD) concentrations ranged from 34 to 1,188 ng per 0.5 mg starting wet weight of tissue. The abundant PrP(CWD) in mule deer tonsil will facilitate development and validation of high-throughput screening tests for CWD in large populations of free-ranging deer.
