Ovine reference materials and assays for prion genetic testing

An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U.S. populations. The samples included two sets of reference sheep: one set for standardizing PRNP genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F141 haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U.S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction.Identifying ovine PRNP polymorphisms in targeted flocks is critical for designing efficient scrapie genetic testing systems. Together with reference DNA panels, this information facilitates training, certification, and development of new tests and knowledge that may expedite the eradication of sheep scrapie.Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurological disorders of humans and other mammals that are characterized by accumulation of an abnormal, protease-resistant isoform of the prion protein in the brain. Naturally occurring prion diseases may have acquired, inherited, or sporadic origins (i.e., no known environmental or genetic cause). TSE outbreaks have arisen in several species and include Creutzfeldt-Jakob disease (CJD) and kuru in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronic wasting disease in deer and elk, feline spongiform encephalopathy in cats, and transmissible mink encephalopathy in farmed mink (for