Background: Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (PRNP). DNA-based tests for scoring PRNP codons are essential tools for eradicating scrapie and for evaluating rare alleles for increased resistance to disease. In addition to those associated with scrapie, there are dozens more PRNP polymorphisms that may occur in various flocks. If not accounted for, these sites may cause base-pair mismatching with oligonucleotides used in DNA testing. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of PRNP polymorphisms in targeted flocks.Results: 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.Conclusion: 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.
|Journal||BMC Veterinary Research|
|State||Published - Apr 30 2010|
Bibliographical noteFunding Information:
We thank J. Carnahan for outstanding technical assistance; M. Wallace and the USMARC sheep crew for production and management of sheep. Drs. Min Lee and Jeff Otto for advice in designing synthetic DNA controls and MALDI-TOF MS assays; L. Flathman, R. Godtel, R. Lee, K. Simmerman for expert technical support; C. Adney, P. Anderson, R. Bradley, M. Friend, D. Light, and J. Wray for database and network support; S. Kluver and J. Rosch for secretarial assistance; M. and S. Wintermute and T. and B. Birkeland for invaluable assistance in identifying and providing sheep with ARK haplotypes. Funding for this research was provided by the USDA Agricultural Research Service. Products and company names are necessary to accurately report the methods and results; however, the USDA neither guarantees nor warrants the standard of the product. Use of names by USDA implies no approval of the product to the exclusion of others that may also be suitable.
ASJC Scopus subject areas
- Veterinary (all)