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ARS Home » Research » Publications at this Location » Publication #246682
Title: Ovine Reference Materials and Assays for Prion Genetic Testing

Author
item Heaton, Michael - Mike
item Leymaster, Kreg
item KALBFLEISCH, THEODORE - University Of Louisville
item Freking, Bradley - Brad
item Smith, Timothy - Tim
item Clawson, Michael - Mike
item LAEGREID, WILLIAM - University Of Illinois

Submitted to: BioMed Central (BMC) Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/30/2010
Publication Date: 4/30/2010
Citation: Heaton, M.P., Leymaster, K.A., Kalbfleisch, T.S., Freking, B.A., Smith, T.P., Clawson, M.L., Laegreid, W.W. 2010. Ovine Reference Materials and Assays for Prion Genetic Testing. BioMed Central (BMC) Veterinary Research [serial online]. 6:23. Available: http://www.biomedcentral.com/1746-6148/6/23.

Interpretive Summary: Scrapie is a transmissible degenerative brain disease in sheep. About 1 in 500 mature culled sheep in the U.S. have scrapie, and the disease is always fatal. The signs of scrapie vary widely among individuals and develop slowly. Signs include: scraping and rubbing off wool, wasting, and loss of coordination. Although scrapie is considered a rare disease, eradicating scrapie is important because it affects sheep production, hurts domestic and export markets, and is undesirable in the food chain. DNA tests are able to identify which sheep are most susceptible to scrapie. Genetic testing and selective breeding are key components of scrapie eradication programs worldwide. These programs represent the first widespread use of DNA testing for livestock disease control. The present report describes strategies, tests, and standardized DNA resources for accurate and efficient DNA testing for scrapie susceptibility in any type of sheep population. The DNA and tissue resources include a set of 28 sheep that represent nearly every possible result a laboratory can get when testing for genetic susceptibility to scrapie. These DNAs provide testing laboratories with a quick and easy way to verify the accuracy of their testing procedures. Resources described in this report also include a set of 96 families of sheep from popular U.S. breeds. All of the DNAs, tissues, and information are available for use without restriction.

Technical Abstract: Background: Genetic predisposition to scrapie in sheep is associated with variation in the peptide sequence of the ovine prion protein encoded by Prnp. Codon variants implicated in scrapie susceptibility or disease progression include those at amino acid positions 112, 136, 141, 154, and 171. Nine single nucleotide polymorphisms (SNPs) determine which residues are encoded by the five implicated codons and accurately scoring these SNPs is essential for eradicating scrapie from diverse populations. In addition, the Prnp coding region contains another 36 known polymorphic sites that may cause base-pair mismatching with oligonucleotides used in DNA testing procedures. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of Prnp SNPs in the target population. Results: A DNA sequencing strategy was developed to determine the full-length Prnp coding sequence for any sheep and, thereby, produce a consensus sequence for any population. The strategy was applied to 953 sheep DNAs, including those from two sets of reference sheep (one set for standardizing Prnp genetic testing and another set for discovering SNPs) estimating allele frequencies and determining haplotype phase. DNA sequencing revealed 16 previously unreported SNPs, including a L237P variant on the F141 haplotype. Two mass spectrometry multi-plex assays were developed to score codons 112, 136, 141, 154, and 171. The predicted amino acids encoded at these sites were 100% concordant with those from Sanger sequencing, and non-Mendelian inheritances were not observed in 96 reference families. Tissues, purified DNA, sequence trace files, and genetic information were made publicly available for use without restriction. Conclusion: Identifying the Prnp polymorphisms in local sheep populations is critical for designing efficient scrapie genetic testing systems. Combined with reference DNA panels, these resources facilitate training, certification, and the development of new tests and knowledge that may expedite the eradication of sheep scrapie.