A sequencing strategy for identifying variation throughout the prion gene of BSE-affected cattle
© Clawson et al; licensee BioMed Central Ltd. 2008
Received: 18 March 2008
Accepted: 23 June 2008
Published: 23 June 2008
Classical and atypical bovine spongiform encephalopathies (BSEs) are cattle prion diseases. Distinct bovine prion gene (PRNP) alleles have been associated with classical and atypical BSE susceptibility. However, the full extent of PRNP allele association with BSE susceptibility is not known. A systematic sequence-based genotyping method that detects variation throughout PRNP would be useful for: 1) detecting rare PRNP alleles that may be present in BSE-affected animals and 2) testing PRNP alleles for an association with either classical or atypical BSE susceptibility.
We improved a Sanger-based sequencing strategy for detecting bovine PRNP variation through all exons, introns, and part of the promoter (25.2 kb). Our current method can detect 389 known and other potentially unknown PRNP polymorphisms that may be present in BSE-affected cattle. We determined PRNP genotypes for the first U.S. BSE case and her sire. Previously unknown PRNP polymorphisms were not detected in either animal and all PRNP genotypes support the sire-daughter relationship.
The methodologies described here characterize variation throughout PRNP. Consequently, rare PRNP alleles that may be present in BSE-affected cattle can be detected.
Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a class of neurodegenerative disorders that occur in humans, ruminants, cats, and mink . Three distinct TSEs afflict cattle: classical bovine spongiform encephalopathy (BSE), atypical H-type BSE, and atypical L-type BSE [2–4]. Bovine prion gene (PRNP) alleles are associated with classical and atypical BSE susceptibility [5–8]. However, the extent of bovine PRNP alleles that predict BSE susceptibility has not yet been determined.
Classical BSE is acquired by cattle through the consumption of meat and bone meal contaminated with the infectious prion agent . Classical BSE susceptibility is associated with the deletion alleles of two bovine PRNP insertion/deletion polymorphisms, one within the promoter region and the other in intron 1 [5–7]. Both of these polymorphisms have alleles in linkage disequilibrium (LD) with the alleles of at least 43 other PRNP polymorphisms in Bos taurus cattle . Thus, additional PRNP alleles are likely to associate with classical BSE susceptibility with comparable if not greater significance than the indel alleles.
Atypical BSEs have recently been identified in Asian, North American, and European cattle . Two PRNP alleles are implicated with atypical BSE susceptibility. A non-synonymous polymorphism (E211K) was found by one of us (J.A.R.) within the prion coding region of an H-type atypical BSE case identified in the U.S. (2006). The E211K polymorphism is homologous to the human E200K polymorphism, a risk factor for genetic Creutzfeldt-Jakob disease , and is the suspected cause of the atypical BSE case. The 211 K allele is exceedingly rare and has not been found in other atypical BSE cases or healthy cattle [10, 13, 14]. However, a PRNP haplotype that is associated with atypical BSE susceptibility was found in H- and L-type atypical cases from Canada, France, and the U.S., and may have widespread involvement with atypical BSE . The haplotype spans part of PRNP intron 2, the entire coding region of exon 3, and part of the three prime untranslated region of exon 3 (13 kb) [8, 10]. Alleles that may be causative for atypical BSE susceptibility, including those that may be within PRNP, are thought to be linked with the implicated haplotype in atypical BSE cases .
Efforts to identify PRNP alleles that predict classical or atypical BSE susceptibility require improved methods to: 1) efficiently extract DNA from available tissue samples and 2) identify variation throughout PRNP. The brainstem obex is the specified sample for BSE testing . If an obex sample is BSE positive and the carcass from which it originated is destroyed before additional samples are collected, the obex becomes the sole source of transmissible material and DNA from the BSE-afflicted animal. Thus, the ability to obtain DNA from obex tissue is a critical prerequisite for PRNP genotyping. The ability to accurately genotype known PRNP polymorphisms and identify additional PRNP polymorphisms that may be present in BSE-afflicted animals is also important. Here, we provide an efficient method of DNA extraction from bovine obex and report a Sanger-based sequencing strategy that comprehensively identifies variation throughout PRNP (25.2 kb). We determined the PRNP sequence and haplotypes of the U.S. case of classical BSE (2003) and her sire, show that both animals do not have previously unrecognized PRNP variation, and that their parent-offspring relationship is supported throughout PRNP.
DNA isolation method
Cattle obex DNA yield and quality
DNA Yield (μg)
DNA concentration (μg/mL)b
Development of oligonucleotides for PRNPamplification and sequence genotyping
Additional PRNP variation is predicted to exist in cattle and some of it could affect the hybridization of oligonucleotides within the set. This likelihood is accounted for by overlapping PRNP amplicons coupled with overlapping PRNP nucleotide coverage by sequencing oligonucleotides (Figure 2). Thus, the set will facilitate the detection of both known and unknown PRNP polymorphisms, including those within primer hybridization sites.
Complete PCR coverage of PRNP (25.2 kb) from a BSE-positive animal can be conducted in a single 96-well plate using a thermocycler capable of zoned heating cycles (see additional file #4 for a 96-well PCR design). Two 55-μL PCRs for each of the 24 PRNP amplicons yield enough template to sequence PRNP at least twice with each of the 189 oligonucleotides on an ABI capillary 3730 sequencer. This coverage can yield approximately eight-fold coverage of each PRNP nucleotide base throughout the 25.2-kb region, except for several small problematic regions (Figure 2). Each amplification reaction requires 50 ng of DNA. Consequently, thorough coverage of PRNP can be achieved with 2.4 μg of genomic DNA. All of the sequencing reactions required for duplicate coverage of PRNP can fit on a single 384-well plate.
Complete PRNPsequences of the U.S. classical BSE case of 2003 and her sire
The oligonucleotide set was used to amplify and sequence PRNP from: 1) the U.S. case of classical BSE (2003), a Holstein cow imported from Canada, and 2) her Holstein sire. All sequence nucleotides were scored with Phred, aligned with Phrap, edited in Consed, and genotyped with Polyphred software . Unambiguous genotypes for 388 PRNP polymorphisms were scored for the U.S. BSE case and her sire (see additional file #5 for a complete list of genotypes and GenBank files EU130450 and EU130451). Sequences from both animals did not reveal any previously unknown PRNP polymorphisms. Both animals are homozygous for the deletion alleles of the two InDels previously associated with classical BSE susceptibility. All of the PRNP polymorphism genotypes support the sire-offspring relationship.
PRNPhaplotypes of the BSE case and her sire
PRNP haplotypes of the classical BSE case and her sire were compared to each other and to those from 86 U.S. Holsteins, of which 82 were part of the USMARC Dairy Cattle Panel (MDCP1.5)  and 4 were part of the USMARC Beef Cattle Discovery Panel 2.1 (MBCDP2.1) . The classical BSE case and her sire were both homozygous for a haplotype in network one that is common in U.S. Holstein (haplotype A, Figure 3). The two InDel deletion alleles that associate with BSE susceptibility are present on this haplotype. However, while the sire was homozygous for a common PRNP haplotype in network two (haplotype b), the classical BSE case was heterozygous for the same haplotype and one much less common (haplotype e). The frequencies of haplotype combinations "Ab" and "Ae" in the Holstein DNAs are 0.264 and 0.030, respectively. The significance of these haplotype combinations and classical BSE susceptibility is unknown. PRNP haplotype combinations such as those described here can be tested for association with either classical or atypical BSEs in larger studies.
BSE has grown in complexity with the identification of classical and atypical types. PRNP alleles associate with classical and atypical BSE susceptibility. The methodologies described here characterize variation throughout PRNP. Consequently, rare PRNP alleles that may be present in BSE-affected cattle can be detected.
We thank Gennie Schuller-Chavez for outstanding technical support; Dr. Mohammad Koohmaraie for coordinating the collection of cattle obex samples at a Nebraska slaughter plant; Drs. Shuna Jones and Dee Griffin for collecting the obex samples; Tammy Sorensen and Jacky Carnahan for technical support; Drs. Jeff Gawronski and Dan Nonneman for critically reviewing the manuscript; and Joan Rosch for secretarial support. This research was supported by the USDA National Research Initiative, Competitive Grant No. 2005-35212-15890 (MLC, MPH, WWL) and the Agricultural Research Service (all authors).
- Prusiner SB: Prions. Proc Natl Acad Sci U S A. 1998, 95: 13363-13383. 10.1073/pnas.95.23.13363.PubMed CentralView ArticlePubMedGoogle Scholar
- Wells GA, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M, Dawson M, Bradley R: A novel progressive spongiform encephalopathy in cattle. Vet Rec. 1987, 121: 419-420.View ArticlePubMedGoogle Scholar
- Biacabe AG, Laplanche JL, Ryder S, Baron T: Distinct molecular phenotypes in bovine prion diseases. EMBO Rep. 2004, 5 (1): 110-114. 10.1038/sj.embor.7400054.PubMed CentralView ArticlePubMedGoogle Scholar
- Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, Monaco S, Caramelli M: Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci U S A. 2004, 101 (9): 3065-3070. 10.1073/pnas.0305777101.PubMed CentralView ArticlePubMedGoogle Scholar
- Sander P, Hamann H, Pfeiffer I, Wemheuer W, Brenig B, Groschup MH, Ziegler U, Distl O, Leeb T: Analysis of sequence variability of the bovine prion protein gene (PRNP) in German cattle breeds. Neurogenetics. 2004, 5 (1): 19-25. 10.1007/s10048-003-0171-y.View ArticlePubMedGoogle Scholar
- Juling K, Schwarzenbacher H, Williams JL, Fries R: A major genetic component of BSE susceptibility. BMC Biol. 2006, 4: 33-10.1186/1741-7007-4-33.PubMed CentralView ArticlePubMedGoogle Scholar
- Haase B, Doherr MG, Seuberlich T, Drogemuller C, Dolf G, Nicken P, Schiebel K, Ziegler U, Groschup MH, Zurbriggen A, Leeb T: PRNP promoter polymorphisms are associated with BSE susceptibility in Swiss and German cattle. BMC Genet. 2007, 8: 15-10.1186/1471-2156-8-15.PubMed CentralView ArticlePubMedGoogle Scholar
- Clawson ML, Richt JA, Baron T, Biacabe AG, Czub S, Heaton MP, Smith TPL, Laegreid WW: Association of a bovine prion gene haplotype with atypical BSE. PLoS ONE. 2008, 3(3): e1830-10.1371/journal.pone.0001830.PubMed CentralView ArticleGoogle Scholar
- Wilesmith JW, Ryan JB, Atkinson MJ: Bovine spongiform encephalopathy: epidemiological studies on the origin. Vet Rec. 1991, 128: 199-203.View ArticlePubMedGoogle Scholar
- Clawson ML, Heaton MP, Keele JW, Smith TPL, Harhay GP, Laegreid WW: Prion gene haplotypes of U.S. cattle. BMC Genet. 2006, 7: 51-10.1186/1471-2156-7-51.PubMed CentralView ArticlePubMedGoogle Scholar
- Ducrot C, Arnold M, de Koeijer A, Heim D, Calavas D: Review on the epidemiology and dynamics of BSE epidemics. Vet Res. 2008, 39: 15-10.1051/vetres:2007053.View ArticlePubMedGoogle Scholar
- Kovacs GG, Puopolo M, Ladogana A, Pocchiari M, Budka H, van Duijn C, Collins SJ, Boyd A, Giulivi A, Coulthart M, Delasnerie-Laupretre N, Brandel JP, Zerr I, Kretzschmar HA, de Pedro-Cuesta J, Calero-Lara M, Glatzel M, Aguzzi A, Bishop M, Knight R, Belay G, Will R, Mitrova E: Genetic prion disease: the EUROCJD experience. Hum Genet. 2005, 118 (2): 166-174. 10.1007/s00439-005-0020-1.View ArticlePubMedGoogle Scholar
- Heaton MP, Leymaster KA, Freking BA, Hawk DA, Smith TPL, Keele JW, Snelling WM, Fox JM, Chitko-McKown CG, Laegreid WW: Prion gene sequence variation within diverse groups of U.S. sheep, beef cattle, and deer. Mamm Genome. 2003, 14 (11): 765-777. 10.1007/s00335-003-2283-y.View ArticlePubMedGoogle Scholar
- Heaton MP, Keele JW, Harhay GP, Richt JA, Koohmaraie M, Wheeler TL, Shackelford SD, Casas E, King DA, Sonstegard TS, Van Tassell CP, Neibergs HL, Chase CC, Kalbfleisch TS, Smith TP, Clawson ML, Laegreid WW: Prevalence of the prion protein gene E211K variant in U.S. cattle. BMC Vet Res. 2008, 4: 25-10.1186/1746-6148-4-25.PubMed CentralView ArticlePubMedGoogle Scholar
- USDA: Procedures Manual Bovine Spongiform Encephalopathy (BSE) Ongoing Surveillance Plan. 2006, U.S. Department of Agriculture Animal and Plant Health Inspection Service Veterinary ServicesGoogle Scholar
- Heaton MP, Chitko-McKown CG, Grosse WM, Keele JW, Keen JE, Laegreid WW: Interleukin-8 haplotype structure from nucleotide sequence variation in commercial populations of U.S. beef cattle. Mamm Genome. 2001, 12: 219-226. 10.1007/s003350010269.View ArticlePubMedGoogle Scholar
- Stephens M, Sloan JS, Robertson PD, Scheet P, Nickerson DA: Automating sequence-based detection and genotyping of SNPs from diploid samples. Nat Genet. 2006, 38 (3): 375-381. 10.1038/ng1746.View ArticlePubMedGoogle Scholar
- Stephens M, Smith NJ, Donnelly P: A new statistical method for haplotype reconstruction from population data. Am J Hum Genet. 2001, 68: 978-989. 10.1086/319501.PubMed CentralView ArticlePubMedGoogle Scholar
- Stephens M, Scheet P: Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. Am J Hum Genet. 2005, 76: 448-462. 10.1086/428594.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.