Cytogenet Genome Res 102:85–88 (2003) DOI: 10.1159/000075730 Identification of a novel ovine PrP polymorphism and scrapie-resistant genotypes for St. Croix White and a related composite breed C.M. Seabury and J.N. Derr Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station TX (USA) Abstract. Susceptibility to scrapie is primarily controlled by a region which exhibits extreme conservation across mammali- polymorphisms in the ovine prion protein gene (PRNP). Here, an taxa. The relatively high frequency (0.75) of resistant ARR we report a novel ovine exon three PRNP polymorphism (SNP alleles and the absence of ARQ alleles for the SCW ewes used as G346C; P116), its association with the ovine ARQ allele breeding stock for CMP resulted in significant genic differenti- (P116 A136 R154 Q171 ), and two new genotypes (PARQ/ARR; ation (P = 0.0123; S.E. = 0.00113). Additionally, the majority PARQ/ARQ) for the St. Croix White (SCW) breed and a relat- of the SCW (66.7%) and CMP (65.4%) sampled possessed ed composite (CMP) breed developed for meat production. genotypes considered resistant or nearly resistant to scrapie and The (P116 ) polymorphism occurs between the N-terminal cleav- experimental BSE (bovine spongiform encephalopathy. age site and the hydrophobic region of the ovine prion protein, Copyright © 2003 S. Karger AG, Basel Scrapie is an inevitably fatal transmissible spongiform en- A136 R154 R171 (hereafter ARR), ARQ, VRQ, AHQ, and ARH cephalopathy (TSE) affecting sheep and goats. Polymorphisms (Belt et al., 1995). It should also be noted that seven additional within exon three of the ovine host-encoded prion protein gene ovine PRNP polymorphisms, exhibiting little or no association (PRNP) at codons 136 (Alanine or Valine; A,V), 154 (Histidine with the phenotypic expression of scrapie, have been described or Arginine; H,R), and 171 (Glutamine, Arginine, or Histidine; at codons 112, 127, 137, 138, 141, 151, and 211 (as referenced Q, R, or H) are associated with variation in the phenotypic in Bossers et al., 2000). High susceptibility to scrapie is associ- expression of scrapie including incubation period, clinical ated with the ovine VRQ allele, while the ARR allele is associ- signs, and pathology (Bossers et al., 1996, 2000; reviewed ated with resistance (Westaway et al., 1994; Belt et al., 1995; by Hunter, 1997). Of the twelve possible alleles derivable Hunter et al., 1996; Sabuncu et al., 2003). The AHQ allele may from these polymorphisms, only five are commonly seen: be associated with resistance in some ovine breeds, but not oth- ers, while the ARH allele is likely to be neutral (Dawson et al., 1998; Baylis et al., 2002a). In the U.S. sheep population scrapie has only been con- firmed in sheep homozygous for the PRNP allele encoding glu- tamine at codon 171 (Q/Q), regardless of breed (Westaway et al., 1994; O’Rourke et al., 1996, 1997, 2002). Moreover, the Supported by Texas A&M University and the Texas Agricultural Experimnent Sta- ovine PRNP genotype ARR/ARR is known to confer global tion. resistance to scrapie and experimental BSE (for review of geno- Received 25 May 2003; manuscript accepted 30 July 2003. types see Belt et al., 1995; Hunter, 1997; Baylis et al., 2002a, Request reprints from James N. Derr, Department of Veterinary Pathobiology 2002b). The ARR/AHQ and ARR/ARQ genotypes are associ- College of Veterinary Medicine, Texas A&M University College Station, TX 77843-4467 (USA); telephone: +1 979-862-4775 ated with nearly complete resistance to scrapie worldwide as fax: +1 979-845-9972; e-mail: [email protected] well as incubation periods of 15 years following intracerebral Fax + 41 61 306 12 34 © 2003 S. Karger AG, Basel Accessible online at: ABC E-mail [email protected] 0301–0171/03/1024–0085$19.50/0 www.karger.com/cgr www.karger.com challenge (IC) with BSE (Foster et al., 2001; Jeffrey et al., 2001; Table 1. PRNP allele frequencies and observed genotype frequencies Baylis et al. 2002a, 2002b). The ARQ/ARQ genotype is gener- Breeda PRNPb Total Frequency Genotype (obs.) Total Frequency ally associated with increased risk of scrapie worldwide al- allele % though some breeds (e.g. Cheviot Sheep, UK) are relatively resistant (Baylis et al., 2002b). Sheep possessing the ARQ/VRQ WD ARQ 2 1.0000 ARQ/ARQ 1 100 WD Sum 2 1.0000 1 100 genotype are at high risk of scrapie and experimental BSE (Bay- lis et al., 2002b). The ARR/VRQ genotype, somewhat variable SCW ARR 9 0.7500 ARR/ARR 3 50.0 ARQ 0 0.0000 P116ARQ/ARR 1 16.7 by breed, is generally associated with rare to slightly elevated VRQ 1 0.0833 ARR/AHQ 1 16.7 risk of scrapie as well as incubation periods of 15 years follow- AHQ 1 0.0833 ARR/VRQ 1 16.7 ARH 0 0.0000 ing IC with BSE (Belt et. al., 1995; Foster et al., 2001; Jeffrey et c P116ARQ 1 0.0833 al., 2001; Baylis et al., 2002a, 2002b). SCW Sum 12 1.0000 6 100 The St. Croix White (SCW) and White Dorper (WD) breeds CMP ARR 22 0.4231 ARR/ARR 4 15.4 are members of a larger group of sheep commonly referred to as ARQ 23 0.4423 ARR/ARQ 12 46.2 VRQ 2 0.0385 P ARQ/ARQ 4 15.4 hair sheep or hair breeds. Collectively, hair breeds make up a 116 AHQ 1 0.0192 ARQ/ARQ 3 11.5 relatively small portion of the overall world sheep population ARH 0 0.0000 ARR/AHQ 1 3.8 c and as a result have often escaped studies pertaining to scrapie P116ARQ 4 0.0769 ARR/VRQ 1 3.8 or the PRNP locus in general, yet they are the predominant ARQ/VRQ 1 3.8 CMP Sum 52 1.0000 26 100 breed type found throughout the Caribbean and other tropical regions (Shelton, 1991; Godfrey and Collins, 1999). Addition- a WD, White Dorper; SCW, St. Croix White; CMP, composite breed. b ally, hair breeds are commonly utilized in tropical regions Ovine PRNP exon 3. c Alanine is the wild-type amino acid at ovine position 116. worldwide for meat production and are valued for their resis- tance to Trichostrongyle (Mazzola, 1990; Godfrey and Collins 1999). In this study we investigated exon three PRNP geno- types and allelic variants for SCW as well as a related composite breed (CMP) developed for commercial meat production. at 96 °C; 35 cycles × 30 s at 96 ° C, 20 s at 54 ° C, 4 min at 60° C; 5 min at 60 °C. Each 10-Ìl sequencing reaction included: 60 ng purified PCR product, 2 Ìl Big DyeTM, 0.8 ÌM primer and 0.5× MasterAmpTM PCR Enhancer. Materials and methods Reactions were purified with G-50 sephadex columns (Biomax, Odenton, MD). Sequence fragments were separated and analyzed using an ABI 3100 Study animals Genetic Analyzer (Applied Biosystems, Foster City, CA), and are available A total sampling of 33 sheep from Dorpcroix Sheep Farm (Hermleigh, through GenBank accession no. (AY350241–AY350275). TX USA) consisted of the following: six unrelated adult SCW (ewes) pre- viously utilized as breeding stock for CMP, one full-blooded adult WD (ram) Validation techniques utilized as breeding stock for CMP, and 26 CMP (20 adult ewes, three ewe- Most samples were directly sequenced more than once. Representative lambs, and three adult rams). Composite animals (26 of n = 500 total farm) alleles from each genotypic class with more than one single nucleotide poly- were developed for commercial meat production in 1998 and represent a morphism (SNP) were validated through cloning using a TOPO TA Cloning synthetic breed resulting from an initial cross (SCW ewes × WD rams) fol- kit according to the manufacturer’s recommendations (Invitrogen, Carlsbad lowed by selection and crossing of animals exhibiting economically impor- CA). Plasmid DNA was isolated and purified using a Qiagen Plasmid Mini tant traits such as overall hardiness and robust body stature. The WD and Kit as directed by the manufacturer (Qiagen Inc., Valencia CA). Insert SCW sampled do not represent the sole founding stock for CMP. Study ani- sequencing for 12 clones was carried out via the sequencing method pre- mals had no previous history or symptoms of scrapie at the time of publica- viously described with the following exceptions: 400 ng/reaction plasmid tion. DNA, 50 °C anneal temperature, and (6.2 pmol/reaction) M13 forward and reverse primers. DNA isolation and PRNP amplification Genomic DNA was isolated from whole blood samples either by spotting Computer software and analysis whole blood on Whatman Bioscience FTA® Classic Cards, and following the Ovine PRNP exon three genotypes and allelic variants were visualized preparation protocol provided by the manufacturer (Whatman Inc., Clifton, using ABI PRISM SeqScape SNP Discovery and Validation Software ver- NJ), or through utilization of the SUPER QUICK-GENE DNA Isolation kit sion 1.01 (Applied Biosystems, Foster City, CA). Allele frequencies and tests (Analytical Genetic Testing Center, Denver, CO). of genic differentiation were calculated in GENEPOP (Raymond and Rous- The entire coding region for exon three of the ovine PRNP gene was set, 1995). amplified via PCR with the flanking synthetic oligonucleotides SAF1 and SAF2 (Prusiner et al., 1993). Thermal cycling parameters, as optimized in our laboratory, were as follows: 2 min at 96 °C; 4 cycles × 30 s at 96 °C, 30 s at 58 °C (–1 °C/cycle), 90 s at 65 °C; 31 cycles × 30 s at 96 ° C, 30 s at 54 °C, Results and discussion 90 s at 65 ° C; 15 min at 65 ° C.