Prion gene (PRNP) haplotype variation in United States goat breeds (Open Access publication)

Scrapie eradication efforts cost 18 million dollars annually in the United States and rely heavily upon PRNP genotyping of sheep. Genetic resistance might reduce goat scrapie and limit the risk of goats serving as a scrapie reservoir, so PRNP coding sequences were examined from 446 goats of 10 breeds, 8 of which had not been previously examined at PRNP. The 10 observed alleles were all related to one of two central haplotypes by a single amino acid substitution. At least five of these alleles (M142, R143, S146, H154, and K222) have been associated with increased incubation time or decreased odds of scrapie. To the best of our knowledge, neither S146 nor K222 has been found in any goats with scrapie, though further evaluation will be required to demonstrate true resistance. S146 was more common, present in several breeds at widely varying frequencies, while K222 was observed only in two dairy breeds at low frequency. Overall, this study provides frequency data on PRNP alleles in US goats, shows the pattern of relationships between haplotypes, and demonstrates segregation of multiple scrapieassociated alleles in several breeds not examined before at PRNP.


INTRODUCTION
Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Sequence changes in the PRNP gene that encodes prion protein have been associated with differential resistance and susceptibility to scrapie.
In particular, sheep have substitutions in amino acids 136, 154, and 171 with well-established allelic and genotypic effects on resistance to common scrapie strains [11]. This is consistent with genetic relationships to a wide variety of TSEs, including kuru and sporadic Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and chronic wasting disease in elk and deer [3,[12][13][14]18,19]. Scrapie eradication programs worldwide involve breeding sheep based upon the most resistant alleles, and scrapie prevalence has been falling using such measures [16].
This study examined sequence variants in PRNP present in 10 breeds of US goats; 8 of these breeds had never been examined at PRNP. In addition, the present study used allele discovery and frequency information to assess the potential for selective breeding of goats to augment scrapie eradication efforts.

Animals
Blood was collected from 10 breeds of goats commonly found in the United States: Alpine, Angora, Boer, LaMancha, Myotonic (Tennessee Fainting Goat), Nubian, Oberhasli, Pygmy, Saanen, and Toggenburg. Approximately 30 private producers and university herds were solicited either directly or at goat shows for samples of US animals. When more than one animal was included from the same herd, producers and/or pedigree records assured the animals were unrelated.

Genotyping
DNA was isolated from 600 lL aliquots of blood using the PureGene Kit (Gentra Systems, Minneapolis, MN), following the manufacturer's protocols.

Statistical analysis
Haplotypes were analyzed using PHASE v2 [22,23], with a 95% confidence threshold for haplotype predictions for individual animals. If a haplotype had not been previously reported, a PCR product was cloned to verify the predicted haplotype. Linkage disequilibrium was assessed using Haploview [4]. Simultaneous confidence intervals for allele frequencies were calculated using a published macro program [17] in SASÒ v9.1 (SASÒ Institute, Cary, NC).

RESULTS
Ten polymorphisms were observed in caprine PRNP, including eight that encode amino acid substitutions and two silent polymorphisms. The amino acid substitutions in codons 127, 142, 143, 146, 154, 211, 222, and 240 and their haplotypic configurations are shown in Table I. The silent polymorphisms were PRNP variation in US goats a G-A substitution in the third nucleotide positions of codon 42 and a T-C substitution in the third nucleotide position of codon 138 as previously described [8], and they were in tight linkage disequilibrium with the amino acid substitution in codon 240 (D' 0.988 and 0.983, respectively). All polymorphisms with at least 5% minor allele frequency within breed were found to be in agreement with Hardy-Weinberg proportions within breed.
The haplotype distributions are shown by breed with 95% confidence intervals in Table II (online). More than 75% of animals had haplotype confidence scores of 1, indicating known haplotypes with no prediction necessary. All the remaining animals had confidence scores of at least 95% for haplotype assignment computed by PHASE from population genotype frequency data using established methods [22,23]. Haplotype 5 was verified by cloning and sequencing since it had not been observed in previous reports, and the clones verified the haplotype predictions from PHASE. Haplotype 1 was the only haplotype observed in every breed examined. Haplotype 2 was common in all breeds except the Toggenburg and Myotonic breeds. Haplotypes 3, 4, 5, 8, 9, and 10 were observed only within dairy breeds.

DISCUSSION
Natural scrapie is found in both goats and sheep, and since goats and sheep often cohabitate, goats may serve as a scrapie reservoir for sheep. This is especially concerning since goats can have more subtle scrapie symptoms than sheep [6,21,25]. Sheep PRNP genetic testing has greatly aided sheep scrapie eradication efforts in the US. Goat monitoring efforts will be phased into the US scrapie eradication program, and this study addressed the question of whether goat genetics can be used as a tool in such efforts. As a first step, this study identified 10 different PRNP haplotypes and their frequencies in 10 goat breeds. At least two variants have never been found in goats with scrapie. Three other variants have been associated with scrapie impact but not complete resistance, and breed differences were observed for all five scrapie-associated alleles. Ten haplotypic alleles were observed in this sample of US goats with relationships suggesting a simple evolutionary history. Figure 1 shows two central haplotypes, and all other alleles related to one of these two by only a single amino acid substitution. The high frequency of these two haplotypes and their central position in the haplotype diagram suggest that the other alleles arose by mutation on the background of these two central haplotypes. The one exception is haplotype 5, which has been experimentally verified by cloning. Haplotype 5 probably arose by recombination to combine the M142 and S240 variants on one haplotype (see Fig. 1), and to the best of our knowledge this is the first report of a recombinant PRNP haplotype in goats. Several haplotypes were only observed in dairy breeds, indicating low levels of historical gene flow from the dairy breeds into other breed types.
The S146 and K222 alleles (found on haplotypes 7 and 10, respectively) are especially promising since relatively large numbers of animals with each allele PRNP variation in US goats 557 have been present in scrapie-exposed herds but no scrapie positive animal has been reported with even a single copy of either allele [1,20,24]. The S146 allele was a relatively common minor allele at 12.3% frequency overall, and it was found in 7 of 10 breeds examined including breeds used primarily for dairy, meat, and other purposes. This study added Alpine, LaMancha, Nubian, Myotonic, and Pygmy goats to the list of breeds in which S146 has been observed. In these US goats, S146 was always the minor allele, but its within breed frequency ranged from 35.2% (Boer) to 1.4% (Alpine). In contrast, the K222 allele was only observed in two breeds at low frequencies (5.4% in Toggenburg and 1.0% in LaMancha). This study adds these two breeds to the list of known carriers of K222.
Three other PRNP variants have been associated with incomplete scrapie resistance and were observed in US goats. The M142 variant has been associated with extended scrapie incubation time [8] and had previously been found in Saanen and mixed-breed dairy goats [1,7,8,15]. The M142 variant (observed on haplotypes 4 and 5) was present at 6.7% frequency overall and was found only in dairy animals. These results added Alpine, Oberhasli, Toggenburg, and LaMancha as known breeds in which this variant has been observed, with frequencies ranging from 43.2% in Toggenburgs to 4.0% in LaMancha goats. Also, both R143 and H154 were observed in US goats (on haplotypes 6 and 8, respectively) and have been associated with reduced scrapie [2,5,24], but not with complete resistance. This study demonstrates for the first time segregation of R143 in Nubian, Pygmy, and Angora goats, and confirms a previous finding of segregation in Boer goats [27]. This study also adds Saanen goats as a breed in which H154 is known to segregate.
In summary, this study documents the presence of 10 PRNP alleles in US goats and shows the relationships between segregating haplotypes. Eight of the ten breeds examined had not previously been examined for PRNP allelic variation, and several scrapie-associated alleles were found to segregate in these breeds. However, additional data are necessary to demonstrate full genetic scrapie resistance before genetic approaches to goat scrapie can serve as a useful adjunct to current efforts in scrapie eradication.

ONLINE MATERIAL
The supplementary file (Tab. II) supplied by the authors is available at: http://www.gse-journal.org. Table II. PRNP haplotype frequencies with 95% confidence intervals for alleles by breed.