1 US Department of Agriculture, Agricultural Research Service, Animal Disease Research Unit, 3003 ADBF, Pullman, WA 99164, USA
2 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA
3 Colorado State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Colorado State University, Fort Collins, CO, USA
Correspondence
Katherine I. O'Rourke
korourke{at}vetmed.wsu.edu
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ABSTRACT |
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INTRODUCTION |
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The TSEs are a diverse family of fatal neurodegenerative diseases characterized by accumulation of PrPd, an abnormal disease-specific isoform (Prusiner, 1982) of the normal host protein PrPc (Basler et al., 1986
). Transmission and pathogenesis of the TSEs appear to involve a novel mechanism in which PrPd, a major protein component in tissue extracts from infected animals, catalyses the conversion of PrPc to the pathogenic PrPd molecule through a series of aggregation and post-translational secondary changes (Caughey et al., 1990
; Horiuchi & Caughey, 1999
; Safar et al., 1994
). The protein only model is supported by experiments demonstrating that expression of PrPc in the host is a necessary prerequisite for disease (Bueler et al., 1993
) and that the primary amino acid sequence of PrPc is associated with relative susceptibility to natural TSE infection in some species. Residues 136, 154 and 171, alone or in combination, control relative susceptibility and incubation time in sheep (Belt et al., 1995
; Bossers et al., 1996
; Clouscard et al., 1995
; Goldmann et al., 1994a
, b
; Hunter et al., 1992
, 1994
; O'Rourke et al., 1997
; Westaway et al., 1994
). Predisposing genotypes in elk with CWD (O'Rourke et al., 1999
) and in humans with iatrogenic, sporadic and variant CreutzfeldtJakob disease (Collinge et al., 1991
, 1996
; Palmer et al., 1991
) are associated with mutations at a single site (elk codon 132 and the corresponding human codon 129). Genetic analysis of susceptible or predisposing prion protein precursor gene (PRNP) alleles in mule deer is complicated by the nearly universal presence of an unexpressed processed pseudogene (PRNP
) in which exon 3 encodes a polymorphic open reading frame (ORF) (Brayton et al., 2004
). In this study, we examined the PRNP functional gene and pseudogene alleles in a herd of white-tailed deer with a high prevalence of naturally occurring CWD to investigate the possibility of predisposing genotypes in this species.
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METHODS |
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PCR and DNA sequence analysis.
DNA was extracted from frozen brain tissue using a commercial kit (Q-Biogene) following the manufacturer's instructions. Primer pairs specific for amplification of PRNP and PRNP were derived from the corresponding sequences from mule deer (GenBank accession nos AY330343 and AY371694). Intronexon borders were derived from the sequences of bovine PRNP cDNA and genomic DNA (GenBank accession nos AB001468 and AJ298878) and cDNA from mule deer (K. I. O'Rourke, unpublished data). PCR amplification was performed using a universal prion primer pair amplifying an 800 bp fragment including the entire ORF in exon 3 of PRNP and PRNP
(forward primer 247 5'-atggtgaaaagccacataggcag-3', beginning with the initiation site for PrP translation and reverse primer 224 5'-agaagataatgaaaacaggaag-3', beginning 8 bp after the termination codon); a primer pair specific for the functional gene (forward primer 223 5'-acaccctctttattttgcag-3' in intron 2 and reverse primer 224), yielding an 830 bp product; and two primer pairs specific for the pseudogene (forward primer 379 5'-aagaaaattcctgagagagcat-3' containing the pseudogene flanking repeat sequence and the adjacent 8 bp from exon 1 or forward primer 369 5'-caaccaagtcgaagcatct-3' from exon 2; reverse primer 224 was used in both cases). Amplification of DNA from deer with PRNP
yielded a product of 950974 bp with primers 379/224 and 875899 bp with primers 369/224, depending on the octapeptide repeat number. Although primers 369/224 annealed to PRNP and to PRNP
, the intervening intron in PRNP (deleted in PRNP
) precluded efficient amplification of the functional gene and therefore the primer pair selectively amplified PRNP
under the conditions used. All PCR reactions were as follows: 95 °C for 5 min, followed by 30 cycles of denaturation (95 °C, 30 s), annealing (54 °C, 30 s) and extension (72 °C, 59 s) followed by an extension cycle (72 °C, 7 min) under standard buffer conditions with 2·5 mM MgCl2 (Qiagen). PCR products were analysed on 1 % agarose ethidium-bromide-stained gels. PRNP
amplification reactions were scored for the presence or absence of a pseudogene-specific band.
PCR products were purified by Exo/SAP (USB Corporation) to remove unincorporated dNTPs and primers, then sequenced on an ABI Prism 377 DNA Sequencer with Big Dye Terminator chemistry (PE-Applied Biosystems) using forward primer 245 (5'-gccaaccgctatccacctca-3') and reverse primer 12 (5'-ggtggtgactgtgtgttgcttga-3') (Amplicon Express). PCR products were cloned for sequencing of each PRNP allele and all PRNP products using a TOPO TA cloning system (Invitrogen) according to the manufacturer's instructions and sequenced with primers T3 and T7 (MWG Biotech). In this study, PRNP alleles were identified by the deduced amino acids at positions 95 (glutamine, Q, or histidine, H), 96 (glycine, G, or serine, S), 116 (alanine, A, or glycine, G) and 138 (serine, S, or asparagine, N), sites of the three coding changes in the functional gene, and the PRNP/PRNP
marker at codon 138 of Odocoileus spp. (S in PRNP and N in PRNP
). PRNP
alleles were identified by the number of octa/nonapeptide repeat units (five or six).
CWD diagnosis.
CWD was diagnosed by monoclonal antibody immunohistochemistry as described previously (Spraker et al., 2002a). Deer were considered CWD-positive if PrPd was detected in the dorsal motor nucleus of the vagus nerve (DMNV), the tonsil or the retropharyngeal lymph node. Animals were considered to be in early disease if the tonsil or retropharyngeal node was positive but no immunostaining was evident in the DMNV (Spraker et al., 2002b
). Animals were considered to be in late disease if PrPd was detected in brain as well as lymphoid tissue. Deer with no detectable PrPd in brain or nodes were considered No PrPd. These animals were not considered CWD-free because of the long undefined incubation period between infection and accumulation of detectable PrPd.
Statistical analysis.
Frequencies of CWD associated with specific PRNP alleles were assessed by 2 analysis of contingency tables, using Yates continuity correction where appropriate (for comparisons with a single degree of freedom). Confidence intervals were calculated for odds ratios of CWD in the major genotypes detected in the study population, using the presumed wild-type genotype QGAS QGAS as the reference genotype (Armitage & Berry, 1994
).
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RESULTS |
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The frequencies of CWD differed among deer with the five major diploid genotypes (i.e. the first five rows of Table 2) (
2=12·2, 4 df, P=0·016). The other genotypes were too rare for meaningful analysis. The strongest association detected was between CWD occurrence and carriage of the QGAS allele (
2=11·3, 1 df, P<0·001). Deer either haploid (
2=6·52, 1 df, P=0·01) or diploid (
2=9·36, 1 df, P<0·01) for the QGAS allele were more frequently affected by CWD compared with deer lacking this allele. Correspondingly, a negative association was observed between CWD and carriage of one or two QSAS alleles (
2=6·98, 2 df, P<0·05). No association was detected between carriage of a PRNP pseudogene allele and CWD (
2=0·003, P>0·05). Sample sizes were too small for analysis of all genotypes. Odds ratios and 95 % confidence intervals (CI) for the occurrence of CWD in the most frequently occurring diploid genotypes, using the presumed wild-type allele QGAS homozygous deer as the reference group, are shown in Table 2
. In spite of the small sample size, the 95 % CI showed a decrease in the odds ratio for deer with the QSAS allele and lacking the QGAS allele when compared with deer with the presumed wild-type QGAS homozygous genotype. Homozygous QSAS deer had a lower odds ratio (0·089, 95 % CI 0·0160·502) than did heterozygous QSAS QGGS deer (0·167, CI 0·0350·789). The presence of the QGAS allele resulted in similar odds ratios for heterozygous QSAS (0·564, 95 % CI 0·2121·501) or QGGS (0·444, 95 % CI 0·1331·489) deer.
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DISCUSSION |
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The ORF of the PRNP gene was more variable in this group of deer than was reported previously for free-ranging elk (O'Rourke et al., 1999) or mule deer (Brayton et al., 2004
) and was duplicated in the cervid prion pseudogene PRNP
as described previously in mule deer (Brayton et al., 2004
). The presumed wild-type allele (QGAS) and three alleles with coding changes at codons 95 (H), 96 (S) or 116 (G) were identified in the functional gene. In addition, a coding change at codon 138 distinguished PRNP (138S) from PRNP
(138N) in this sample set. Both the location and potential charge shift of the three amino acid replacements in the functional gene are of interest. The H/Q polymorphism at position 95 results in substitution of a neutral for a polar residue but is commonly reported in the deduced amino acid sequences from the PRNP gene among and within a large number of species (Schätzl et al., 1995
; Van Rheede et al., 2003
; Wopfner et al., 1999
). Residue 96, encoding G or S, is adjacent to the final octapeptide repeat in a region poorly conserved among the mammalian species examined, with insertions and single base changes reported (Van Rheede et al., 2003
). Although there was no evidence for resistance to disease in deer with any of the major genotypes in this study, deer lacking the QGAS allele and carrying one or two copies of the allele encoding 96S (QSAS) were less likely to be found in the CWD population than deer with the QGAS allele on one or both chromosomes. This finding is consistent with data from deer in Wisconsin (Johnson et al., 2003
), although in that study universal prion primers were used and the functional genotype could not be determined for some animals from the data shown. The sample sizes in both studies are relatively small (26 in Wisconsin, 67 in this study). If larger sample sizes support these observations, however, the pattern of genetic susceptibility in white-tailed deer would be similar to that observed in Rocky Mountain elk, in which a predisposing genotype is reported (O'Rourke et al., 1999
), although all major genotypes are susceptible to CWD (Spraker et al., 2004
). In contrast, PRNP polymorphisms in sheep result in the lack of disease in virtually all sheep with resistant genotypes (Belt et al., 1995
; Bossers et al., 1996
; Clouscard et al., 1995
; Goldmann et al., 1994a
, b
; Hunter et al., 1992
, 1994
, 1997
; O'Rourke et al., 1997
; Westaway et al., 1994
).
The polymorphism at codon 116 of the functional gene is of interest. The alanine (A) residue at this position is conserved across a large number of eutherian orders (Van Rheede et al., 2003) and previously reported to be variant only in the tenrec, an insectivorous mammal found only on the island of Madagascar. Residue 116 (human 113) is in a potential membrane-spanning domain (Hegde et al., 1998
) of PrPc, immediately adjacent to a proposed cleavage site for cytosolically derived protease but protected from proteolysis by its insertion through the membrane of the endoplasmic reticulum (Hegde et al., 1998
). The A
G mutation at residue 116 in white-tailed deer results in replacement of a hydrophobic, aliphatic residue with a polar residue and may affect the intracellular processing and transport of the PrP precursor. However, in this study, the 116G allele was found in 13 CWD-positive heterozygous deer, demonstrating that the allele does not provide protection under these conditions. Two 116G homozygous animals were identified (one adult and one fawn), arguing against an early lethal effect of the mutation. Neither homozygous deer had detectable PrPd or neurodegenerative changes.
The cervid prion pseudogene in white-tailed deer in this study appeared at low frequency in comparison with the pseudogene in O. hemionus, the mule deer (Brayton et al., 2004) and black-tailed deer (subspecies of O. hemionus) (unpublished data). PRNP
was characterized by a polymorphism not detected in the functional gene and heterogeneity was limited to a variable octapeptide repeat number. The low frequency and lack of heterogeneity of the white-tailed deer pseudogene could be due to inbreeding within this confined herd, to polymorphisms in the primer binding sites resulting in detection of a limited subset of alleles or to a relatively recent introduction of the pseudogene into the genome of white-tailed deer. Although the founder group in the study herd was estimated at fewer than 20 animals, loss of heterogeneity in the pseudogene due to inbreeding is an unlikely explanation because all functional alleles identified in the Wisconsin (Johnson et al., 2003
), Mississippi (unpublished data) and Wyoming (Heaton et al., 2003
) groups are represented in this herd. Furthermore, using the 138N polymorphism as a marker for PNRP
in the Wisconsin and Wyoming studies, pseudogene frequencies are similar among the groups. Polymorphisms in the PCR primer-binding sites cannot be ruled out. However, the pseudogene was detected with two primer pairs in all deer in which the 138N mutation was detected with the universal prion primer pair, suggesting that failure to amplify a novel PRNP
allele is not likely. The third explanation is the appearance of the pseudogene in O. hemionus (mule deer and other black-tailed deer) after divergence from white-tailed deer, with limited and relatively recent introgression into the white-tailed deer genome through hybridization of ancestral sympatric populations. Although the evolution and taxonomy of the dozens of subspecies of white-tailed and black-tailed deer in the Americas are complex, examination of mitochondrial DNA and Y-linked genes supports the model that white-tailed deer and black-tailed deer diverged on the North American continent during the Pleistocene, with subsequent hybridization of sympatric populations resulting in appearance of mule deer (reviewed by Geist & Francis, 1999
). Subsequent hybridization between mule deer and white-tailed deer probably occurred (and continues to occur) infrequently, with a low level of introgressive hybridization of maternal DNA from mule deer and black-tailed deer into white-tailed deer populations within limited geographic areas (Cronin, 1991
).
The PRNP alleles in this study and the previous study of mule deer (Brayton et al., 2004
) invariably encoded 138N and an expansion of the octapeptide repeat number was observed in some deer. These changes were not seen in the functional gene, leaving unresolved the question of whether the changes occurred in the ancestral functional gene before the duplication event or in the processed pseudogene after duplication. The substitution of N for S in the prion protein is common within and among species and functional alleles encoding 138N are common in fallow deer (Dama dama) and reindeer (Rangifer tarandus) (unpublished data). Likewise, a variable octapeptide repeat number is common in some breeds of cattle. There are several dozen subspecies of Odocoileus in the Americas and genetic examination of additional populations should demonstrate whether the changes noted in the pseudogene in this study are characteristic of the processed pseudogene or are found in functional alleles as well. No evidence of the pseudogene was found in small populations of New World moose (Alces alces), holoarctic reindeer, Old World Rocky Mountain elk (C. elaphus) or captive Asian fallow deer (unpublished data). If PRNP
arose after evolutionary radiation of Odocoileus species in the New World, this pseudogene is not likely to be found in more distantly related members of the order Artiodactyla such as sheep, cattle or goats. Novel pseudogenes may yet be found in some populations of domestic livestock through judicious selection of primer pairs optimized for pseudogene discovery. However, processed pseudogenes are not typically transcribed or translated (Mighell et al., 2000
) and are therefore not expected to affect susceptibility to a protein-based disorder.
All major PRNP diploid genotypes were represented in the CWD group in this study. We addressed the possibility of genetically controlled differences in incubation time (the period from infection until accumulation of detectable PrPd in the brain) by examining fawns (animals under 1 year of age) for late disease (PrPd in the brain). Using this crude measure, we identified three fawns (QGAS QSAS and QGAS QGGS genotypes) with late disease, demonstrating an extremely rapid course in those animals. PrPd is thought to accumulate first in the lymphoid tissues of the alimentary tract (Sigurdson et al., 1999), with subsequent neuroinvasion and transport through the splenic or vagus nerves to the brain. In experimental rodent models, the incubation time and neuroinvasion rate are associated with a number of variables, including PRNP polymorphisms (Dickinson & Outram, 1973
), innervation and PrPc production in lymphoid tissues (Mabbott et al., 2000
; Glatzel et al., 2001
), and route of infection (Bartz et al., 2003
). Transgenic mice with cervid PRNP genes may be an appropriate laboratory model for examining the extremely rapid neuroinvasion seen in some genotypes.
CWD in mule deer is typically associated with weight loss, ataxia, hypersalivation, polyuria and polydipsia (Williams & Miller, 2002) and similar signs have been reported in captive white-tailed deer held in research settings (M. W. Miller, personal communication). In spite of an infection rate of nearly 50 % in this herd, no clinical evidence of CWD was noted in live deer by the property owner or hunters or at necropsy. Losses to intercurrent viral diseases were extensive during some years and the terrain provided adequate cover for sick animals to evade detection prior to death, possibly masking the presence of a TSE in this herd. Alternately, the clinical course in these white-tailed deer may have been more acute than described in mule deer due to differences in the biology of the cervid host, the strain of TSE agent in this herd or the dose of the disease-causing agent in this confined facility. Additional observations on clinical CWD in white-tailed deer held in captive settings will be necessary to address the spectrum of phenotypes of TSE infection in cervids.
PRNP alleles and diploid genotypes of white-tailed deer with CWD identify those associated with CWD susceptibility. In contrast, disease resistance is considerably more challenging to define in free-ranging populations for several reasons. First, equivalent exposure to the disease-causing agent cannot be demonstrated across the population. Although the deer in this study were removed from a single fenced area, it is likely that the herd spent part of the year in small maternally related groups, congregating at feed plots and at natural watering areas. CWD transmission routes are not known and the relative efficiency of transmission through deer-to-deer contact or through shared feed and water sources cannot be determined. Microsatellite analysis of DNA from the affected animals needs to be performed to determine whether maternal contact increases the probability of infection. Secondly, some of the alleles are relatively rare and additional study populations from the endemic areas or from selectively bred captive white-tailed deer will be needed to increase the sample size in these genotypes. Thirdly, the interval from infection to the accumulation of detectable PrPd levels is not known. CWD was diagnosed in deer at 810 months of age in this herd, establishing a minimum incubation period. The maximum incubation period cannot be identified if infection times occur outside the perinatal period (Miller & Williams, 2003). Some older adult deer had detectable PrPd restricted to the lymphoid tissue. If the temporal pattern of PrPd accumulation in white-tailed deer is similar to that in sheep (Hadlow et al., 1982
) or experimentally infected mule deer (Sigurdson et al., 1999
), this observation indicates that white-tailed deer can be infected as adults, as reported in mule deer (Miller & Williams, 2003
) or that the incubation period can be as long as 5 years. Failure to detect PrPd in some deer is less likely to be due to genetic differences than to the termination of the entire herd for regulatory reasons while some or all deer were in the very early stage of the disease, before PrPd is detectable. Long-term observations of heavily exposed herds or direct oral challenge of captive deer will be needed to establish relative disease resistance in deer with the rare genotypes. However, the presence of a relatively resistant but rare genotype may not confer a selective advantage sufficient to reduce the prevalence of CWD in free-ranging herds.
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ACKNOWLEDGEMENTS |
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Received 5 November 2003;
accepted 23 December 2003.