Journal of Heredity Advance Access originally published online on November 21, 2007
Journal of Heredity 2007 98(7):678-686; doi:10.1093/jhered/esm091
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Molecular Characterization of the Rocky Mountain Elk (Cervus elaphus nelsoni) PRNP Putative Promoter
From the Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467 (Seabury, Templeton, Derr, Owens, Davis, and Womack); Department of Animal Science, Texas A&M University, College Station, TX 77843-2471 (Gill and Adelson); Cervid Research and Recovery Institute, Ignacio, CO 81137 (Dyar); and Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466 (Kraemer)
Address correspondence to C. M. Seabury at the address above, or e-mail: cseabury{at}cvm.tamu.edu.
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting deer (Odocoileus spp.), moose (Alces alces), and Rocky Mountain elk (Cervus elaphus nelsoni). Leucine homozygosity at elk PRNP codon 132 has been associated with reduced CWD susceptibility. However, naturally acquired CWD has been detected in elk possessing the 132 Leu/Leu genotype. Recent human and bovine studies indicate that PRNP regulatory polymorphisms may also influence TSE occurrence. Therefore, we generated sequences for the elk PRNP putative promoter (2.2 kb), exon 1 (predicted; 54 bp), intron 1 (predicted; 193 bp), and exon 3 (771 bp). Promoter prediction analysis using CpGProD yielded a single elk PRNP promoter that was homologous to regions of known promoter activity in cow and sheep. Molecular interrogation of the elk PRNP putative promoter revealed 32 diallelic single-nucleotide polymorphisms (SNPs). No variation was detected within the predicted exon 1 or intron 1 sequences. Evaluation of elk PRNP exon 3 revealed 3 SNPs (63Y, 312R, 394W
Met/Leu). Bayesian haplotype reconstruction resulted in 3 elk PRNP haplotypes, with complete linkage disequilibrium observed between all PRNP putative promoter SNPs and codon 132. The results of this study provide the initial genomic foundation for future comparative and haplotype-based elk PRNP studies.
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of inevitably fatal neurodegenerative diseases of humans and animals often generally characterized by progressive dementia and/or ataxia (Prusiner 1998; Collinge 2001). The pathogenic agents of prion diseases are infectious, protease-resistant proteins (PrPSc) generated via aberrant refolding of the normal host-encoded cellular prion protein (PrPC) in a susceptible host (Prusiner 1982; Prusiner 2004). Notably, prion diseases may occur as genetic, infectious, or sporadic disorders (for review, see Prusiner 2004). Presently, no overwhelming consensus exists regarding the precise function of PrPC. However, previous studies suggest that PrPC may play a role in immunoregulation, signal transduction, copper binding, synaptic homeostasis, apoptosis, and antiapoptosis (for review, see Aguzzi and Polymenidou 2004; Aguzzi and Heikenwalder 2006).
Chronic wasting disease (CWD) is a TSE occurring in both captive and free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni) primarily within the western United States of America (Williams 2005). Recent hunter-harvest surveillance efforts in the state of Colorado have also detected CWD in wild moose (Alces alces; http://wildlife.state.co.us/news/press.asp?pressid=3645). Clinical signs of CWD in elk include polydipsia, polyuria, increased salivation, weight loss, teeth grinding, wide body stance, and behavioral changes (Williams and Young 1982; Williams 2005). Although the precise origin and natural history of CWD is largely unknown, CWD differs from scrapie and bovine spongiform encephalopathy (BSE) in that it readily occurs among nondomestic free-ranging species detached from modern agricultural practices (Williams 2005). However, it should also be noted that many similarities exist between CWD and scrapie, including the apparent efficiency of horizontal transmission (for review, see Williams 2005; Mathiason et al. 2006).
To date, several nonsynonymous single-nucleotide polymorphisms (SNPs) within the prion protein gene (PRNP) have been associated with enhanced putative resistance, prolonged incubation periods, and/or enhanced susceptibility to TSEs in humans, sheep, goats, deer, and elk (for review, see Belt et al. 1995; O'Rourke et al. 1999; Collinge 2001; Billinis et al. 2002; O'Rourke et al. 2004; Jewell et al. 2005; Williams 2005; Hamir et al. 2006; Johnson et al. 2006; Vaccari et al. 2006). However, unlike several other TSE-susceptible mammals, only one nonsynonymous SNP and corresponding amino acid substitution (MetLeu; elk PRNP codon 132) has been detected in Rocky Mountain elk (C. elaphus nelsoni; hereafter elk; O'Rourke et al. 1999). Both case–control (O'Rourke et al. 1999) and experimental oral infection studies (Hamir et al. 2006) provide evidence of enhanced CWD susceptibility among elk possessing the Met/Met PRNP codon 132 genotype and enhanced putative resistance (or prolonged incubation periods) among elk possessing the Leu/Leu codon 132 genotype. In addition to associations between nonsynonymous PRNP polymorphisms and TSE occurrence, an inverse relationship between TSE incubation period and the level of host PRNP gene expression has also been established (Vilotte et al. 2001; Castilla et al. 2004; Safar et al. 2005; Sander et al. 2005; Scott et al. 2005). Therefore, enhanced susceptibility and/or resistance to TSEs in mammals is likely influenced by at least 4 genetic factors: 1) genetic variation within the PRNP coding region and the associated genotypes, 2) genetic variation within the PRNP regulatory regions and the associated genotypes, 3) the combined qualitative and quantitative effects of genetic variation within the PRNP coding and regulatory regions, and 4) genetic variation within genes other than PRNP. Although many of these genetic factors have been explored with respect to TSEs in sheep, cattle, mice, and humans, no such basic information currently exists regarding the sequence composition and/or the frequency of genetic polymorphism within the putative regulatory regions of the elk PRNP gene. A recent study demonstrated that bovine PRNP expression in vitro is modulated by genetic variation within the bovine PRNP promoter (Sander et al. 2005). To date, insertion–deletion (indel) polymorphisms within the bovine PRNP promoter and intron 1 have been associated with BSE incidence in several domestic cattle populations (Sander et al. 2004, 2005; Juling et al. 2006). In addition, previous studies indicate that human PRNP polymorphisms in both the intronic and upstream regulatory regions may be associated with sporadic Creutzfeldt-Jakob disease (CJD; McCormack et al. 2002; Bratosiewicz-Wasik et al. 2007). Therefore, mutations potentially influencing the level of elk PRNP expression may also influence CWD incubation period and thus overall resistance and/or susceptibility.
In this study, we utilized a comparative genomics approach employing both the cow (Bos taurus) PRNP sequence (Genbank AJ298878 [GenBank] ; Hills et al. 2001) as well as bovine oligonucleotide polymerase chain reaction (PCR) primers (Sander et al. 2004) to facilitate the development and characterization of the elk PRNP putative promoter sequence. Herein, we also provide the first detailed polymorphism study, corresponding haplotype analysis, and subsequent comparative analysis with respect to the elk PRNP putative promoter. The results of this study provide a robust framework for future comparative and evolutionary analyses while also aiding in the characterization of genomic regions potentially influencing the regulation of elk PRNP.
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Materials and Methods |
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Study Animals and DNA Isolation
To characterize and evaluate the frequency of genetic polymorphism within the elk PRNP gene, we developed and utilized a DNA panel consisting of n = 29 elk. Eighteen of the study elk (10 cows and 8 bulls) were obtained from the Cervid Research and Recovery Institute (CRRI, Durango, CO). Information regarding the PRNP codon 132 genotypes for all CRRI elk was available (see O'Rourke et al. [1999], for a description of the 3 elk codon 132 genotypes). Therefore, efforts were made to select unrelated elk representative of the 3 PRNP codon 132 genotypic classes. DNA was isolated and purified from whole blood using the MasterPure Genomic DNA Purification Kit (Epicentre, Madison, WI) according to the manufacturer's recommended protocol. Additionally, 11 DNA samples from an unrelated wild elk population (11 cows; Yellowstone National Park, Northwest Wyoming and South-Central Montana) were randomly selected from a local repository and included in our DNA panel. None of the elk utilized in this study had any known history or clinical symptoms of CWD.
Elk PRNP Amplification
Oligonucleotide primers flanking the bovine PRNP putative promoter (PRNP 47784F and PRNP 49673R; Sander et al. 2004) were utilized in 50-µl PCRs to generate a single 2447-bp amplicon for the homologous region of the elk PRNP gene. All 50-µl PCRs were carried out using GeneAmp 9700 PCR Systems (Applied Biosystems, Foster City, CA) and consisted of the following: 125 ng genomic DNA, 0.2 mM each deoxynucleoside triphosphate, 0.8 µM each primer (PRNP 47784F and PRNP 49673; Sander et al. 2004), 1x Master Amp PCR Enhancer (Epicentre), 1x GeneAmp PCR Gold Buffer with MgCl2 (1.5 mM MgCl2; Applied Biosystems), and 3.0 units of AmpliTaq Gold DNA polymerase (Applied Biosystems). Thermal cycling parameters, as optimized in our laboratory, were as follows: 5 min at 95 °C; 8 cycles x 30 s at 95 °C, 30 s at 60 °C (–1 °C/cycle), 2.20 min at 72 °C; 45 cycles x 30 s at 95 °C, 30 s at 52 °C, 2.20 min at 72 °C; 10 min at 65 °C. Additionally, flanking primers SAF1 and SAF2 (Prusiner et al. 1993) were utilized in 25-µl PCRs to amplify PRNP exon 3 for all study elk as previously described (Seabury and Derr 2003). All elk PRNP amplicons were examined via agarose gel electrophoresis and subsequently purified using the Qiagen QIAquick PCR Purification Kit (Qiagen, Valencia, CA) according to the manufacturer's recommended protocol.
Elk PRNP Sequencing
All purified elk PRNP amplicons were directly sequenced using Big Dye Terminator Cycle Sequencing technology in conjunction with GeneAmp 9700 PCR Systems (Applied Biosystems) in 10-µl reaction volumes. Elk PRNP putative promoter amplicons were initially sequenced using the primers PRNP 47784F and PRNP 49673R (Sander et al. 2004). Thereafter, internal sequencing primers were designed using the online utility Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and utilized in a stepwise fashion to generate the remaining elk PRNP promoter sequences. Elk PRNP putative promoter primers used for amplicon sequencing are depicted in Table 1. Internal sequencing primers were designed to facilitate generous overlaps, thereby enabling both correct assemblies and verification of observed SNPs. For the elk PRNP putative promoter amplicons, each 10-µl sequencing reaction consisted of the following: 150 ng purified amplicon, 2 µl Big Dye, 2 µl HalfBD (Genetix, Boston, MA), 1 µM primer, and 0.5x Master Amp PCR Enhancer. Thermal cycling parameters followed those previously described (Seabury and Derr 2003) with the exception that a 50 °C annealing temperature was utilized in combination with 50 total cycles. Likewise, purified elk PRNP exon 3 amplicons were also directly sequenced using the aforementioned thermal cycling parameters with reaction concentrations as previously described (Seabury and Derr 2003).
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Sequence Analysis and Validation Techniques
Most elk PRNP regions were sequenced more than once to ensure correct assemblies and confirm observed SNPs. Likewise, this strategy also allowed for confirmation of PRNP codon 132 genotypes in CRRI elk. All elk PRNP sequences were assembled and analyzed within the program Sequencher 4.7 (Gene Codes Corporation, Ann Arbor, MI) using the more rigorous default assembly parameters. Sequence quality was examined by manual inspection of electropherograms and confirmed via Sequencher 4.7 quality analysis score. Heterozygous nucleotides were flagged manually during initial electropherogram inspection and subsequently confirmed by Sequencher 4.7 analysis of multiple overlapping sequences. All heterozygous nucleotides were annotated with the appropriate International Union of Pure and Applied Chemistry–International Union of Biochemistry and Molecular Biology code for heterozygosity within Sequencher 4.7, and the final consensus sequences were exported for further analysis.
Haplotype Analysis
PRNP genotypes (unphased) for all elk were assembled into a single data set. Each variable site was enumerated based on its position in the putative elk PRNP promoter sequence developed herein and/or the elk PRNP exon 3 reference sequence (Genbank AF016227
[GenBank]
; coding sequence). Because deviations from Hardy–Weinberg equilibrium (HWE) may enhance the error rate associated with haplotype inference algorithms (Niu et al. 2002; Stephens and Donnelly 2003), HWE for the entire elk PRNP data set was assessed via the exact test of Guo and Thompson (1992) online (GENEPOP; http://wbiomed.curtin.edu.au/genepop/) and/or the chi-square test (http://www.genes.org.uk/software/hardy-weinberg.shtml). Estimates for the elk PRNP haplotype frequencies and the most likely pairs of haplotypes for each individual were reconstructed using a Bayesian statistical method implemented within the software package PHASE 2.1 (Stephens et al. 2001; Stephens and Donnelly 2003). Only elk PRNP SNPs with genotype distributions similar to HWE expectations and minor allele frequencies greater than 0.10 were included in haplotype reconstruction analyses. Estimates of recombination across the elk PRNP putative promoter and exon 3 were computed using PHASE 2.1 as previously described (Li and Stephens 2003). The physical distance (base pairs) between variable sites within the elk PRNP putative promoter and exon 3 was estimated using the mule deer PRNP sequence (GenBank AY330343). Moderate changes in distance approximations did not change the outcome of PHASE 2.1 analyses.
Promoter Prediction and Comparative Analysis
Two approaches were utilized to predict the elk PRNP putative promoter regions via the primary sequence data generated. The first analysis was conducted using the online utility PROSCAN version 1.7, an application for identifying mammalian promoters based on homology scoring with putative eukaryotic Pol II promoter sequences (Prestridge 1995; http://thr.cit.nih.gov/molbio/proscan/). Additionally, because approximately 60% of all promoters in humans and mice colocalize to CpG islands (Bird 2002; Antequera 2003), a second promoter analysis was performed using the mammalian-specific program CpGProD online (Ponger and Mouchiroud 2002; http://pbil.univ-lyon1.fr/software/cpgprod_query.html). The results of both analyses were compared for consistency. Likewise, both PROSCAN 1.7 and CpGProD were utilized to predict PRNP promoter regions for mule deer (O. hemionus; GenBank AY330343; Brayton et al. 2004), cow (B. taurus; GenBank AJ298878
[GenBank]
; Hills et al. 2001), and sheep (Ovis aries; GenBank DQ077504; Green et al. 2006) for comparison with elk. Because PRNP regions possessing promoter activity in cattle and sheep have previously been identified (Inoue et al. 1997; O'Neill et al. 2003; Sander et al. 2005), promoter prediction analysis for these species was useful for tentatively assessing the relative accuracy of PROSCAN 1.7 and CpGProD. The online BLAST utilities bl2seq and blastn (http://www.ncbi.nlm.nih.gov/blast/) were routinely employed for comparative analysis of the elk PRNP putative promoter sequences with the PRNP sequence for cow (GenBank AJ298878
[GenBank]
; DQ457195), sheep (GenBank DQ077504), and mule deer (GenBank AY330343). Elk and mule deer (AY330343) PRNP sequences were also evaluated for the presence of 4 conserved motifs previously identified within the PRNP promoter sequences for human, Syrian golden hamster, sheep, mouse, rat, and cow (Westaway et al. 1994; Saeki et al. 1996; Inoue et al. 1997).
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Collectively, we generated and analyzed more than 93 000 bp of PRNP nucleotide sequence data for 29 elk derived from 2 populations. Elk PRNP amplification success was 100% using sheep and cattle PRNP primers previously described (Prusiner et al. 1993; Sander et al. 2004). BLASTN alignment (bl2seq) of the elk PRNP sequence (2447 bp) generated herein with the complete mule deer PRNP sequence (AY330343; Brayton et al. 2004) resulted in significant similarity beginning 2.2 kb upstream of mule deer PRNP exon 1, extending through exon 1 (54 bp), and terminating 193 bp into intron 1. The maximum sequence identity (blastn) between the 2447-bp elk PRNP sequence and the corresponding mule deer (AY330343), cow (DQ457195), and sheep (DQ077504) PRNP sequences were 94%, 91%, and 92%, respectively. Molecular interrogation of the 2.2-kb putative elk PRNP promoter sequences resulted in the identification of 32 previously unreported diallelic SNPs (Table 2), indicating an average density of one SNP for every 69 bp sequenced. The predicted elk PRNP exon 1 (54 bp) and partial intron 1 (193 bp) sequences were monomorphic among the study samples. Notably, the predicted elk PRNP exon 1 sequences were identical to those previously reported for mule deer (AY330343; Brayton et al. 2004). Pairwise sequence identities between the cow (AJ298878 [GenBank] ) and sheep (DQ077504) PRNP exon 1 sequences and the predicted elk exon 1 sequence were 92% and 94%, respectively. Analysis of electropherograms and sequence alignments for all elk PRNP sequences yielded no evidence of indel polymorphism among the study samples. Molecular interrogation of elk PRNP exon 3 resulted in the identification of 1 nonsynonymous (394W; codon 132 Met/Leu) and 2 synonymous SNPs (63Y; 312R; Table 2). The synonymous SNP at position 312 was observed in a single elk derived from Yellowstone National Park. Elk PRNP exon 3 SNPs at positions 312 and 394 have previously been reported (O'Rourke et al. 1998, 1999; Hamir et al. 2006).
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Of the 35 diallelic elk PRNP SNP loci observed, only one (PRNP exon 3 position 312; Table 2) displayed a minor allele frequency less than 0.10 (observed allele frequencies: 312A = 0.02; 312G = 0.98) and was therefore excluded from haplotype reconstruction analysis. The major and minor allele frequencies observed for all elk PRNP SNPs are depicted in Table 2. Of the 35 SNPs observed within the elk PRNP gene, 34 had genotype distributions similar to HWE. The exact test for HWE (Guo and Thompson 1992) could not be performed for SNP variation associated with PRNP exon 3 position 312 using GENEPOP online. Three elk PRNP haplotypes were predicted via the PHASE 2.1 best reconstruction and best pairs analysis. The 3 predicted elk PRNP haplotypes and estimates of the sample haplotype frequencies are depicted in Table 3. Phase probabilities for all elk PRNP variable sites were 1.0. Likewise, probabilities for the best pairs of PRNP haplotypes for all study elk also were 1.0.
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No evidence of recombination was detected using the general model for varying recombination rate (Li and Stephens 2003) for the elk PRNP region spanning the putative promoter (2.2 kb), exon 1 (54 bp; predicted), intron 1 (193 bp; predicted), and exon 3 (771 bp) for our study samples. Moreover, complete linkage disequilibrium (LD) was observed for all diallelic SNPs within the elk PRNP promoter, resulting in the segregation of 2 high-frequency promoter haplotypes (see Table 3). Additionally, complete LD was also noted between all elk PRNP promoter SNPs and nonsynonymous exon 3 variation (394W codon 132 Met/Leu; O'Rourke et al. 1998, 1999) for all study elk (n = 29). Therefore, the PRNP exon 3 codon 132 genotypes (Met/Met, Met/Leu, Leu/Leu) for all study elk could be retrospectively ascertained with 100% accuracy by genotyping any one of the 32 diallelic promoter SNPs (see Table 2).
The 4 conserved PRNP promoter motifs previously described for mouse, sheep, human, Syrian golden hamster, rat, and cow (Westaway et al. 1994; Saeki et al. 1996; Baybutt and Manson 1997; Inoue et al. 1997) were also conserved within the elk and mule deer (AY330343) PRNP sequences (Figure 1). No genetic variation was observed within the 4 elk PRNP promoter motifs. PRNP promoter prediction analyses for elk, mule deer (AY330343), sheep (DQ077504), and cow (AJ298878 [GenBank] ) are depicted in Table 4. Notably, CpGProD predicted a single plus-strand PRNP promoter for all species evaluated, whereas PROSCAN 1.7 routinely predicted multiple PRNP promoter regions on both the plus and minus strands (see Table 4). The results of PRNP promoter prediction analyses using both CpGProD and PROSCAN 1.7 were not congruent for all species evaluated (see Table 4). The elk PRNP promoter region predicted by CpGProD included 2 potential binding sites for the transcription factor AP-2 (Mitchell et al. 1987) and 4 consensus Sp1 transcription factor–binding sites in the following orientation—AP-2: CCCCGGGC 2104–2111, putative promoter; Sp1: TCCCCGCCCC 2152–2161, putative promoter; Sp1: CCGCCC 2175–2180, putative promoter; Sp1: CCGCCC 2191–2196, putative promoter; Sp1: CCGCCC 2293–2298, predicted intron 1; and AP-2: CCCCGGGC 2323–2330, predicted intron 1. The corresponding elk PRNP promoter region predicted by PROSCAN 1.7 did not include the second potential binding site for AP-2 (2323–2330; Table 4). Moreover, PROSCAN 1.7 also predicted a second PRNP promoter (minus strand) for elk possessing the codon 132 MM and 132 LM genotypes (Table 4). Similar to the elk, 2 potential binding sites for AP-2 (Mitchell et al. 1987) and 3 consensus Sp1 binding sites, arranged in the same general orientation as the elk, were also noted within the mule deer PRNP promoter regions predicted by CpGProD and PROSCAN 1.7 (Table 4). PRNP promoter regions predicted by CpGProD for cow and sheep included regions of known promoter activity (see Table 4). Cow and sheep PRNP promoters predicted by PROSCAN 1.7 were not homologous with sequence regions known to possess transcriptional activity (see Table 4). The elk and mule deer PRNP promoter regions homologous with regions of known promoter activity in both cow and sheep are defined in Table 4. Likewise, the elk PRNP promoter region corresponding to the CpGProD-predicted promoter for mule deer is also presented in Table 4. Nucleotide sequence alignment of the predicted elk PRNP promoter (CpGProD; Table 4) with the PRNP region of known promoter activity in sheep (O'Neill et al. 2003) revealed 8 total diallelic SNPs (sheep DQ077504: 5354M, 5525M, 5622S, 5700Y, 5739K; elk: 1772R, 1946S, 2059K). Similarly, comparative alignments made between the PRNP regions of known promoter activity in cow (Inoue et al. 1997) and the predicted elk PRNP promoter (CpGProD; Table 4) revealed 7 total diallelic SNPs (cow DQ457195: 3966R, 4065K, 4186Y, 4262R; elk: 1772R, 1946S, 2059K). No PRNP SNPs were in common among sheep, cow, and elk. No corresponding mule deer and/or white-tailed deer (O. virginianus) PRNP polymorphism data were available for similar comparisons.
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Discussion |
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Herein, we have provided the first molecular characterization and polymorphism analysis for the elk PRNP putative promoter, thereby also representing the most comprehensive assemblage of elk PRNP data to date. Collectively, 32 novel SNPs were identified within the genomic region encompassing the elk PRNP putative promoter. Additionally, one novel synonymous SNP (63Y) was also identified within exon 3 of the elk PRNP gene. Comparative sequence analyses between the 2447-bp elk PRNP sequences and the PRNP sequences of mule deer, cow, and sheep yielded evidence of both good conservation (>90% sequence identity) as well as regions of elk-specific PRNP sequence (data not shown). Conservation of the 4 distinct PRNP promoter motifs (Westaway et al. 1994) across 8 divergent mammalian taxa (Figure 1) implies some level of biological significance despite the corresponding lack of transcriptional activity noted during reporter gene deletion studies in mouse, cow, and rat (Saeki et al. 1996; Baybutt and Manson 1997; Inoue et al. 1997). Given the known limitations of reporter gene deletion studies, previous authors have suggested that the conserved promoter region may regulate PRNP expression in different tissues and/or during different stages of development (McCormack et al. 2002).
Identification of LD within the elk PRNP putative promoter was not completely unexpected given recent evidence of high LD within the promoter of both cow and sheep (Clawson et al. 2006; Green et al. 2006). However, detection of complete LD between all elk PRNP putative promoter SNPs (n = 32; Table 2) for elk obtained from 2 distinct populations was not anticipated. Moreover, the identification of complete LD between all elk PRNP putative promoter SNPs (n = 32) and nonsynonymous exon 3 variation (394W; codon 132 Met/Leu; Table 2) is unprecedented among PRNP studies of artiodactyls. Interestingly, elk is the only TSE-susceptible artiodactyl known to posses an amino acid substitution equivalent to the human PRNP codon 129 polymorphism (Owen et al. 1990; O'Rourke et al. 1999; note, human codon 129 = elk codon 132). Similar to the results presented here, previous studies of human PRNP noted complete LD between 2 upstream regulatory SNPs and human codon 129 (129 Met/Val; McCormack et al. 2002; Bratosiewicz-Wasik et al. 2007). Specifically, the –101G SNP, previously determined to be in complete disequilibrium with methionine at human codon 129, has been associated with sporadic CJD in humans (McCormack et al. 2002; Bratosiewicz-Wasik et al. 2007). Comparative alignment of the human PRNP promoter sequence (AF315723; McCormack et al. 2002) with the elk PRNP sequence revealed a proximal relationship between the elk 2059K SNP (Table 2) and the human –101G SNP (AF315723; McCormack et al. 2002). The functional significance of the 32 elk PRNP putative promoter SNPs, and/or the 2 corresponding high-frequency PRNP haplotypes (Table 3), remains to be determined.
PRNP promoter prediction results generated using the program CpGProD consistently appeared more plausible and potentially more accurate than PROSCAN 1.7 based on a priori knowledge of PRNP regions possessing promoter activity in cow and sheep (Inoue et al. 1997; O'Neill et al. 2003; Table 4). Moreover, a CpG island–related PRNP promoter was predicted for all mammalian species analyzed. Notably, the PRNP promoters predicted by CpGProD for elk and mule deer (AY330343) were homologous with sequence regions known to possess promoter activity in both cow and sheep (Inoue et al. 1997; O'Neill et al. 2003; Table 4). Therefore, additional studies are necessary to ascertain whether the PRNP promoter regions predicted for elk and mule deer possess significant transcriptional activity. Additionally, given the relative density of SNPs previously reported within regions of known promoter activity for cow and sheep, future studies are generally needed to assess the significance of the relationship between PRNP regulatory SNPs and relevant TSEs among ruminant species.
Herein, we report 33 novel elk PRNP polymorphisms and 2447 bp of previously unreported elk PRNP genomic sequence. The nucleotide sequence data and corresponding haplotype analysis reported in this study provide the initial foundation for further characterizing elk PRNP haplotype structure while also providing a natural segue toward future haplotype-based case–control studies with respect to CWD in elk. Additionally, the prediction of PRNP regions potentially possessing promoter activity for both elk and mule deer provides an immediate opportunity for reporter gene assays. Future studies targeting the entire elk PRNP gene for additional populations are currently needed to comprehensively evaluate the overall patterns of polymorphism while also further assessing the distribution and magnitude of LD.
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Funding |
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Cervid Research and Recovery Institute, Texas A&M College of Veterinary Medicine, Department of Veterinary Pathobiology (to C.M.S.), and the Texas Agricultural Experiment Station TAES 404951 (to C.M.S.).
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Acknowledgments |
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Nucleotide sequence data reported herein are available in the GenBank database (accession numbers EU082234–EU082291).
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Footnotes |
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Corresponding Editor: Stephen J. O'Brien
Received February 13, 2007
Accepted September 24, 2007
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Aguzzi A, Heikenwalder M. Pathogenesis of prion diseases: current status and future outlook. Nature (2006) 4:765–775.
Aguzzi A, Polymenidou M. Mammalian prion biology. One century of evolving concepts. Cell (2004) 116:313–327.[CrossRef][Web of Science][Medline]
Antequera F. Structure, function and evolution of CpG island promoters. Cell Mol Life Sci (2003) 60:1647–1658.[CrossRef][Web of Science][Medline]
Baybutt H, Manson J. Characterization of two promoters for prion protein (PrP) gene expression in neuronal cells. Gene (1997) 184:125–131.[CrossRef][Web of Science][Medline]
Belt PB, Muileman IH, Schreuder BE, Bos-de Ruijter J, Gielkens AL, Smits MA. Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol (1995) 76:509–517.
Billinis C, Panagiotidis CH, Psychas V, Argyroudis S, Nicolaou A, Leontides S, Papadopoulos O, Sklaviadis T. Prion protein gene polymorphisms in natural goat scrapie. J Gen Virol (2002) 83:713–721.
Bird A. DNA methylation patterns and epigenetic memory. Genes Dev (2002) 16:6–21.
Bratosiewicz-Wasik J, Liberski PP, Golanska E, Jansen GH, Wasik TJ. Regulatory sequences of the PRNP gene influence susceptibility to sporadic Creutzfeldt-Jakob disease. Neurosci Lett (2007) 411:163–167.[CrossRef][Web of Science][Medline]
Brayton KA, O'Rourke KI, Lyda AK, Miller MW, Knowles DP. A processed pseudogene contributes to apparent mule deer heterogeneity. Gene (2004) 326:167–173.[CrossRef][Web of Science][Medline]
Castilla J, Gutierrez-Adan A, Brun A, Pintado B, Parra B, Ramirez MA, Salguero FJ, Díaz San Segundo F, Rábano A, Cano MJ, et al. Different behavior toward bovine spongiform encephalopathy infection of bovine prion protein transgenic mice with one extra repeat octapeptide insert mutation. J Neurosci (2004) 24:2156–2164.
Clawson ML, Heaton MP, Keele JW, Smith TPL, Harhay GP, Laegreid WW. Prion gene haplotypes of U.S. cattle. BMC Genet (2006) 7:51.[CrossRef][Medline]
Collinge J. Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci (2001) 24:519–550.[CrossRef][Web of Science][Medline]
Green BT, Heaton MP, Clawson ML, Laegreid WW. Linkage disequilibrium across six prion gene regions spanning 20 kbp in U.S. sheep. Mamm Genome (2006) 17:1121–1129.[CrossRef][Web of Science][Medline]
Guo SW, Thompson EA. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics (1992) 48:361–372.[CrossRef][Web of Science][Medline]
Hamir AN, Gidlewski T, Spraker TR, Miller JM, Creekmore L, Crocheck M, Cline T, O'Rourke KI. Preliminary observations of genetic susceptibility of elk (Cervus elaphus nelsoni) to chronic wasting disease by experimental oral inoculation. J Vet Diagn Invest (2006) 18:110–114.
Hills D, Comincini S, Schlaepfer J, Dolf G, Ferretti L, Williams JL. Complete genomic sequence of the bovine prion gene (PRNP) and polymorphism in its promoter region. Anim Genet (2001) 32:231–232.[CrossRef][Web of Science][Medline]
Inoue S, Tanaka M, Horiuchi M, Ishiguro N, Shinagawa M. Characterization of the bovine prion protein gene: the expression requires interaction between the promoter and intron. J Vet Med Sci (1997) 59:175–183.[CrossRef][Web of Science][Medline]
Jewell JE, Conner MM, Wolfe LL, Miller MW, Williams ES. Low frequency of PrP genotype 225SF among free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease. J Gen Virol (2005) 86:2127–2134.
Johnson C, Johnson J, Vanderloo JP, Keane D, Aiken JM, McKenzie D. Prion protein polymorphisms in white-tailed deer influence susceptibility to chronic wasting disease. J Gen Virol (2006) 87:2109–2114.
Juling K, Schwarzenbacher H, Williams JL, Fries R. A major genetic component of BSE susceptibility. BMC Biol (2006) 4:33.[CrossRef][Medline]
Li N, Stephens M. Modeling linkage disequilibrium and identifying recombination hotspots using single-nucleotide polymorphism data. Genetics (2003) 165:2213–2233.
Mathiason CK, Powers JG, Dahmes S, Osborn DA, Miller KV, Warren RJ, Mason GL, Hays SA, Hayes-Klug J, Seelig DM, et al. Infectious prions in saliva and blood of deer with chronic wasting disease. Science (2006) 314:133–135.
McCormack JE, Baybutt HN, Everington D, Will RG, Ironside JW, Manson JC. PRNP contains both intronic and upstream regulatory regions that may influence susceptibility to Creutzfeldt-Jakob disease. Gene (2002) 17:139–146.[CrossRef]
Mitchell PF, Wang C, Tjian R. Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell (1987) 50:847–861.[CrossRef][Web of Science][Medline]
Niu T, Qin ZS, Xiping X, Liu JS. Bayesian haplotype inference for multiple linked single-nucleotide polymorphisms. Am J Hum Genet (2002) 70:157–169.[CrossRef][Web of Science][Medline]
O'Neill GT, Donnelly K, Marshall E, Cairns D, Goldmann W, Hunter N. Characterization of ovine PrP gene promoter activity in N2a neuroblastoma and ovine foetal brain cell lines. J Anim Breed Genet (2003) 120:114–123.[CrossRef][Web of Science]
O'Rourke KI, Baszler TV, Miller JM, Spraker TR, Sadler-Riggleman I, Knowles DP. Monoclonal antibody F89/160.1.5 defines a conserved epitope on the ruminant prion protein. J Clin Microbiol (1998) 36:1750–1755.
O'Rourke KI, Besser TE, Miller MW, Cline TF, Spraker TR, Jenny AL, Wild MA, Zebarth GL, Williams ES. PrP genotypes of captive and free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease. J Gen Virol (1999) 80:2765–2769.
O'Rourke KI, Spraker TR, Hamburg LK, Besser TE, Brayton KA, Knowles DP. Polymorphisms in the prion precursor functional gene but not the pseudogene are associated with susceptibility to chronic wasting disease in white-tailed deer. J Gen Virol (2004) 85:1339–1346.
Owen F, Poulter M, Collinge J, Crow TJ. Codon 129 changes in the prion protein gene in Caucasians. Am J Hum Genet (1990) 46:1215–1216.[Web of Science][Medline]
Ponger L, Mouchiroud D. CpGProD: identifying CpG islands associated with transcription start sites in large genomic mammalian sequences. Bioinformatics (2002) 18:631–633.
Prestridge DS. Predicting Pol II promoter sequences using transcription factor binding sites. J Mol Biol (1995) 249:923–932.[CrossRef][Web of Science][Medline]
Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science (1982) 216:136–144.
Prusiner SB. Prions. Proc Natl Acad Sci USA (1998) 95:13363–13383.
Prusiner SB. An introduction to prion biology and diseases. In: Prion biology and diseases—Prusiner SB, ed. (2004) 2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. 1–87.
Prusiner SB, Füzi M, Scott M, Serban D, Serban H, Taraboulos A, Gabriel JM, Wells GAH, Wilesmith JW, Bradley R, et al. Immunologic and molecular biologic studies of prion proteins in bovine spongiform encephalopathy. J Infect Dis (1993) 167:602–613.[Web of Science][Medline]
Saeki K, Matsumoto Y, Matsumoto Y, Onodera T. Identification of a promoter region in the rat prion protein gene. Biochem Biophys Res Commun (1996) 219:47–52.[CrossRef][Web of Science][Medline]
Safar JG, DeArmond SJ, Kociuba K, Deering C, Didorenko S, Bouzamondo-Bernstein E, Prusiner SB, Tremblay P. Prion clearance in bigenic mice. J Gen Virol (2005) 86:2913–2923.
Sander P, Hamann H, Drogemuller C, Kashkevich K, Schiebel K, Leeb T. Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility. J Biol Chem (2005) 280:37408–37414.
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) 5:19–25.[CrossRef][Web of Science][Medline]
Scott MR, Peretz D, Nguyen H-O, DeArmond SJ, Prusiner SB. Transmission barriers for bovine, ovine, and human prions in transgenic mice. J Virol (2005) 79:5259–5271.
Seabury CM, Derr JN. Identification of a novel ovine PrP polymorphism and scrapie-resistant genotypes for St. Croix White and a related composite breed. Cytogenet Genome Res (2003) 102:85–88.[CrossRef][Web of Science][Medline]
Stephens M, Donnelly P. A comparison of Bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet (2003) 73:1162–1169.[CrossRef][Web of Science][Medline]
Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet (2001) 68:978–989.[CrossRef][Web of Science][Medline]
Vaccari G, Di Bari MA, Morelli L, Nonno R, Chiappini B, Antonucci G, Marcon S, Esposito E, Fazzi P, Palazzini N, et al. Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie. J Gen Virol (2006) 87:1395–1402.
Vilotte JL, Soulier S, Essalmani R, Stinnakre MG, Vaiman D, Lepourry L, Da Silva JC, Besnard N, Dawson M, Buschmann A, et al. Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine Prp. J Virol (2001) 75:5977–5984.
Westaway D, Cooper C, Turner S, Da Costa M, Carlson GA, Prusiner SB. Structure and polymorphism of the mouse prion protein gene. Proc Natl Acad Sci USA (1994) 91:6418–6422.
Williams ES. Chronic wasting disease. Vet Pathol (2005) 42:530–549.
Willams ES, Young S. Spongiform encephalopathy of Rocky Mountain elk. J Wildl Dis (1982) 18:465–471.[Abstract]
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