Published as doi: 10.1096/fj.06-7522com.
(The FASEB Journal. 2007;21:1547-1555.)
© 2007 FASEB
Functional relevance of DNA polymorphisms within the promoter region of the prion protein gene and their association to BSE infection
Kseniya Kashkevich*,
Andreas Humeny*,
Ute Ziegler
,
Martin H. Groschup,
Petra Nicken
,1,
Tosso Leeb,2,
Christine Fischer
,
Cord-Michael Becker* and
Katrin Schiebel*,3
* Institute for Biochemistry, Emil-Fischer-Center, University of Erlangen-Nürnberg, Erlangen, Germany;
Friedrich-Loeffler-Institut, Institute for Novel and Emerging Infectious Diseases, Insel Riems, Germany;
Institute for Animal Breeding and Genetics, University of Veterinary Medicine, Hannover, Germany; and
Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany
3Correspondence: Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany. E-mail: katrin.schiebel{at}biochem.uni-erlangen.de
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ABSTRACT
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Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that can occur spontaneously or can be caused by infection or mutations within the prion protein gene PRNP. Nonsynonymous DNA polymorphisms within the PRNP gene have been shown to influence susceptibility/resistance to infection in sheep and humans. Analysis of DNA polymorphisms within the core promoter region of the PRNP gene in four major German bovine breeds resulted in the identification of both SNPs and insertion/deletion (indel) polymorphisms. Comparative genotyping of both controls and animals that tested positive for bovine spongiform encephalopathy (BSE) revealed a significantly different distribution of two indel polymorphisms and two SNPs within Braunvieh animals, suggesting an association of these polymorphisms with BSE susceptibility. The functional relevance of these polymorphisms was analyzed using reporter gene constructs in neuronal cells. A specific haplotype near exon 1 was identified that exhibited a significantly lower expression level. Genotyping of nine polymorphisms within the promoter region and haplotype calculation revealed that the haplotype associated with the lowest expression level was underrepresented in the BSE group of all breeds compared to control animals, indicating a correlation of reduced PRNP expression and increased resistance to BSE.—Kashkevich, K., Humeny, A., Ziegler, U., Groschup, M. H., Nicken, P., Leeb, T., Fischer, C., Becker, C.-M., Schiebel, K. Functional relevance of DNA polymorphisms within the promoter region of the prion protein gene and their association to BSE infection.
Key Words: PRNP • German bovine breeds • haplotype • expression • single nucleotide polymorphism
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INTRODUCTION
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STRUCTURAL ALTERATIONS WITHIN THE PRION protein are thought to underlay the molecular pathology of spongiform encephalopathies. In humans, the heritable forms [Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker and fatal familiar insomnia(FFI)], are caused by mutations within the prion protein gene, PRNP. Phenotypic variations of the disease depend on mutated amino acids but in addition are influenced by the M129V polymorphism. Depending on the allele encoding M129V, the mutation D178N results in either CJD or FFI (1
, 2)
. In both spontaneous and iatrogenic forms the M129V polymorphism modulates the age of onset, duration, and susceptibility to the disease.
The association of polymorphisms within the PRNP gene and resistance/susceptibility to scrapie has been well documented for sheep (3)
. In cattle, however, no clear association between nonsynonymous polymorphisms and BSE susceptibility has so far been identified (4
, 5)
. Recently, several studies characterized the genomic region of the bovine PRNP gene for polymorphisms and extended the analysis to different breeds (6
7
8
9)
. The first report on German BSE animals indicated a possible effect of PRNP promoter variations on BSE susceptibility (10)
. In particular, the frequency of a 23 bp indel polymorphism located 1.6 kbp upstream of exon 1 and a 12 bp indel within intron 1 were significantly different between control and BSE animals across breeds (10)
.
In this study, the potential promoter region upstream of exon 2 was analyzed for DNA polymorphisms in the German breeds Schwarzbunt (SB), Rotbunt (RB), Fleckvieh (FV), and Braunvieh (BV). A panel of nine polymorphisms was used for genotyping and haplotype calculation in 127 healthy breeding bulls and 293 German cows tested positive for BSE. The promoter activity of major haplotypes was investigated by luciferase reporter gene experiments in neuronal cells and the association of differentially expressed haplotypes to BSE susceptibility was analyzed by comparing the representation of these haplotypes in the BSE and control groups.
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MATERIALS AND METHODS
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DNA preparation
In Germany, animals older than 24 mo of age were routinely tested for BSE when slaughtered. Tissue of each positive sample was collected and confirmed at the Friedrich-Loeffler-Institut, Insel Riems (Germany). DNA was extracted as described previously (11)
. Samples were taken from BSE animals classified according to the farmers declaration into each of the four major breeds as SB (Holstein Friesian or black-and-white German Holstein; 35.8%), RB (Red Holstein or red-and-white German Holstein; 12%), FV (Fleckvieh; 30.5%) and BV (Braunvieh; 12%) or unknown (9.5%). Sperm DNA from German breeding bulls used for artificial insemination of the corresponding breeds served as controls (11)
.
Identification of DNA polymorphisms within the PRNP gene region
To identify DNA polymorphisms within different German bulls, polymerase chain reaction (PCR) amplified DNA fragments (12)
were directly sequenced after cleaning up of the amplification products using GE Healthcare GFX PCR DNA and Gel Band Purification Kit, and DYEnamic ET terminator cycle sequencing premix kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s protocol. To identify polymorphic positions, the resulting DNA sequences were compared to Genbank sequence AJ298878, which was used as a reference for numbering all positions (allele 1). Primers used for primary PCR, position and PCR conditions are given in Table 1
.
Genotyping of the PRNP gene
Genotyping of polymorphic positions was predominantly done by allele specific primer extension followed by MALDI-TOF-MS analysis of extended oligonucleotides: primer bovPRNPI1G242Afor (49834G
A, 5' GAA TCG GAC CGG TAG AGG 3', 58°C, dGTP, ddATP, ddCTP; primer bovPRNPI1–278for (49871G A), 5' TGG GGA GCT CAG AAG 3', 48°C, dGTP, ddATP; primer bovPRNPG405Afor (50000G A), 5' GAG GAG TGC CGG AGC 3', 52°C, dGTP, ddTTP, ddATP; bovPRNPC449Tfor (50044C T), 5' AAC TCC TCC CGA GAG G 3', 41°C, dTTP, dGTP, ddCTP; bovPRNPI1–543for (50138G A) 5' CGG CTC TGG CGG GCG T 3', 58°C, dGTP, ddCTP, and ddATP, as described previously (6
, 13)
or by direct sequencing of PCR fragments. Genotyping of the 12 bp and 23 bp indel polymorphisms was analyzed by fragment length polymorphism of PCR fragments (Table 1)
and separation of fragments on 2% agarose gels. Because of the limited amount and sporadically poor quality of the tissue for DNA isolation of BSE animals, not all animals could be tested for all markers. As SNPs 50138, 50297, 50308, and 50313 were in strong linkage disequilibrium, not all polymorphic positions were genotyped.
Statistical methods of genotype and haplotype analysis
Genotyping results were subjected to statistical analysis comparing the control and BSE group of each breed separately using 
2-tests (www.georgetown.edu/faculty/ballc/webtools/web_chi.html). Probabilities of haplotypes were estimated using an algorithm described by Stephens et al. (14
, 15)
implemented in the PHASE program (version 2.0.2), Department of Statistics, University of Washington, Seattle (www.stat.Washington.edu/stephens/software/html). Haplotype distribution differences were tested with a permutation procedure implemented in PHASE. Test results are not adjusted for multiple comparisons.
Generation of reporter plasmids
Various test plasmids containing the putative promoter region of the PRNP gene were prepared (
Fig. 3A
). The 5.2-kbp constructs, including either the ins or the del allele of both indel polymorphisms have been described earlier (16)
. Using these clones as templates, we amplified the 1.9-kbp construct with either variant of 12-bp indel and subcloned into the BglII site of a pGL3-Enhancer luciferase vector. Major haplotype constructs were engineered by PCR amplification of individual samples and restriction fragment cloning. Primers for amplification are given in Table 1
and polymorphic positions of the major haplotypes in Fig. 3B
. The constructs 1.9-kbp included an artificial EcoRI site that was used for ligation of a 1.15 kbp polymorphic and a 0.7 kbp constant part of the promoter. The constructs 1.9-kbp include the most relevant promoter region identified by Inoue et al. (17)
.
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Figure 3. Expression analysis of luciferase reporter constructs. A) Luciferase reporter gene constructs used for transfection assays. Cloning of the 5.2-kbp constructs has been previously described (16)
. The 1.9-kbp plasmids were created by PCR amplification of the initial plasmids, according to a high-expressing construct (pPrPCAT-4) described by Inoue et al. (17)
. Variable polymorphic positions were introduced by PCR amplification of individual animal DNAs and combinations of restriction fragments. The 1.1-kbp construct was not sufficient to act as a promoter for the expression of the reporter gene (data not shown). B) Polymorphic position of 1.9-kbp constructs representing the major haplotypes (A–E). Haplotype F represents a rare haplotype, differing from the most prominent haplotype A only by the 12-bp indel (A=del, F=ins). Shading marks haplotype positions that were not included in the haplotype calculation but could be deduced due to their strong association to SNP 50138. Therefore, the SNPs are given in parenthesis. C) Expression of 5.2-kbp constructs, including all four combinations of 23-bp and 12-bp indel polymorphisms (upper panels). Luciferase reporter gene constructs containing either the 23-bp del/12-bp ins allele, the 23-bp del/12-bp del allele, the 23-bp ins/12-bp del or the 23-bp ins/12-bp ins allele were transfected into Neuro-2A (left) or primary hippocampal cells (right). Relative light units (RLU) were normalized to cotransfected ß-galactosidase. Results of four to five parallel assays of two (hippocampal cells) and three (Neuro-2A) independent transfections, respectively, are summarized. Nested two-way ANOVA demonstrated a significant difference between constructs in both cell lines. The differences in constructs were due to the 12-bp and not the 23-bp polymorphism. Constructs 1.9 kbp in length representing the five major haplotypes were analyzed for their expression in 3–4 (Neuro-2A, lower left panel) and 2 (hippocampal cells, lower right panel) transfection assays with 3–5 replicants. In addition, the haplotype F was transfected in order to demonstrate that the influence of the 12-bp indel alone on the expression level was lower than the influence of the surrounding SNPs. Haplotype B resulted in a significantly lower expression level than all other haplotypes in both Neuro-2A and hippocampal cells. The numbers of repeated analysis are given in parenthesis. Error bars indicate standard deviation.
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Transient transfection and luciferase assays
Reporter plasmids containing luciferase under the control of various promoter constructs were transfected into cells of the mouse neuroblastoma cell line Neuro-2A (18)
using calcium phosphate precipitation (19)
or into primary hippocampal neuronal cells using LipofectamineTM 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol. Neuro-2A and hippocampal cells were isolated and cultivated as described (20
, 21)
. Cells were plated in six-well dishes at a concentration of 5 x 105 (Neuro-2A) or 2.5 x 105 cells/well (hippocampal neurons). Cells were transfected with 3 µg/well plasmid constructs and cotransfected with 2 µg/well of a pSV40 ß-galactosidase plasmid (Promega, Mannheim, Germany) to determine the transfection efficiency. After transfection cells were grown for 48 h, and ß-galactosidase and luciferase activities were determined by ß-Gal and Luciferase Reporter Gene Assay Kits (Roche, Mannheim, Germany) according to the manufacturer’s protocol. Relative light units were calculated by dividing the ratios of luciferase of the constructs to the ß-galactosidase of the construct and the ratio of luciferase of the control to the ß-galactosidase of the control (pGL3-Control vector, Promega, Mannheim, Germany). Statistical analyses were performed using the unpaired Student’s t test, the Kolmogorov-Smirnov test or by ANOVA between groups (ANOVA) at www.physics.csbsju.edu/stats/. Significance level was taken as P < 0.05.
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RESULTS
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Polymorphism identification
Sequences of sperm DNA from 10 breeding bulls from each of the four different breeds were analyzed for DNA polymorphisms, indels and single nucleotide polymorphisms (SNPs). As shown in Fig. 1
, 25 polymorphisms were identified in the region flanking exon 1 (GenBank Accession no. AJ298878, positions 48238 to 50379). Sequencing of 1171 bp upstream of exon 1 resulted in the isolation of 11 polymorphisms (Fig. 1)
. Nine polymorphisms, including two indel polymorphisms residing upstream of the previously identified promoter region were in linkage disequilibrium, whereas SNP 48700G A was not. Some polymorphisms were not found in all breeds, e.g., the T-allele of SNP 49493G T was exclusively found in BV, while the A-alleles of SNPs 48700G A and 49542G A (previously termed G914A (6)
) were rarely found in Holstein breeds (SB, RB), and the T-allele of SNP 50311C T was specific for these breeds (controls in Table 2
).
Genotyping of German breeds
Depending on localization within the potential core promoter region described by Inoue et al. (17)
, as well as their allele frequency, breed specificity, and linkage disequilibrium, nine polymorphisms were selected for indepth analysis in a total of 144 breeding bull DNAs of SB, RB, FV and BV origin (21–42 individuals per breed). In addition, the 23 bp indel polymorphism, previously described to be significantly differentially distributed in a small group of control and BSE animals was included in the analysis (10)
. The results for the control data are summarized in Table 2
. The allele frequencies in BV were clearly different from these in SB, RB, and FV breeds. In all breeds, the frequency of the 12-bp indel differed significantly from BV (P<0.001). Allele frequencies of SNPs 49871A G and 50000G A were also significantly different in SB and FV compared to BV (P<0.001). RB was only significantly different for the allele frequency of 49871A G (P<0.01). Therefore, comparative analysis of control and BSE animals was provided within each breed separately.
Genotyping of animals tested positive for BSE
Genotyping of the 10 polymorphisms (see above) in up to 144 control and up to 319 BSE animals yielded significantly different genotype distributions for both the 23-bp and the 12-bp indel polymorphisms in BV animals (P<0.01 and P<0.025). Because of the limited amount and sporadically poor quality of the tissue for DNA isolation, however, not all animals were tested for all markers. In BSE animals of the BV breed, the 12-bp del-allele was more frequent than in the control group (34.9%/16.6%). In addition, allele frequencies of SNPs 50000G A and SNP 50138G A were distributed differently in BV (P<0.05 and P<0.025; Table 2
and Fig. 2
A). Variation for the other SNPs did not reach statistical significance across breeds.
Haplotype analysis
On the basis of genotyping for nine polymorphisms located in the region surrounding exon 1 (Table 2
, excluding the 23-bp indel) haplotype distributions were calculated and compared using a permutation test for each breed separately. Significantly different haplotype distributions were found in all breeds, except RB (SB P<0.0052; RB P<0.1975; FV P<0.017: BV P<0.049). Haplotype frequencies exceeding 1% in either control or BSE animals are summarized in Table 3
. In BSE and control animals of SB, RB, and FV breeds haplotype A (111111111; identical to sequence AJ298878) was the most prominent. This haplotype occurred at a frequency of 40–65% in these breeds. However, BV animals exhibited three major haplotypes (occurring in 15–30%) due to the higher frequency of the 12-bp ins- and the 49871G-allele. In SB, haplotype A had a lower frequency in BSE animals (58.4%) compared to control animals (63.7%) but was found at higher frequency in all BSE animals of other breeds (Fig. 2B
, Table 3
). Haplotype B (211221222), the second most frequent haplotype in SB, RB, and FV controls showed a reduced frequency in BSE animals of all breeds, especially FV (3.6%/9.4%; Fig. 2B
). This suggested that haplotype B is associated with resistance to BSE infection.
Expression assays
The influence of 12-bp and 23-bp indels on gene expression was investigated in neuronal cells. Four DNA luciferase reporter constructs including either the ins- or the del-allele of both 23-bp and 12-bp indel polymorphisms (16)
were transfected into Neuro-2A and primary hippocampal cells. For both cell types a statistically significant difference between the expression of all four reporter gene constructs was observed (ANOVA P<0.05 for hippocampal and P<0.01 for Neuro-2A cells). Moreover, statistical analysis suggested that the difference between constructs depends on the 12-bp indel but not on the 23-bp indel (Fig. 3
C upper panels).
To study the influence of SNPs in the region surrounding the 12-bp indel representing the core promoter region (16)
, 5.2-kbp constructs (16)
were reduced to 1.1-kbp constructs and subcloned into the promoter-analyzing luciferase vector pGL3-Enhancer. The 1.1-kbp construct (Fig. 3A
) included the 5'-upstream region of exon 1, exon 1 and the 5'-part of intron 1 (nt 49247 – nt 50398). With this construct the expression was only slightly above the level of the control (pGL3-Enhancer without an insert, data not shown). In contrast, a 1.9-kbp construct, which included additionally exon 2, as well as 630 bp of the 3'-part of intron 1 resulted in expression levels similar to a 3.7-kbp construct described previously (16)
. Therefore exon 2 and/or the 3'-part of intron 1 were required for expression. To analyze the influence of the 12-bp indel, as well as neighboring SNPs on expression six different 1.9-kbp constructs, were created by amplification of individuals with corresponding haplotypes. In Neuro-2A cells, the highest mean expression level of 9.5 relative light units (RLUs) was observed in the haplotype F (111211111) representing the 12-bp ins version of the major haplotype A (Fig. 3C
, lower left panel). Nevertheless, the 12-bp ins-allele itself is not always associated with an increased expression level, as the construct with the significantly lowest expression level was found for haplotype B (211221222) in both Neuro-2A and hippocampal cells (Fig. 3C
, lower panel). All other constructs were not significantly different from the major haplotype A. However, no single SNP was identified to be responsible for the reduced expression level of haplotype B.
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DISCUSSION
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In both humans and sheep, a clear correlation of specific genotypes of the PRNP gene and resistance to TSE infection is known (22
, 23)
. Until recently, very limited information was available regarding bovine PRNP polymorphisms and their association to BSE. The identification of DNA polymorphisms in the bovine PRNP gene region and the availability of DNA samples from more than 290 German cows tested positive for BSE offered the possibility to analyze these polymorphisms for an association with BSE resistance/susceptibility (6
7
8)
.
The analysis of sequence variability in the bovine PRNP gene in German cattle by Sander et al. indicated an association of a 23-bp and a 12-bp indel polymorphism in the 5'-region of PRNP and susceptibility to BSE (10)
. The Sander et al. (10)
study was limited by the small number of control animals (8/breed) and the lack of breed differentiation within the 43 BSE animals. Very recently, two further studies were published investigating the association of microsatellite and indel polymorphisms in the genomic region of the PRNP gene and the 23-bp and 12-bp indels, respectively (24
, 25)
. Both groups used 250–280 animals out of the 400 animals tested positive for BSE in Germany and two different control animal groups of 376 and 584 individuals for breed-specific comparisons. Geldermann et al. (24)
genotyped three polymorphic loci in intron 1 and 2 of PRNP, the 12-bp indel (named REG2), R16, and R18, which revealed significantly different distribution in BSE and control animals, whereas two further microsatellites located upstream of the 23-bp indel showed no significant differences. The 23-bp and the 12-bp indel polymorphisms were used for an association study by Juling et al. (25)
, resulting in an association of both polymorphisms to BSE susceptibility. In accordance with our study, the main effect was found for the 12-bp indel. Microsatellites and other markers used for association studies frequently do not represent the underlying cause of differences in gene function observed. This implies that the polymorphic regions have to be characterized in more detail to isolate the functional polymorphisms. Our more refined analysis of SNPs in the surrounding of the 12 bp indel polymorphism fulfills these requirements. The replication of association studies using three different control groups strengthen the suggestion that specific haplotypes in the promoter region are involved in resistance/susceptibility to BSE infection.
In mice, transcript levels of the Prnp gene are correlated with susceptibility to prion infection. Transgenic mice expressing higher amounts of the prion protein have a higher incidence and shorter incubation times following inoculation with infectious prion protein PrPsc (26)
. In contrast, heterozygous knockout mice (Prnp+/–) exhibit a prolonged incubation time and Prnp–/– animals are resistant to the disease (27
, 28)
. Transferring these results to cattle, low-expressing promoter variants of PRNP lead to a decreased expression of the PRNP gene and less PrP protein. A reduced amount of endogenous PrPc protein diminished the chance to get converted into PrPsc and resulted in a prolonged incubation time. Thus, low-expressing promoter variants are expected to occur less frequently in BSE animals compared to controls.
For the 12-bp indel polymorphism we previously have shown that the ins-allele binds to the transcription factor SP1, which is known as a ubiquitously expressed activating transcription factor. In contrast to the hypothesis that an additional SP1 binding site would increase susceptibility to BSE by increasing the expression of the PRNP gene, the nonbinding 12 bp del-allele was overrepresented in BSE animals (this study and (24
, 25)
). Previously, reporter gene constructs with 5.2-kbp inserts, including the 23-bp in addition to the 12-bp indel polymorphisms in all four possible combinations transfected into bovine kidney and esopharyngeal cells had revealed lower levels of expression for the 23-bp ins/12-bp ins haplotypes compared to the del/del constructs (16)
. Analysis of the identical constructs in the mouse neuroblastoma cell line (Neuro-2A) or in rat primary hippocampal neurons revealed significant differences between groups in both cases (ANOVA, P<0.01; P<0.05). Nevertheless, statistical analysis suggested that the difference between constructs depends on the 12-bp indel but not on the 23-bp indel polymorphism. The discrepancy between recent and previous results (16)
may be due to the different origin and types of cells used for transfection assays (i.e., bovine vs. rat and mouse; kidney/esophagus vs. neuronal). Neuronal cells are the cell type affected by prion diseases; therefore, we suggest that expression of the PRNP gene in these cells may be more relevant. Thus, in this study, we focused on the core promoter region defined by Inoue et al. (17)
surrounding the 12-indel polymorphism, 200 bp upstream of exon 1, part of intron 1 and exon 2. All SNPs located in this core promoter region and identified in at least three breeds were genotyped for haplotype analysis and tested in the corresponding promoter constructs.
This study demonstrates that the 12-bp indel and its flanking SNPs are important for the PRNP expression. The reference sequence (AJ298878) proved to be the most prominent haplotype in all German breeds. The comparison of calculated haplotype distributions in control and BSE animals yielded significant differences in all breeds except RB. Indeed, promoter constructs of the five most frequent haplotypes exhibited differential expression levels. The highest expression levels in Neuro-2A cells were found in the rare haplotype F (11121111), which differs from the most prominent haplotype A only by the ins version of the 12-bp indel. The haplotype B (211221222), which also includes the 12-bp ins version but differs in 5 SNPs, exhibited the lowest expression level in both Neuro-2A cells and hippocampal cells. In agreement with the hypothesis above, the frequency of this haplotype was reduced in all breeds in the BSE group compared to the controls (Fig. 2B
). Two different transcription element search tools (TESS and MatInspector, data not shown) were used to predict possible binding sites for the alternative allelic versions of haplotype A and B, but the results they gave were not consistent. It is likely that the combination of certain SNPs rather than a single SNP is responsible for the regulation of transcription.
Currently, the pathological mechanisms of oral infection in BSE have not been elucidated. Nothing is known as to whether elevated expression of PRNP in brain is correlated with a higher risk for developing the disease or if an elevated expression in the gut-associated lymphoid or neuronal tissue is more relevant. Therefore, several tissues from animals with different haplotypes have to be investigated for in vivo expression to identify the relevant tissue. Quantitative RT-polymerase chain reaction (RT-PCR) amplifications of PRNP in 96 bovine tissue samples revealed no significant difference in the mean expression level of brain stem, spleen, and liver samples with respect to the genotype of the 23-bp and the 12-bp indel polymorphisms. In contrast, lymph node samples of animals heterozygous for the 23-bp indel and homozygous for the 12-bp ins allele had decreased PRNP expression compared to the other genotypes of the indel polymorphism, however, SNPs were not analyzed (16)
.
In comparison to sheep, BSE susceptibility of different bovine breeds and their association to polymorphisms is much weaker. The comparison of haplotype distribution of the four major German breeds clearly demonstrated that the BV breed had a different gene pool with breed-specific SNPs characterizing special haplotypes. Because of this observed difference in geno- and haplotype distributions, breed-specific association studies are required. Furthermore, epidemiology data also argue for a differential susceptibility between bovine breeds. Specifically, of all German cattle, 56.4% are SB, while only 35.8% of all BSE animals were classified as SB. The data presented here suggests that polymorphisms may be involved in the variability of susceptibility, although further genetic factors over and above the PRNP gene may also play a role. Analysis of the PRND gene, which is located directly adjacent to the PRNP gene, showed a weak association of two SNPs with BSE susceptibility. This may be due to linkage disequilibrium rather than being causal (11)
. A whole genome search for QTLs involved in BSE susceptibility identified two significant and four suggestive regions that were not identical to the localization of the PRNP gene (29)
. One or more of these loci may be involved additionally in breed-specific differences observed in the susceptibility of German BSE animals.
In this study, we have identified DNA polymorphisms in the promoter region of the bovine PRNP gene. Haplotype analysis of nine genotyped polymorphisms resulted in five haplotypes that were found frequently in four different German bovine breeds. The promoter constructs of these major haplotypes were tested for their expression, and haplotype B (21122122) was found to be the least expressed haplotype in both neuronal cell types. Interestingly, haplotype distribution was significantly different within all breeds except the RB animals. In agreement with the hypothesis that a low level of expression may be associated with a reduced susceptibility to BSE, the haplotype B was reduced in the BSE group of all breeds.
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ACKNOWLEDGMENTS
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We thank B. Orlicz-Welcz, I. Henz, P. Wenzeler, and S. Beck for excellent technical assistance and S. Neumann for support with the transfection experiments. For the donation of bovine sperm we are grateful to C. Leiding, Besamungsverein, (Neustadt/Aisch, Germany), D. Fresen, Rinderproduktion Niedersachsen, (Verden, Germany), W. Schütz, Rinderbesamungsstation, (Memmingen, Germany), and B. Wollgarten, Top Genetic, (Greifenberg, Germany). S. Stamm, Institute for Biochemistry (Erlangen, Germany) is acknowledged for providing the cell lines. We are grateful to Dr. Markus Kostrzewa and Bruker Daltonik GmbH for helpful discussions and for providing the MALDI-TOF mass spectrometer. K. Becker and S. Seeber are acknowledged for their critical reading of the manuscript. This work was supported by grants from the Bayerisches Staatsministerium für Umwelt, Gesundheit und Verbraucherschutz as part of the Bayerischer BSE-Forschungsverbund forprion (Erl-2) and the DFG (Schi 451/5–2).
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FOOTNOTES
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1 Present address: Department of Food Toxicology, University of Veterinary Medicine Hannover, Hannover, Germany. 
2 Present address: Institute of Genetics, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Received for publication October 18, 2006.
Accepted for publication December 14, 2006.
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ABSTRACT
INTRODUCTION
