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ANIMAL GENETICS |
Departamento de Producción Animal I, Universidad de León, 24071 León, Spain
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: prion protein gene • scrapie susceptibility • sheep • single nucleotide polymorphism detection
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
Genetic susceptibility to scrapie seems to be influenced by the host genotype at the PrP encoding locus. The PRNP gene has shown polymorphism at different amino acid positions, but susceptibility to scrapie in sheep is linked to variations at codons 136 (A/V), 154 (R/H), and 171 (Q/R/H), where alternative amino acids are indicated by single letter symbols. Not all theoretically possible haplotypes have been found; they are mainly restricted to ARQ, ARR, AHQ, ARH, and VRQ, resulting in 15 genotypes (Baylis and Goldmann, 2004). Recently, new haplotypes (VRR, AHR, and AHQ) have been identified at low frequency in some breeds, but their association with scrapie is unknown (Goldmann et al., 2005).
The ARR/ARR genotype is associated with resistance to conventional scrapie, whereas animals carrying the VRQ allele are the most susceptible. In breeds with low frequencies of the VRQ allele, the wild type ARQ is associated with the highest susceptibility to scrapie. The effect of the other alleles is subtle and differs for different populations. Recently, rare and atypical forms of scrapie have been reported; these strains primarily affect sheep that rarely succumb to classical strains (Baylis and McIntyre, 2004). This association between the PRNP genotype and susceptibility to scrapie is the basis of breeding programs for scrapie resistance in different countries (European Commission, 2003).
We describe the method for genotype identification used with 2 Spanish dairy sheep breeds, Churra and Castellana, to ascertain their protective status against scrapie. In our screening, we found a new allele at codon 154 that may be linked to host susceptibility to scrapie.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The sampling procedure for Churra sheep included 325 rams used in the AI program and 3,316 animals belonging to 55 flocks distributed as follows: the complete set of rams used for natural mating in these flocks (1,175 animals ranging between 2 and 9 yr old), a random sample of 743 young male lambs 1 to 9 mo old (8 to 25 per flock), and a random sample of 1,398 ewes (15 to 30 from each flock) with ages ranging between 1.5 and 8 yr.
For the Castellana breed, 35 AI rams were sampled, and following the same strategy as above, the rest of animals were sampled in 26 flocks as follows: the complete set of rams used for natural mating (683 animals ranging from 2 to 10 yr old), a random sample of 491 (12 to 28 by flock) young male lambs 1 to 11 mo old, and a random sample of 380 ewes (8 to 25 in each flock) ranging from 1.5 to 8 yr old.
DNA Extraction and PCR Amplification
The DNA was extracted from 5 mL of whole EDTA-treated blood (Venoject tubes, Terumo Europe, Leuven, Belgium) using the salting-out isolation procedure (Miller et al., 1988).
A fragment of 356 bp of the open reading frame of the PRNP gene was amplified by PCR with primers Scrapcr_UP and Scrapcr_DN (sequences shown in Table 1). These primers encompassed codons 95 to 212 and were designed from the PRNP sheep gene (GenBank accession number U67922). The PCR amplification was carried out in a 10-µL reaction volume containing 50 ng of genomic DNA, PCR Gold Buffer (Applied Biosystems, Foster City, CA), and 0.5 U of AmpliTaq Gold (Applied Biosystems, Foster City, CA). The temperature profile of PCR consisted of denaturation at 95°C for 5 min, followed by 35 cycles of 30 s at 94°C, 40 s at 62°C, and 40 s at 72°C. Finally, an extension step of 10 min at 72°C was performed.
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Minisequencing or Primer Extension Technique
For the minisequencing reaction, 4 primers were designed to detect the polymorphisms at codons 136 (scragen1), 154 (scragen2), and the 2 polymorphisms at codon 171 [second position called 171a (scragen3) and third position or 171b (scragen4)]. The sequence of each primer is given in Table 1
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The single-base extension reaction used an internal extension primer that was designed so that its 3' end annealed adjacent to the polymorphic base pair. The reaction was essentially a sequencing reaction containing only dye-terminator dideoxynucleotides that could not be extended further. The identity of the base added (or bases in the case of a heterozygote) was determined by measuring fluorescence polarization. The dideoxynucleotides were labeled with different fluorochromes. The temperature profile of PCR consisted of denaturation at 96°C for 5 min, followed by 25 cycles of 10 s at 96°C and 35 s at 60°C.
After the minisequencing reaction, 1 U of shrimp alkaline phosphatase (USB Corp.) was added to the tube and incubated at 37°C for 1 h, and the enzyme was inactivated at 80°C for 15 min. The product was electrophoresed in an ABIPRISM 377 automatic sequencer (Applied Biosystems). Fragments were analyzed using GeneScan and Genotyper software (v. 3.7; Applied Biosystems).
Genotyping using this procedure allows for an accurate identification in a one-tube reaction of the PRNP 136-154-171 genotypes most frequent in European sheep breeds. The primers detect the mutated nucleotide at each codon as follows: the second position in codon 136 (GCC or GTC), which produces an amino acid substitution A to V; the central nucleotide at codon 154 (CGT or CAT), which encodes the amino acid A or H; and the second and third positions at codon 171 (CGG; CAG or CAT), which carry amino acids A, Q, or H at this position, respectively. To enhance the reaction, 2 of the primers were designed complementary to the direct strand (scragen1 at codon 136 and scragen3 at codon 171a), whereas the other 2 were designed complementary to the reverse strand (scragen2 at codon 154 and scragen4 at codon 171b). Furthermore, primers named scragen2, scragen3, and scragen4 were modified by adding a poly-TA tail at the 5' end (shown as underlined in Table 1) allowing their identification by size differences through a single electrophoresis by an automatic sequencer.
Validation by Sequencing
To evaluate the reliability of the primer extension assay, and to confirm the phase in animals heterozygous at 2 of the 3 codons, we carried out a sequencing validation study.
A total of 140 animals were chosen for direct sequencing of the 356-bp fragment of the PRNP gene. The number selected per genotype ranging from 4 to 15. Direct sequencing of both strands of the PRNP fragment (codon 95 to 212) was achieved with scrapcr_UP and scrapcr_DN primers. Sequencing was performed in a 10-µL reaction with Big-Dye terminator kit v3.1 in a DNA Thermal Cycler 9700 (Applied Biosystems) and an ABI PRISM 377 automatic sequencer (Applied Biosystems). Data were collected using Sequencing Analysis software v.3.7 (Applied Biosystems) and analyzed with the Staden sequence analysis package (version 1.5; Staden, 1996).
To determine the possibility of the presence of rare haplotypes, for animals heterozygous in 2 of the 3 codons (ARR/AHQ, ARR/VRQ, AHQ/ARH, AHQ/VRQ, and ARH/VRQ), the PCR fragments of selected animals (4 to 15 animals for each genotype) were cloned into the pCR2.1 vector using the TOPO TA-cloning kit (Invitrogen, Paisley, UK). Eight clones of each animal were selected and sequenced in both strands as indicated above. Furthermore, the progeny from animals carrying these genotypes (heterozygous in 2 positions) were analyzed to determine the phase. Paternity in all mates (85) was checked with a set of 10 microsatellite markers.
For the new codon 154-leucine variant, a PCR fragment belonging to animals with this allele was cloned and sequenced on both DNA strands to demonstrate the presence of the nucleotide thymine at codon 154. Eight clones were obtained from each animal.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To provide a proof-of-principle of the genotyping procedure, 140 sheep associated with 15 possible variants (4 to 15 per genotype) were analyzed by direct PCR sequencing. Typing results were identical in both methods, confirming the reliability of primer extension in SNP genotyping.
In the case of detection of rare haplotypes from the 2 approaches, sequencing of cloned PCR fragments and phase determination in the pedigree showed no deviation from the main 5 haplotype patterns. All animals that displayed infrequent haplotypes were analyzed by both procedures.
A New Leucine Allele at Codon 154
In the genotyping process, we found a rare allele at codon 154 in 2 sheep from the same Churra flock, a ewe and her lamb. The genotype indicated that codon 154 had a thymine in the second position, giving a CTT triplet that codes for leucine. Results for the ewe were similar to lamb but with a heterozygous VRQ/ALQ genotype. To verify the relationship between these 2 animals, they and the supposed ram were subjected to paternity control with 10 microsatellite markers, which confirmed the kinship of the 3 animals.
The PCR fragments for the ewe and lamb were cloned and sequenced as indicated in the Materials and Methods section. Figure 1 shows the amino acid sequence represented by the single letter code, inferred from translation of 2 cloned fragments from the heterozygous ARQ/ALQ lamb. The 2 alternative sequences at codon 154 are the wild type CGT (Arg or R) and the new allele CTT (Leu or L). The 2 haplotypes, ARQ and ALQ, show polymorphism in different positions: in codon 101, a transition produces a change from arginine to glutamine (R101N); in codon 176, a transversion A to C produces a change from asparagine to lysine (N176K). These mutations have been described previously in European breeds (Goldmann et al., 2005). Furthermore, 2 silent mutations have been detected in codons 231 and 237.
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), with the exception of described R154L mutation.
Allele and Genotype Frequencies
Table 2 shows the genotype frequencies for 3,641 Churras and 1,589 Castellanas. Frequencies for the 2 new genotypes found in Churra sheep (VRQ/ALQ and ARQ/ALQ) are not shown.
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General figures were very similar for Castellana sheep, where the wild type ARQ/ARQ reached 47.9%, and ARR/ARQ animals represented 28.4% of the analyzed population. Apart from the new ALQ allele, all the possible genotype combinations were present, with 4% of the animals carrying the VRQ allele. None of the AI rams of the Castellana breed belonged to the scrapie-resistant group, and 18 animals (72%) showed the ARQ/ARQ wild type.
The PRNP allele frequencies are summarized in Table 3. Frequencies are presented by group of sampled animals: AI rams, natural mating rams, lambs, ewes, and in the overall population for each breed. No differences were observed between the 4 groups analyzed within each breed. One important point is the situation of the AI rams that are progeny tested (Churra sheep only).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Several methods are currently available for SNP genotyping of the PRNP gene, including direct sequencing and matrix assisted laser desorption ionization time-of-flight mass spectrometry (Humeny et al., 2002), single strand conformation polymorphisms (Zhou et al., 2005), amplification refractory mutation system (Buitkamp and Semmer, 2004), PCR-restriction fragment length polymorphisms and TaqMan assay Minor Groove binding (García-Crespo et al., 2004), dual multiprobe assay (Van Poucke et al., 2005), and others based on the same principle as proposed here; that is, minisequencing or primer extension assay (Zsolnai et al., 2003; Vaccari et al., 2004). The sequencing method is the best for giving the most detailed information not only for target codons, but also for describing all possible PRNP polymorphisms. Nevertheless, sequencing is very time consuming and is not appropriate for screening a large number of animals.
Herein we have developed a reliable procedure based on multiplex minisequencing using SNaPshot (Applied Biosystems), similar to those proposed by Zsolnai et al. (2003) and Vaccari et al. (2004). This technique is easy to standardize and interpret, has a high automation potential, and is an optimum medium-throughput approach. However this procedure has lower throughput than other genotyping procedures based on gel-free methodologies such as matrix assisted laser desorption ionization time-of-flight mass spectrometry, pyrosequencing, Taqman, or melting curve analyses (Van Poucke et al., 2005) that are more reliable for screening of large populations. The accuracy of the procedure allows for the unambiguous description of the genetic variability associated with proneness to scrapie in sheep, and its reliability has been demonstrated in all samples checked by a perfect match with the results obtained by direct sequencing.
A potential shortcoming of minisequencing is that, in the case of animals heterozygous for more than one position, it does not permit the identification of the phase of alleles, making it necessary to clone in order to unequivocally verify their phase. This limitation could be important in some specific populations in which uncommon haplotypes are known to occur, as in breeds from Germany and Slovakia displaying AHR and VRR genotypes (Kutzer et al., 2002) or the VHQ genotype founded in Oklahoma sheep (DeSilva et al., 2003). In most ovine populations, this drawback should be negligible because these new variants have not been described. This is the case for the Churra and Castellana breeds analyzed in this study, with all the haplotypes described belonging to the 5 classical variants. In any event, it is necessary to bear these new alleles in mind when screening a population never before analyzed, and to screen by cloning and sequencing some individuals heterozygous at 2 codons or by using another SNP diagnostic method allowing for the determination of the haplotype phase, such as pyrosequencing (Syvanen, 2001).
On the other hand, minisequencing is very reliable and allows distinction of the exact base mutation and detection of the appearance of new alleles in the nucleotides analyzed. This was demonstrated in the determination of the ALQ variant because the new leucine allele at codon 154 was detected owing to the flexibility of minisequencing. Classical procedures like PCR-RFLP or frequently used high-throughput methods as Taqman assay will skip these newly mutated forms.
Genotype frequencies found in Churra and Castellana sheep are similar to those described in other Spanish sheep (Acin et al., 2004; García-Crespo et al., 2004). In most populations, the most widespread genotypes are those carrying ARQ, with an allele frequency of nearly 70%. The prevalence of scrapie in Spain may be considered low with 48 positive sheep in 27,780 animals sampled during the monitoring program for transmissible spongiform encephalopathies in small ruminants in 2004 (European Commission, 2005). The high frequency of the wild type ARQ allele may be linked to the development of autochthonous populations in environments where there has been no scrapie challenge. The low frequency of the resistant ARR allele (lower than 0.25) could limit the establishment of a selection program for resistance to scrapie without affecting the dairy selection schemes presently underway. One important point to verify will be the possible association between selection traits and PRNP or closely linked genes (De Vries et al., 2005; our unpublished observations). However, the main problem will be a reduction in the number of animals available for selection as progenitors of the next generation if selection pressure is high during the first steps.
For the Churra breed analyzed here, only one resistant ram (ARR/ARR) belonging to the AI program with positive EBV for milk was detected. Furthermore, 38% of the rams with positive EBV were heterozygous, and the 4 top rams for milk production were homozygous ARQ/ARQ. One possibility for the use of these top animals should be mating with homozygous ARR/ARR elite females to have heterozygous lambs that could be good candidates for the progeny test. Therefore, initial selection for scrapie resistance must be carefully performed in order to prevent possible bottlenecks or inbreeding problems in the future.
The occurrences of new haplotypes such as ALQ in this study, or the ARK haplotype described by Guo et al. (2003), are not widespread in the sheep populations studied to date. The new ALQ allele has been detected in 3 different breeds belonging to the same phylogenetic trunk (Churro, of long coarse-wool breeds) at frequencies below 1%. Research into the behavior of these new variants and the new haplotypes described in European breeds regarding susceptibility to scrapie is yet to be done. One of the most important points in this regard is the role of different polymorphisms on the conformational conversion of cellular protein (PrPC) into a pathogenic "scrapie" isoform (PrPSC; Prusiner, 2004). This feature is the fundamental event in the pathogenicity of transmissible spongiform encephalopathies (Prusiner, 2004).
The role played by codon 154 and its significance in scrapie resistance are controversial. In some scrapie strains such as type C, susceptibility is linked only to polymorphism at codon 171, and the amino acid at position 154 is not considered (O’Rourke et al., 1997). Consequently, in some countries where type C is the most prevalent (e.g., the United States), codon 154 is not examined in eradication programs. Nevertheless, other research shows that the R154 (arginine) allele is associated with rapid onset of symptoms, whereas H154 (histidine) involves a delayed onset of symptoms for classical scrapie strains (Hunter, 1997). Furthermore, the role of this protein region in susceptibility to scrapie has been strengthened by a recently discovered strain of scrapie, called Nor98, that primarily affects animals carrying histidine at codon 154 (AHQ), which is generally associated with resistance (or, at most, low susceptibility) to conventional scrapie (Moum et al., 2005).
This new genetic variant might have a different susceptibility to scrapie because codon 154 belongs to the helix H1 region of the mature protein, which is considered to play a major role in the conformational conversion of cellular to pathogenic protein (Kozin et al., 2001). Furthermore, in a recent study, Megy et al. (2004) showed that the potential nucleation site for the molecular rearrangement of the prion protein might be localized to a region spanning residues 152 to 156. In the new haplotype described here a nonpolar aliphatic-hydrophobic amino acid (leucine) replaced the wild-type polar hydrophilic, positively charged arginine. The consequence of this mutation for conformation of the mature protein and for the susceptibility to scrapie needs to be investigated.
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Footnotes |
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2 Corresponding author: jjarranz{at}unileon.es
Received for publication May 13, 2005. Accepted for publication September 28, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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