- Email: [email protected]
Simultaneous detection of eight single nucleotide polymorphisms in the ovine prion protein gene
ARTICLE IN PRESS
Molecular and Cellular Probes 21 (2007) 363–367 www.elsevier.com/locate/ymcpr
Simultaneous detection of eight single nucleotide polymorphisms in the ovine prion protein gene Bernhard F. Benkela,, Edith Valleb, Nathalie Bissonnettec, A. Hossain Farida a
Department of Plant and Animal Sciences, Nova Scotia Agricultural College, Truro, NS, Canada B2N 5E3 Bioproducts and Bioprocesses Program, Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1 c Bioproducts and Bioprocesses Program, Dairy and Swine Research and Development Centre, Lennoxville, QC, Canada J1M 1Z3
b
Received 8 January 2007; accepted 1 May 2007 Available online 18 May 2007
Abstract Amino acid polymorphisms in the prion protein gene (PrP) affect the susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy (TSE). In particular, amino acid substitutions at codons 136, 154 and 171 of the ovine PrP gene are associated with different degrees of susceptibility to the classical form of scrapie, caused by ‘typical’ scrapie strains. Existing genotyping tests for scrapie susceptibility normally interrogate only the single nucleotide polymorphisms (SNPs) most relevant to ‘typical’ strains. Recently, however, a number of novel variants of the scrapie agent have been discovered. The ability of these new, ‘atypical’ scrapie variants to infect sheep that are resistant to ‘typical’ variants has raised concerns about the reduction in genetic variability that may result from intense selection for resistance to classical scrapie. Furthermore, a growing interest in a potential role for specific PrP genotypes in modulating performance traits is also driving a move toward more extensive characterization of haplotypes at the PrP locus. Here, we describe a single-tube method for the interrogation of eight SNPs within seven codons (112, 136, 141, 154, 171, 231 and 241) of the ovine PrP gene. This method is as accurate as sequencing, yet more affordable, and can easily be automated for high-throughput sample screening. Moreover, it can be modified to accommodate genetic variations that are found in local and heritage breeds. r 2007 Elsevier Ltd. All rights reserved. Keywords: Genotyping assay; Scrapie resistance; Single-base extension
1. Introduction With over 30 SNPs already reported, the ovine prion gene (PrP) shows an unusually high level of genetic variation (reviewed in [1,2]). Of particular interest to the sheep industry are mutations at codons 136, 154 and 171 which are associated with susceptibility to natural and experimental scrapie, i.e. the haplotype A136R154R171 has been shown to confer resistance to typical scrapie strains whereas V136R154Q171 is common amongst susceptible sheep [3]. The demonstrated association between PrP haplotype and scrapie susceptibility has led a number of countries to adopt selection programs designed to increase the frequency of genotypes that show resistance to typical scrapie. However, concerns Corresponding author. Tel.: +1 902 893 6165; fax: +1 902 895 6734.
E-mail address: [email protected] (B.F. Benkel). 0890-8508/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2007.05.002
have been raised regarding the intense selection for resistant genotypes which has been standard practice in many countries. The reasons for these concerns are outlined below. Firstly, sheep that show resistance to classical scrapie based on their genotypes at codons 136, 154 and 171 are susceptible to atypical variants [4,5]. Thus, selection for resistance to typical scrapie agents could put sheep populations at risk to infections by atypical variants such as Nor98, for example, the susceptibility to which is affected by a polymorphism at codon 141 [5]. Secondly, intense selection for a single PrP haplotype will cause a reduction in genetic variability overall and an increase in inbreeding, particularly in breeds where resistant genotypes are present at a low frequency [6,7]. Thirdly, in addition to the well established link between PrP genotypes and scrapie resistance, there is also evidence, albeit less compelling, for associations between
ARTICLE IN PRESS B.F. Benkel et al. / Molecular and Cellular Probes 21 (2007) 363–367
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haplotypes at the PrP locus and performance traits of importance to sheep breeders, mediated either through pleiotrophic effects of the PrP gene or through linkage of specific alleles at the PrP gene and production trait genes [8–12]. The fact that only a limited amount of information is currently available to support such associations may be due, at least in part, to a lack of resolution in PrP haplotype discrimination resulting from the use of the common 3-codon PrP genotyping assays. More extensive genotyping, beyond the standard 3-codon analysis, using the assay described here for example, may well reveal associations that have so far remained obscured. These issues speak to the need for high-throughput methods that yield more information on PrP alleles than the standard 3-codon assays provide. A variety of methods have been developed for restricted genotyping sheep at the PrP locus. However, gel-based assays such as restriction fragment length polymorphism [13], denaturing gradient gel electrophoresis [14,15], allele-specific hybridization [16], single-strand conformational polymorphism analysis [17], and allele-specific amplification [15] do not lend themselves to automation and are time consuming since they require a separate assay to be performed for each SNP. Non-gel-based methods, such as TaqMan/real time PCR [18], DNA sequencing, and single-base extension [2,19,20] are more suited for large-scale genotyping. The latter represents a reliable and inexpensive (especially in comparison to DNA sequencing), high-throughput method for the determination of haplotypes of the PrP gene, which has been used previously for 3-codon genotyping. Here, we describe an extension of this method for the simultaneous identification of eight SNPs in seven codons of the ovine PrP gene.
2. Materials and methods 2.1. Primers and PCR amplification A 1126 bp long segment that spans the entire coding region of the PrP gene was amplified by the polymerase chain reaction (PCR) using a forward primer (50 -GAGGAA GAGTTGTGTTACTACT) and a reverse primer (50 -GTCTGCTTGTCATTTCCCAGTG). Amplification was performed in 10.0 mL reactions containing 1 PCR buffer (Applied Biosystems Inc., Foster City, CA), 0.2 mM each dNTP, 500 nM each primer, 0.625 unit of AmpliTaq Gold (Applied Biosystems), 2.5 mM MgCl2 and 50–100 ng of genomic DNA (final concentration). Amplification was performed in an Eppendorf Mastercycler S (Hamburg, Germany), which was programmed for an initial denaturation for 5 min at 94 1C, followed by 35 cycles of denaturation at 94 1C for 30 s, annealing at 58 1C for 30 s, and extension at 72 1C for 60 s, followed by a 6 min final extension at 72 1C. The thermal cycler’s ramping speed was set at 18% of maximum. PCR products were purified by Exo SAP-IT (Amersham) according to the manufacturer’s instructions. 2.2. SNP interrogation SNP interrogation was carried out using a single-base extension (SNaPshot) method essentially as recommended by the manufacturer (Applied Biosystems). Briefly, an interrogation primer was designed for each of the eight target SNPs (Table 1). Each primer consists of a segment designed to anneal immediately 50 or 30 to the polymorphic position in the PrP gene, and a variable-length nonhomologous polynucleotide tail with a minimal secondary
Table 1 Interrogation primers for the detection of eight SNPs at seven codons of the PrP gene Codon no.
Wild type
Interrogation primers (50 –30 )a
Mutant
Codon
aa
Codon
aa
112
ATG
M
ACG
T
GTAAGCCAAAAACCAACA
136
GCC
A
GTC ACCc
V T
aacccgacccaaagacGCTACATGCTGGGAAGTG
141b
CTT
L
TTT
F
aaacccgactaaaaaccccgactacccaaagactaaaTAGTCATTGCCAAAATGTATAA
154
CGT
R
CAT CTT
H L
aaaaaaaaaaaaaaTATGAGGACCGTTACTATC
171A
CAG
Q
CGG AAGc
R K
gactgactgactgactgactACTACAGACCAGTGGATC
171Bb 231 241b
CAG AGG CCT
Q R P
CAT CGG TCT
H R S
gactaaaagactaaaagactACAAAGTTGTTCTGGTTACTATA gactaaaagactgactaaaaaaaagactAATCCCAGGCTTATTACCAA aaaaacccccaaaaaccccccccccaaaaacGATGAGGAGGATCACAGGAG
a
Underlined sequences indicate the region with homology to the PrP gene. Sequences shown in the lowercase are nonhomologous tails. These nucleotides are detected on the complementary DNA strand. c Rare variants, not investigated. b
ARTICLE IN PRESS B.F. Benkel et al. / Molecular and Cellular Probes 21 (2007) 363–367
structure. SNP interrogation is achieved by primer extension using fluorescently labeled dideoxy nucleotide triphosphates (ddNTPs). The concentration of each of the SNP interrogating primers in the SNaPshot reaction was adjusted in order to equalize SNP peak heights. Final primer concentrations were 0.125 mM, except for primers 171B, 154 and 231, whose concentrations were adjusted to 0.625, 0.0625, and 0.225 mM, respectively. A reaction mixture containing 0.5 mL of primer mix (see above), approximately 100 ng of PCR product, 2.5 mL SNaPShot Multiplex reagent (Applied Biosystems), and 1.5 mL ddH2O was prepared and subjected to 25 cycles of denaturation at 96 1C for 10 s, annealing at 50 1C for 5 s and extension at 60 1C for 30 s in an Eppendorf Mastercycler S. Unincorporated ddNTPs were removed by treating the reactions with shrimp alkaline phosphatase (SAP-IT; Amersham) in 96-well plates using an epMotion 5075 liquid handling robot (Eppendorf). Following SAPIT treatment, 1 mL of the single-base extension reaction was added to 10 mL of a 40:1 mixture of Hi-Di fomammide and GeneScan 120 Liz size standard (Applied Biosystems), loaded into an optical 96-well plate, and resolved on an ABI 3130 capillary DNA analyzer with POP7 polymer (Applied Biosystems). The results were analyzed using GeneMapper software and further interpreted using a program written in-house in Visual Basic to convert SNaPshot results to haplotyes and genotypes at the PrP locus. 2.3. DNA samples DNA samples used for validation were selected from a DNA bank of over 7000 individuals from more than 50 primarily Canadian sheep breeds. DNA was extracted from fresh blood using the high salt procedure of Montgonery and Sise [21]. The complete coding sequence of the PrP gene of approximately 70 sheep from various breeds was sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and the products resolved on an ABI 3130 Genetic Analyzer according to the manufacturer’s instructions. From these 70 sheep, a panel of 25 sheep with 22 different genotypes that covered all of the polymorphisms at the seven codons interrogated in the test was selected for developing and validating the assay. The 400 animals used to further confirm the accuracy of the SNaPshot assay, compared to 3-codon test, in a highthroughput setting had been previously typed at three codons within the PrP gene using a standard RFLP-PCR assay (A. Farid, unpublished). 3. Results and discussion 3.1. Assay design The procedure described here involves the amplification by PCR of a single amplicon followed by a single, multiplexed base extension reaction, to facilitate the rapid
365
and accurate determination of eight SNPs per individual at the PrP gene. A similar method has been used previously to simultaneously genotype four SNPs at three codons within the PrP gene [2,19,20]. In the assay developed in this study, the increase in information content from four to eight SNPs is achieved without an increase in the cost of the assay, either for materials or labor. Moreover, eight SNPs is not the upper limit for this procedure and it is likely that the SNaPshot approach can be expanded to 12 SNPs per lane without incurring any significant increase in cost per sample. The additional information content afforded by the expanded assay can be employed to address the resistance of sheep to ‘atypical’ scrapie agents, or to gather other information regarding the PrP gene associated with phylogenetic or production parameters. For the test described here, the primary aim was to use some of the additional capacity of the SNaPshot test for the identification of haplotypes within the coding region of the PrP gene that are relevant to the sheep breeds commonly found in Canada. In addition to the usual codons 136, 154 and 171, we included codons 112, 141, 231, and 241 in our test because they have the highest frequency among rare variants of the PrP gene in several British and other European breeds that form the base of the Canadian sheep population [1]. Furthermore, the amino acid phenylalanine (F) at codon 141 increases susceptibility of sheep to Nor98, a strain of the scrapie agent recently discovered in some European countries [10,22–25], whereas substitution of the amino acid threonine (T) with methionine (M) at codon 112 may have some protective effect against the typical scrapie agent [1,26]. The codon 231 variant is a silent mutation. It was included in the assay because it is polymorphic in several European and Asiatic breeds, is found at a high frequency in some of these breeds [1,2,26–31], and our results show that it segregates independently of codon 241 (data not shown). Thus, this selectively neutral nucleotide substitution may be valuable for phylogenetic studies.
3.2. Assay validation Initially, each of the eight SNP interrogation primers was tested individually on a small number of DNA samples from a panel with known nucleotides at the target positions within the PrP gene (see Section 2). Multiplexing of all eight primers in a single reaction necessitated adjustments in the concentrations of individual primers in order to normalize peak heights between SNPs on electropherograms. A typical SNaPshot profile is shown in Fig. 1. Once the assay had been optimized, SNaPshot profiles were derived for a panel of 25 sheep with a wide range of genotypes that had previously been sequenced at the PrP gene as well as over 400 sheep that had previously been genotyped at codons 136, 154 and 171 (see Section 2). The results of the three different tests were identical without exception, indicating the high degree of accuracy of the single-base extension method; which is also in agreement
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B.F. Benkel et al. / Molecular and Cellular Probes 21 (2007) 363–367
Fig. 1. SNaPshot profile analysis. A typical SNaPshot profile is shown. Gray bars above the chromatogram indicate the bins delimiting peak positions within the profile for the eight individual SNPs (one bin for each SNP). Numbers embedded within gray bars indicate codons within the PrP peptide harboring the respective SNPs. Colors of the peaks within the profile are as follows: orange, size standard; red, ‘T’; blue, ‘G’; green, ‘A’; and black, ‘C’ (see Table 1 for interpretation of SNaPshot pattern and conversion of SNP calls to amino acids).
with the reports of Zsolnai et al. [19], Vaccari et al. [20], and Alvarez et al. [2].
3.3. Assay constraints The single-base extension (SNaPshot) method is based on the annealing of SNP interrogation primers to complementary sites on template DNA, and the subsequent extension of the primers by a single fluorescently labeled dideoxy nucleotide triphosphate (ddNTP). On a global basis, the ovine PrP gene is a problematic target for SNaPshot interrogation since it contains an unusually large number of SNPs. This high SNP density can complicate the design of effective, universal SNP interrogation primers. For example, there are rare SNPs in some breeds near the interrogated nucleotides within codons 136 and 171 (see Table 1). Prior knowledge of local variants can be used to modify the assay to take into account such breed-specific SNPs. For example, at codon 136, the interrogating primer used in the current assay was designed to discriminate between the nucleotides at the second position (GCC and GTC), but a ‘G’ to ‘A’ substitution at the first position within codon 136 has been reported in at least one breed [31]. The current (upper strand) primer used to interrogate the second position within codon 136 will yield a ‘null’ result for a haplotype containing the ‘G’ to ‘A’ substitution at the first position. In homozyogous ‘A/A’ individuals this is obvious from the SNaPshot profile. In heterozygous individuals, however, the result will be an incorrect genotype call. Designing a primer for the opposite (lower) strand is not a preferred option because both codons 137 (ATG and ACG) and 138 (AGC and AAC) are polymorphic in some breeds [1]. In order to overcome this problem, a modification could be employed that involves the use of overlapping SNP
interrogation primers targeting different positions within the same codon. For example, in the case of codon 136 in the ovine PrP gene, this problem can be approached by incorporating a pair of SNP interrogation primers for codon 136 into the SNaPshot panel as follows: (1) an oligomer that interrogates the first position within the codon; and (2) a primer that shares an identical core with the first primer, but with a ‘G/A’ degeneracy in its 30 position designed to interrogate the second position of the codon irrespective of the nucleotide at the 1st position. A similar approach can be used in other situations that require genotyping of adjacent nucleotide positions, for example to provide full 3-position genotyping within codon 171. It should be noted that complications arising from primer mismatch are not specific to SNaPshot tests, instead this is a common feature of any test that is based on the use of sequence-specific primer annealing, and it also affects genotyping assays based on other methodologies such as restriction fragment length polymorphism analysis [1]. In addition, there are rare instances where the genotype information provided by SNaPshot tests cannot be resolved into haplotypes. For example, the genotypes CAG/CGT and CGG/CAT code for RQ and RH, respectively, at codon 171. However, the SNaPshot patterns for these two genotypes are identical and the haplotypes cannot be resolved by the single-base extension pattern unless combined with genotype information from related individuals. On the other hand, tri-allelic SNP positions do not pose a problem, and the SNaPshot assay will detect the rare arginine (R) to leucine (L) substitution at codon 154 recently reported by Alvarez et al. [2]. 4. Summary We have developed a single tube method for interrogating eight SNPs within seven codons of the ovine PrP gene.
ARTICLE IN PRESS B.F. Benkel et al. / Molecular and Cellular Probes 21 (2007) 363–367
The method, which relies on single-base extension, is as accurate as DNA sequencing, but provides genotype information at a fraction of the cost, and is amenable to high-throughput screening. This assay is currently in use as the standard for scrapie resistance genotyping by the Atlantic Research Centre for Agricultural Genomics at the Nova Scotia Agricultural College, which has performed the bulk of the PrP genotyping for Canadian sheep to date. Acknowledgments The technical assistance of K. Benkel and T. Crossman is gratefully acknowledged. Financial support for this project was received from Agriculture and Agri-Food Canada through the Agri-Futures Nova Scotia program, the Investment Agriculture Foundation of British Columbia, the Technology Development Program of the NS Department of Agriculture, and the Canada Research Chairs program. References [1] Goldmann W, Baylis M, Chihota C, Stevenson E, Hunter N. Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes. J Appl Microbiol 2005;98: 1294–302. [2] Alvarez L, Arranz JJ, San Primitivo F. Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep. J Anim Sci 2006;84:259–65. [3] Baylis M, Chihota C, Stevenson E, Goldmann W, Smith A, Sivam K, et al. Risk of scrapie in British sheep of different prion protein genotype. J Gen Virol 2004;85:2735–40. [4] Le Dur A, Beringue V, Andreoletti O, Reine F, Lai TL, Baron T, et al. A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes. Proc Natl Acad Sci USA 2005;102:16031–6. [5] Moum T, Olsaker I, Hopp P, Moldal T, Valheim M, Moum T, et al. Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases. J Gen Virol 2005;86:231–5. [6] Drogemuller C, Leeb T, Distl O. PrP genotype frequencies in German breeding sheep and the potential to breed for resistance to scrapie. Vet Rec 2001;149:349–52. [7] Acutis PL, Sbaiz L, Verburg F, Riina MV, Ru G, Moda G, et al. Low frequency of the scrapie resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed. J Gen Virol 2004;85:3165–72. [8] de Vries F, Borchers N, Hamann H, Drogemuller C, Reinecke S, Lupping W Distl O. Associations between the prion protein genotype and performance traits of meat breeds of sheep. Vet Rec 2004;155: 140–3. [9] Brandsma JH, Janss LLG, Visscher AH. Association between PrP genotypes and litter size and 135 days weight in Texel sheep. Livest Prod Sci 2004;85:59–64. [10] Brandsma JH, Janss LLG, Visscher AH. Association between PrP genotypes and performance traits in an experimental Dutch Texel herd. Livest Pro Sci 2005;95:89–94. [11] Alexander BM, Stobart RH, Russell WC, O’Rourke KI, Lewist GS, Logan JR, et al. The influence of genotypes at codon 171 of the prion protein gene (PRNP) in five breeds of sheep and production traits of ewes associated with those genotypes. J Anim Sci 2005;83:455–9.
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[12] Isler BJ, Freking BA, Thallman RM, Heaton MP, Leymaster KA. Evaluation of associations between prion haplotypes and growth, carcass, and meat quality traits in a Dorset X Romanov sheep population. J Anim Sci 2006;84:783–8. [13] Hunter N, Goldmann W, Benson G, Foster JD, Hope J. Swaledale sheep affected by natural scrapie differ significantly in PrP genotype frequencies from healthy sheep and those selected for reduced incidence of scrapie. J Gen Virol 1993;74:1025–31. [14] Laplanche JL, Chatelain J, Westaway D, Thomas S, Dussaucy M, Brugere-Picoux J, et al. PrP polymorphisms associated with natural scrapie discovered by denaturing gradient gel electrophoresis. Genomics 1993;15:30–7. [15] Belt PBGM, Muileman IH, Schreuder BEC, Bos-de-Ruijter J, Gielkens ALJ, Smits MA, et al. Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol 1995;76:509–17. [16] Hunter N, Moore L, Hosie BD, Dingwall WS, Greig A. Association between natural scrapie and PrP genotype in a flock of Suffolk sheep in Scotland. Vet Rec 1997;140:59–63. [17] Zhou H, Hickford JGH, Fang Q. Determination of alleles of the ovine PRNP gene using PCR-single-strand conformational polymorphism analysis. J Anim Sci 2005;83:745–9. [18] Van Poucke M, Vandesompele J, Mattheeuws M, Van Zeveren A, Peelman LJ. A dual fluorescent multiprobe assay for prion protein genotyping in sheep. BMC Infect Dis 2005;5:13–21. [19] Zsolnai A, Anton I, Ku¨hn C, Fe´su¨s L. Detection of single-nucleotide polymorphisms coding for three ovine prion protein variants by primer extension assay and capillary electrophoresis. Electrophoresis 2003;24:634–8. [20] Vaccari G, Conte M, Morelli L, Di Guardo G, Petraroli R, Agrimi U. Primer extension assay for prion protein genotype determination in sheep. Mol Cell Probes 2004;18:33–7. [21] Montgonery GW, Sise J. Extraction of DNA from sheep blood cells. N Z J Agric Res 1990;33:437–41. [22] Benestad SL, Sarradin P, Thu B, Schonheit J, Tranulis MA, Bratberg B. Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec 2003;153:202–8. [23] De Bosschere H, Roels S, Benestad SL, Vanopdenbosch E. Scrapie case similar to Nor98 diagnosed in Belgium via active surveillance. Vet Rec 2004;155:707–8. [24] Gavier-Widen D, Noremark M, Benestad S, Simmons M, Renstrom L, Bratberg B, et al. Recognition of the Nor98 variant of scrapie in the Swedish sheep population. J Vet Diagn Invest 2004;16:562–7. [25] Onnasch H, Gunn HM, Bradshaw BJ, Benestad SL, Bassett HF. Two Irish cases of scrapie resembling Nor98. Vet Rec 1 2004;55:636–7. [26] Ikeda T, Horiuchi M, Ishiguro N, Muramatsu Y, Kai-Uwe GD, Shinagawa M. Amino acid polymorphisms of PrP with reference to onset of scrapie in Suffolk and Corriedale sheep in Japan. J Gen Virol 1995;76:2577–81. [27] Vaccari G, Petraroli R, Agrimi U, Eleni C, Perfetti MG, Di Bari MA, et al. PrP genotype in Sarda breed sheep and its relevance to scrapie. Brief report. Arch Virol 2001;146:2029–37. [28] Heaton MP, Leymaster KA, Freking BA, Hawk DA, Smith TP, Keele JW, et al. Prion gene sequence variation within diverse groups of US sheep, beef cattle, and deer. Mamm Genome 2003;14:765–77. [29] Acin C, Martin-Burriel I, Goldmann W, Lyahyai J, Monzon M, Bolea R, et al. Prion protein gene polymorphisms in healthy and scrapie-affected Spanish sheep. J Gen Virol 2004;85:2103–10. [30] Zhang L, Li N, Fab B, Fang M, Xu W. PRNP polymorphisms in Chinese ovine, caprine and bovine breeds. Anim Genet 2004;35:457–61. [31] Billinis C, Psychas V, Leontides L, Spyrou V, Argyroudis S, Vlemmas I, et al. Prion protein gene polymorphisms in healthy and scrapieaffected sheep in Greece. J Gen Virol 2004;85:547–54.
Molecular and Cellular Probes 21 (2007) 363–367 www.elsevier.com/locate/ymcpr
Simultaneous detection of eight single nucleotide polymorphisms in the ovine prion protein gene Bernhard F. Benkela,, Edith Valleb, Nathalie Bissonnettec, A. Hossain Farida a
Department of Plant and Animal Sciences, Nova Scotia Agricultural College, Truro, NS, Canada B2N 5E3 Bioproducts and Bioprocesses Program, Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1 c Bioproducts and Bioprocesses Program, Dairy and Swine Research and Development Centre, Lennoxville, QC, Canada J1M 1Z3
b
Received 8 January 2007; accepted 1 May 2007 Available online 18 May 2007
Abstract Amino acid polymorphisms in the prion protein gene (PrP) affect the susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy (TSE). In particular, amino acid substitutions at codons 136, 154 and 171 of the ovine PrP gene are associated with different degrees of susceptibility to the classical form of scrapie, caused by ‘typical’ scrapie strains. Existing genotyping tests for scrapie susceptibility normally interrogate only the single nucleotide polymorphisms (SNPs) most relevant to ‘typical’ strains. Recently, however, a number of novel variants of the scrapie agent have been discovered. The ability of these new, ‘atypical’ scrapie variants to infect sheep that are resistant to ‘typical’ variants has raised concerns about the reduction in genetic variability that may result from intense selection for resistance to classical scrapie. Furthermore, a growing interest in a potential role for specific PrP genotypes in modulating performance traits is also driving a move toward more extensive characterization of haplotypes at the PrP locus. Here, we describe a single-tube method for the interrogation of eight SNPs within seven codons (112, 136, 141, 154, 171, 231 and 241) of the ovine PrP gene. This method is as accurate as sequencing, yet more affordable, and can easily be automated for high-throughput sample screening. Moreover, it can be modified to accommodate genetic variations that are found in local and heritage breeds. r 2007 Elsevier Ltd. All rights reserved. Keywords: Genotyping assay; Scrapie resistance; Single-base extension
1. Introduction With over 30 SNPs already reported, the ovine prion gene (PrP) shows an unusually high level of genetic variation (reviewed in [1,2]). Of particular interest to the sheep industry are mutations at codons 136, 154 and 171 which are associated with susceptibility to natural and experimental scrapie, i.e. the haplotype A136R154R171 has been shown to confer resistance to typical scrapie strains whereas V136R154Q171 is common amongst susceptible sheep [3]. The demonstrated association between PrP haplotype and scrapie susceptibility has led a number of countries to adopt selection programs designed to increase the frequency of genotypes that show resistance to typical scrapie. However, concerns Corresponding author. Tel.: +1 902 893 6165; fax: +1 902 895 6734.
E-mail address: [email protected] (B.F. Benkel). 0890-8508/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2007.05.002
have been raised regarding the intense selection for resistant genotypes which has been standard practice in many countries. The reasons for these concerns are outlined below. Firstly, sheep that show resistance to classical scrapie based on their genotypes at codons 136, 154 and 171 are susceptible to atypical variants [4,5]. Thus, selection for resistance to typical scrapie agents could put sheep populations at risk to infections by atypical variants such as Nor98, for example, the susceptibility to which is affected by a polymorphism at codon 141 [5]. Secondly, intense selection for a single PrP haplotype will cause a reduction in genetic variability overall and an increase in inbreeding, particularly in breeds where resistant genotypes are present at a low frequency [6,7]. Thirdly, in addition to the well established link between PrP genotypes and scrapie resistance, there is also evidence, albeit less compelling, for associations between
ARTICLE IN PRESS B.F. Benkel et al. / Molecular and Cellular Probes 21 (2007) 363–367
364
haplotypes at the PrP locus and performance traits of importance to sheep breeders, mediated either through pleiotrophic effects of the PrP gene or through linkage of specific alleles at the PrP gene and production trait genes [8–12]. The fact that only a limited amount of information is currently available to support such associations may be due, at least in part, to a lack of resolution in PrP haplotype discrimination resulting from the use of the common 3-codon PrP genotyping assays. More extensive genotyping, beyond the standard 3-codon analysis, using the assay described here for example, may well reveal associations that have so far remained obscured. These issues speak to the need for high-throughput methods that yield more information on PrP alleles than the standard 3-codon assays provide. A variety of methods have been developed for restricted genotyping sheep at the PrP locus. However, gel-based assays such as restriction fragment length polymorphism [13], denaturing gradient gel electrophoresis [14,15], allele-specific hybridization [16], single-strand conformational polymorphism analysis [17], and allele-specific amplification [15] do not lend themselves to automation and are time consuming since they require a separate assay to be performed for each SNP. Non-gel-based methods, such as TaqMan/real time PCR [18], DNA sequencing, and single-base extension [2,19,20] are more suited for large-scale genotyping. The latter represents a reliable and inexpensive (especially in comparison to DNA sequencing), high-throughput method for the determination of haplotypes of the PrP gene, which has been used previously for 3-codon genotyping. Here, we describe an extension of this method for the simultaneous identification of eight SNPs in seven codons of the ovine PrP gene.
2. Materials and methods 2.1. Primers and PCR amplification A 1126 bp long segment that spans the entire coding region of the PrP gene was amplified by the polymerase chain reaction (PCR) using a forward primer (50 -GAGGAA GAGTTGTGTTACTACT) and a reverse primer (50 -GTCTGCTTGTCATTTCCCAGTG). Amplification was performed in 10.0 mL reactions containing 1 PCR buffer (Applied Biosystems Inc., Foster City, CA), 0.2 mM each dNTP, 500 nM each primer, 0.625 unit of AmpliTaq Gold (Applied Biosystems), 2.5 mM MgCl2 and 50–100 ng of genomic DNA (final concentration). Amplification was performed in an Eppendorf Mastercycler S (Hamburg, Germany), which was programmed for an initial denaturation for 5 min at 94 1C, followed by 35 cycles of denaturation at 94 1C for 30 s, annealing at 58 1C for 30 s, and extension at 72 1C for 60 s, followed by a 6 min final extension at 72 1C. The thermal cycler’s ramping speed was set at 18% of maximum. PCR products were purified by Exo SAP-IT (Amersham) according to the manufacturer’s instructions. 2.2. SNP interrogation SNP interrogation was carried out using a single-base extension (SNaPshot) method essentially as recommended by the manufacturer (Applied Biosystems). Briefly, an interrogation primer was designed for each of the eight target SNPs (Table 1). Each primer consists of a segment designed to anneal immediately 50 or 30 to the polymorphic position in the PrP gene, and a variable-length nonhomologous polynucleotide tail with a minimal secondary
Table 1 Interrogation primers for the detection of eight SNPs at seven codons of the PrP gene Codon no.
Wild type
Interrogation primers (50 –30 )a
Mutant
Codon
aa
Codon
aa
112
ATG
M
ACG
T
GTAAGCCAAAAACCAACA
136
GCC
A
GTC ACCc
V T
aacccgacccaaagacGCTACATGCTGGGAAGTG
141b
CTT
L
TTT
F
aaacccgactaaaaaccccgactacccaaagactaaaTAGTCATTGCCAAAATGTATAA
154
CGT
R
CAT CTT
H L
aaaaaaaaaaaaaaTATGAGGACCGTTACTATC
171A
CAG
Q
CGG AAGc
R K
gactgactgactgactgactACTACAGACCAGTGGATC
171Bb 231 241b
CAG AGG CCT
Q R P
CAT CGG TCT
H R S
gactaaaagactaaaagactACAAAGTTGTTCTGGTTACTATA gactaaaagactgactaaaaaaaagactAATCCCAGGCTTATTACCAA aaaaacccccaaaaaccccccccccaaaaacGATGAGGAGGATCACAGGAG
a
Underlined sequences indicate the region with homology to the PrP gene. Sequences shown in the lowercase are nonhomologous tails. These nucleotides are detected on the complementary DNA strand. c Rare variants, not investigated. b
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structure. SNP interrogation is achieved by primer extension using fluorescently labeled dideoxy nucleotide triphosphates (ddNTPs). The concentration of each of the SNP interrogating primers in the SNaPshot reaction was adjusted in order to equalize SNP peak heights. Final primer concentrations were 0.125 mM, except for primers 171B, 154 and 231, whose concentrations were adjusted to 0.625, 0.0625, and 0.225 mM, respectively. A reaction mixture containing 0.5 mL of primer mix (see above), approximately 100 ng of PCR product, 2.5 mL SNaPShot Multiplex reagent (Applied Biosystems), and 1.5 mL ddH2O was prepared and subjected to 25 cycles of denaturation at 96 1C for 10 s, annealing at 50 1C for 5 s and extension at 60 1C for 30 s in an Eppendorf Mastercycler S. Unincorporated ddNTPs were removed by treating the reactions with shrimp alkaline phosphatase (SAP-IT; Amersham) in 96-well plates using an epMotion 5075 liquid handling robot (Eppendorf). Following SAPIT treatment, 1 mL of the single-base extension reaction was added to 10 mL of a 40:1 mixture of Hi-Di fomammide and GeneScan 120 Liz size standard (Applied Biosystems), loaded into an optical 96-well plate, and resolved on an ABI 3130 capillary DNA analyzer with POP7 polymer (Applied Biosystems). The results were analyzed using GeneMapper software and further interpreted using a program written in-house in Visual Basic to convert SNaPshot results to haplotyes and genotypes at the PrP locus. 2.3. DNA samples DNA samples used for validation were selected from a DNA bank of over 7000 individuals from more than 50 primarily Canadian sheep breeds. DNA was extracted from fresh blood using the high salt procedure of Montgonery and Sise [21]. The complete coding sequence of the PrP gene of approximately 70 sheep from various breeds was sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and the products resolved on an ABI 3130 Genetic Analyzer according to the manufacturer’s instructions. From these 70 sheep, a panel of 25 sheep with 22 different genotypes that covered all of the polymorphisms at the seven codons interrogated in the test was selected for developing and validating the assay. The 400 animals used to further confirm the accuracy of the SNaPshot assay, compared to 3-codon test, in a highthroughput setting had been previously typed at three codons within the PrP gene using a standard RFLP-PCR assay (A. Farid, unpublished). 3. Results and discussion 3.1. Assay design The procedure described here involves the amplification by PCR of a single amplicon followed by a single, multiplexed base extension reaction, to facilitate the rapid
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and accurate determination of eight SNPs per individual at the PrP gene. A similar method has been used previously to simultaneously genotype four SNPs at three codons within the PrP gene [2,19,20]. In the assay developed in this study, the increase in information content from four to eight SNPs is achieved without an increase in the cost of the assay, either for materials or labor. Moreover, eight SNPs is not the upper limit for this procedure and it is likely that the SNaPshot approach can be expanded to 12 SNPs per lane without incurring any significant increase in cost per sample. The additional information content afforded by the expanded assay can be employed to address the resistance of sheep to ‘atypical’ scrapie agents, or to gather other information regarding the PrP gene associated with phylogenetic or production parameters. For the test described here, the primary aim was to use some of the additional capacity of the SNaPshot test for the identification of haplotypes within the coding region of the PrP gene that are relevant to the sheep breeds commonly found in Canada. In addition to the usual codons 136, 154 and 171, we included codons 112, 141, 231, and 241 in our test because they have the highest frequency among rare variants of the PrP gene in several British and other European breeds that form the base of the Canadian sheep population [1]. Furthermore, the amino acid phenylalanine (F) at codon 141 increases susceptibility of sheep to Nor98, a strain of the scrapie agent recently discovered in some European countries [10,22–25], whereas substitution of the amino acid threonine (T) with methionine (M) at codon 112 may have some protective effect against the typical scrapie agent [1,26]. The codon 231 variant is a silent mutation. It was included in the assay because it is polymorphic in several European and Asiatic breeds, is found at a high frequency in some of these breeds [1,2,26–31], and our results show that it segregates independently of codon 241 (data not shown). Thus, this selectively neutral nucleotide substitution may be valuable for phylogenetic studies.
3.2. Assay validation Initially, each of the eight SNP interrogation primers was tested individually on a small number of DNA samples from a panel with known nucleotides at the target positions within the PrP gene (see Section 2). Multiplexing of all eight primers in a single reaction necessitated adjustments in the concentrations of individual primers in order to normalize peak heights between SNPs on electropherograms. A typical SNaPshot profile is shown in Fig. 1. Once the assay had been optimized, SNaPshot profiles were derived for a panel of 25 sheep with a wide range of genotypes that had previously been sequenced at the PrP gene as well as over 400 sheep that had previously been genotyped at codons 136, 154 and 171 (see Section 2). The results of the three different tests were identical without exception, indicating the high degree of accuracy of the single-base extension method; which is also in agreement
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Fig. 1. SNaPshot profile analysis. A typical SNaPshot profile is shown. Gray bars above the chromatogram indicate the bins delimiting peak positions within the profile for the eight individual SNPs (one bin for each SNP). Numbers embedded within gray bars indicate codons within the PrP peptide harboring the respective SNPs. Colors of the peaks within the profile are as follows: orange, size standard; red, ‘T’; blue, ‘G’; green, ‘A’; and black, ‘C’ (see Table 1 for interpretation of SNaPshot pattern and conversion of SNP calls to amino acids).
with the reports of Zsolnai et al. [19], Vaccari et al. [20], and Alvarez et al. [2].
3.3. Assay constraints The single-base extension (SNaPshot) method is based on the annealing of SNP interrogation primers to complementary sites on template DNA, and the subsequent extension of the primers by a single fluorescently labeled dideoxy nucleotide triphosphate (ddNTP). On a global basis, the ovine PrP gene is a problematic target for SNaPshot interrogation since it contains an unusually large number of SNPs. This high SNP density can complicate the design of effective, universal SNP interrogation primers. For example, there are rare SNPs in some breeds near the interrogated nucleotides within codons 136 and 171 (see Table 1). Prior knowledge of local variants can be used to modify the assay to take into account such breed-specific SNPs. For example, at codon 136, the interrogating primer used in the current assay was designed to discriminate between the nucleotides at the second position (GCC and GTC), but a ‘G’ to ‘A’ substitution at the first position within codon 136 has been reported in at least one breed [31]. The current (upper strand) primer used to interrogate the second position within codon 136 will yield a ‘null’ result for a haplotype containing the ‘G’ to ‘A’ substitution at the first position. In homozyogous ‘A/A’ individuals this is obvious from the SNaPshot profile. In heterozygous individuals, however, the result will be an incorrect genotype call. Designing a primer for the opposite (lower) strand is not a preferred option because both codons 137 (ATG and ACG) and 138 (AGC and AAC) are polymorphic in some breeds [1]. In order to overcome this problem, a modification could be employed that involves the use of overlapping SNP
interrogation primers targeting different positions within the same codon. For example, in the case of codon 136 in the ovine PrP gene, this problem can be approached by incorporating a pair of SNP interrogation primers for codon 136 into the SNaPshot panel as follows: (1) an oligomer that interrogates the first position within the codon; and (2) a primer that shares an identical core with the first primer, but with a ‘G/A’ degeneracy in its 30 position designed to interrogate the second position of the codon irrespective of the nucleotide at the 1st position. A similar approach can be used in other situations that require genotyping of adjacent nucleotide positions, for example to provide full 3-position genotyping within codon 171. It should be noted that complications arising from primer mismatch are not specific to SNaPshot tests, instead this is a common feature of any test that is based on the use of sequence-specific primer annealing, and it also affects genotyping assays based on other methodologies such as restriction fragment length polymorphism analysis [1]. In addition, there are rare instances where the genotype information provided by SNaPshot tests cannot be resolved into haplotypes. For example, the genotypes CAG/CGT and CGG/CAT code for RQ and RH, respectively, at codon 171. However, the SNaPshot patterns for these two genotypes are identical and the haplotypes cannot be resolved by the single-base extension pattern unless combined with genotype information from related individuals. On the other hand, tri-allelic SNP positions do not pose a problem, and the SNaPshot assay will detect the rare arginine (R) to leucine (L) substitution at codon 154 recently reported by Alvarez et al. [2]. 4. Summary We have developed a single tube method for interrogating eight SNPs within seven codons of the ovine PrP gene.
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The method, which relies on single-base extension, is as accurate as DNA sequencing, but provides genotype information at a fraction of the cost, and is amenable to high-throughput screening. This assay is currently in use as the standard for scrapie resistance genotyping by the Atlantic Research Centre for Agricultural Genomics at the Nova Scotia Agricultural College, which has performed the bulk of the PrP genotyping for Canadian sheep to date. Acknowledgments The technical assistance of K. Benkel and T. Crossman is gratefully acknowledged. Financial support for this project was received from Agriculture and Agri-Food Canada through the Agri-Futures Nova Scotia program, the Investment Agriculture Foundation of British Columbia, the Technology Development Program of the NS Department of Agriculture, and the Canada Research Chairs program. References [1] Goldmann W, Baylis M, Chihota C, Stevenson E, Hunter N. Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes. J Appl Microbiol 2005;98: 1294–302. [2] Alvarez L, Arranz JJ, San Primitivo F. Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep. J Anim Sci 2006;84:259–65. [3] Baylis M, Chihota C, Stevenson E, Goldmann W, Smith A, Sivam K, et al. Risk of scrapie in British sheep of different prion protein genotype. J Gen Virol 2004;85:2735–40. [4] Le Dur A, Beringue V, Andreoletti O, Reine F, Lai TL, Baron T, et al. A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes. Proc Natl Acad Sci USA 2005;102:16031–6. [5] Moum T, Olsaker I, Hopp P, Moldal T, Valheim M, Moum T, et al. Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases. J Gen Virol 2005;86:231–5. [6] Drogemuller C, Leeb T, Distl O. PrP genotype frequencies in German breeding sheep and the potential to breed for resistance to scrapie. Vet Rec 2001;149:349–52. [7] Acutis PL, Sbaiz L, Verburg F, Riina MV, Ru G, Moda G, et al. Low frequency of the scrapie resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed. J Gen Virol 2004;85:3165–72. [8] de Vries F, Borchers N, Hamann H, Drogemuller C, Reinecke S, Lupping W Distl O. Associations between the prion protein genotype and performance traits of meat breeds of sheep. Vet Rec 2004;155: 140–3. [9] Brandsma JH, Janss LLG, Visscher AH. Association between PrP genotypes and litter size and 135 days weight in Texel sheep. Livest Prod Sci 2004;85:59–64. [10] Brandsma JH, Janss LLG, Visscher AH. Association between PrP genotypes and performance traits in an experimental Dutch Texel herd. Livest Pro Sci 2005;95:89–94. [11] Alexander BM, Stobart RH, Russell WC, O’Rourke KI, Lewist GS, Logan JR, et al. The influence of genotypes at codon 171 of the prion protein gene (PRNP) in five breeds of sheep and production traits of ewes associated with those genotypes. J Anim Sci 2005;83:455–9.
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