Abstract
Prion diseases, a protein misfolded disorder (PMD) caused by misfolded prion protein (PrPSc), present in a wide variety of hosts, ranging from ungulates to humans. To date, prion infections have not been reported in horses, which are well-known as prion disease-resistant animals. Several studies have attempted to identify distinctive features in the prion protein of horses compared to prion disease-susceptible animals, without the study on polymorphisms of the horse prion protein gene (PRNP). Since single nucleotide polymorphisms (SNPs) of PRNP in prion disease-susceptible animals are major susceptibility factors, the investigation of SNPs in the horse PRNP gene is important; however, only one study investigated a single horse breed, Thoroughbred. Thus, we investigated genetic polymorphisms and potential characteristics of the PRNP gene in 2 additional horse breeds. To this end, we performed amplicon sequencing of the horse PRNP gene and investigated SNPs in Jeju and Halla horses. We compared genotype, allele and haplotype frequencies among three horse breeds, namely, Thoroughbred, Jeju and Halla horses. In addition, we evaluated the potential influence of the identified nonsynonymous SNPs on the prion protein using PolyPhen-2, PROVEAN, and PANTHER. Furthermore, we measured the aggregation propensity of prion proteins using AMYCO and analyzed linkage disequilibrium (LD) between PRNP and prion-like protein gene (PRND) SNPs. A total of 4 SNPs were found, including two nonsynonymous SNPs (c.301 T > A, c.525 C > A) and three novel SNPs (c.-3A > G, c.301 T > A and c.570 G > A). There were significant differences in genotype, allele and haplotype frequencies among the three horse breeds. The nonsynonymous SNP, c.301 T > A (W101R), was predicted to be benign, deleterious, and possibly damaging by PolyPhen-2, PROVEAN and PANTHER, respectively. In addition, the amyloid propensity of horse prion protein according to 4 haplotypes of nonsynonymous SNPs was predicted to be benign by AMYCO. Finally, we identified weak LD between PRNP and PRND SNPs.
Similar content being viewed by others
Introduction
Prion diseases, which are fatal neurodegenerative diseases and are one type of protein misfolded disorder (PMD) caused by a deleterious form of prion protein (PrPSc) derived from normal prion protein (PrPC), are accompanied by spongiform generation and gliosis in brain lesions1,2. After scrapie was first discovered in sheep in 1732, it was studied for approximately three centuries. Prion diseases showed a broad infection host ranges, including scrapie in sheep and goats; bovine spongiform encephalopathy (BSE) in cattle; chronic wasting disease (CWD) in elk and deer; transmissible mink encephalopathy (TME) in mink; feline spongiform encephalopathy (FSE) in cats, cheetah and pumas; and Creutzfeldt–Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann–Sträussler–Scheinker syndrome (GSS) in humans3,4,5,6,7,8,9,10,11,12. Although prion diseases have been reported in various host species, a natural case of prion disease in horses, has not been reported to date. Previous studies, including molecular dynamics (MD) and nuclear magnetic resonance (NMR) studies, have attempted to understand the mechanism of prion disease resistance. However, these studies have been conducted in nonpolymorphic prion protein genes (PRNPs)13,14. Since polymorphisms in the PRNP gene significantly affect protein structure and are related to the vulnerability to disease, investigating polymorphisms in the horse PRNP gene is very important15,16,17.
In horses, polymorphisms of the PRNP gene have been reported in our previous research. Only one single nucleotide polymorphism (SNP), N175K, was found in the open reading frame (ORF) of the horse PRNP gene in the Thoroughbred breed18. However, since the study was conducted in only Thoroughbred horses, this result is not representative of the genetic diversity of the whole horse species. Thus, we investigated genetic polymorphisms and found genetic characteristics of the PRNP gene in Jeju and Halla horses. The Jeju horse is a Korean native horse, and the Halla horse is hybrid between the Jeju and Thoroughbred breeds that is bred for racing purposes.
To achieve these goals, we carried out amplicon sequencing of the horse PRNP gene and investigated SNPs in Jeju and Halla horses. In addition, we investigated genotype, allele and haplotype frequencies and compared the distributions among three breeds, Thoroughbred, Jeju and Halla horses. Furthermore, we evaluated the potential influence of the identified nonsynonymous SNPs on the prion protein using PolyPhen-2, PROVEAN, and PANTHER and measured the aggregation propensity of prion protein variants using AMYCO19,20,21,22,23,24. Lastly, we analyzed linkage disequilibrium (LD) between PRNP and prion-like protein gene (PRND) SNPs.
Results
The horse PRNP gene is composed of three exons. We amplified the ORF region for exon 3 of the PRNP gene composed of 836 bp and performed automatic amplicon sequencing in 142 Jeju horses and 82 Halla horses. We found a total of 4 SNPs, including c.-3A > G, c.301 T > A, c.525 C > A and c.570 G > A. Of the 4 SNP, c.301 T > A (W101R) and c.525 C > A (N175K) are nonsynonymous SNPs. Except for c.525 C > A, three SNP, including c.-3A > G, c.301 T > A and c.570 G > A, were novel SNPs found in this study (Fig. 1).
To compare the difference in genetic distribution of SNPs in the horse PRNP gene, we performed genotyping and investigated the genotype and allele frequencies of SNPs in this gene (Table 1). Except for c.525 C > A in the Jeju horse, all SNPs are in Hardy-Weinberg Equilibrium (HWE). Then, we compared genotype and allele distributions among the three horse breeds. For c.-3A > G SNP, the Halla horse showed similar genotype and allele distributions as the Jeju horse (P = 1.0). However, Halla horse showed significantly different genotype and allele distributions with Thoroughbred horse (P < 0.0001). For the c.301 T > A SNP, the Jeju horse showed similar genotype and allele distributions with Halla and Thoroughbred horses. Jeju horses showed significantly different genotype and allele distributions with Halla and Thoroughbred horses for c.525 C > A SNP (P < 0.0001). For the 570 G > A SNP, Jeju horses showed significantly different genotype distributions (P = 0.0004) and allele distributions (P = 0.0002) with Halla horses. In addition, Jeju horse also showed significantly different genotype and allele distributions with Thoroughbred horse (P < 0.0001) (Table 1).
Based on the genotype distributions of horse PRNP SNPs, we analyzed the haplotype frequencies of the horse PRNP gene in Jeju and Halla horses. A total of 5 haplotypes were identified (Table 2). Significantly different distributions of the haplotypes between these two breeds were found in Haplotype 1 (P = 0.0002), Haplotype 2 (P = 0.0019) and Haplotype 4 (P = 0.0005).
Next, the extent of LD value was investigated in 4 PRNP SNP using Lewontin’s D’ ( | D’ | ) and r2 values in Jeju and Halla horses. All four SNPs were strongly linked together with a D’ value of 1.0. However, the LD value among 4 PRNP SNPs in the results using the r2 value is notably low (below score 0.3) and showed weak LD in Jeju and Halla horses (Tables 3, 4).
We also estimated the potential impact of nonsynonymous SNPs on horse prion protein using PolyPhen-2, PROVEAN and PANTHER. PolyPhen-2 predicted W101R as “Benign” with a value of 0.033. PROVEAN estimated as “Deleterious” with a −2.569 value. PANTHER was assessed as “Possibly damaging” with 361 values (Table 5).
We investigated the amyloid propensity of highly susceptible (ARQ sheep, VRQ sheep, cattle and deer) and non-susceptible (dogs, rabbits and horses) prion proteins using AMYCO. Ovine prion proteins with 136 A/154 R/171Q haplotype and 136 V/154 R/171Q haplotype were measured with 0.27 and 0.31 values, respectively. Bovine and canine prion proteins were measured with 0.00. Deer and rabbit prion proteins were measured with 0.12 and 0.27 values, respectively. Horse prion protein sequences were classified into 4 haplotypes according to alleles of nonsynonymous SNPs (W101R and N175K), and amyloid propensity was analyzed by AMYCO. 101W/175N and 101R/175N haplotypes were measured with 0.4 values. The 101 W/175 K and 101 R/175 K haplotypes were measured with 0.0 values (Table 6).
To examine whether horse PRND SNPs have genetic linkage with the PRNP SNPs, we analyzed PRND SNPs in 30 Jeju and 30 Halla horses and measured the LD values between SNPs of the PRNP and PRND genes. Notably, all PRND SNP showed weak LD with the PRNP SNPs (r2 value: blow 0.3; Fig. 2).
Discussion
In the present study, we validated the polymorphisms in the ORF of the PRNP gene in a large sampling of outbred horse breeds, Jeju and Halla horses. Unlike inbred Thoroughbred horses, a total of 4 SNPs, including c.-3A > G, c.301 T > A, c.525 C > A and c.570 G > A, were found in the ORF of the horse PRNP gene. Among these SNPs, two (c.301 T > A, c.525 C > A) are nonsynonymous, and three of the four (c.-3A > G, c.301 T > A and c.570 G > A) are novel to this study. Next, we compared genotype and allele distributions among Jeju, Halla and Thoroughbred horses and found significantly different genetic distributions among the horse breeds. Haplotypes of the PRNP gene were also analyzed, and a total of 5 haplotypes were identified. In addition, the genetic distributions of haplotypes were significantly different between Halla and Jeju horses. We investigated the genetic linkage among horse PRNP SNPs in Jeju and Halla horses. Although we analyzed both parameters (D’ and r2 values), D’ value is inflated when one allele is rare. Since 4 PRNP SNPs showed that minor alleles are rare, we interpreted the result using r2 value. Interestingly, weak LD among horse PRNP SNPs was identified in Jeju and Halla horses (Tables 3, 4). In addition, horse showed weak LD between PRNP and PRND SNPs (Fig. 2). Recent studies have reported that prion disease-susceptible animals, including sheep and goats, showed strong LD between PRNP and PRND genes9,25. On the other hand, in prion disease-resistant animals, dogs showed weak LD between PRNP and PRND genes, unlike prion disease-susceptible animals26.
We also found potential effect of W101R to function of horse prion protein using PROVEAN and PANTHER (Table 5). Next, we estimated amyloid propensity of the horse prion protein. Previous studies have reported that horse prion protein presents a highly stable structure that can endure harsh conditions (high temperature and pH changes). It is suggested that the highly stable structure of the horse prion protein contributes to resistance to conformational changes in the abnormal form of the prion protein, which supports prion disease resistance in horses27,28. However, since these studies have not been performed with polymorphisms found in the horse prion protein, these findings are limited. In this study, we performed in silico estimation on the amyloid propensity of horse prion proteins with various combinations of the nonsynonymous SNPs. We classified the prion protein sequence into 4 haplotypes based on alleles of nonsynonymous SNPs (W101R and N175K) and performed an analysis of amyloid propensity in horse prion protein. The horse prion protein sequence registered in GenBank (101 W/175 N) had a value of 0.4. Interestingly, the other three haplotypes showed 0.4 (101 R/175 N) and 0.0 (101 W/175 K and 101 R/175 K) values. This result indicates that polymorphisms of horse prion protein showed no effect on amyloid propensity in the 101 R/175 N haplotype were unlikely to form amyloid while the 101 W/175 K and 101 R/175 K haplotypes. Although amyloid propensity prediction score did not strictly correlate with the susceptibility of prion disease (cattle: 0.00; rabbits: 0.27), the amyloid propensity of equine prion protein was predicted to be different according to alleles of nonsynonymous SNPs (Table 6). Further studies using in vivo and in vitro models are warranted.
In conclusion, we investigated prion genetic characteristics in horses and validated the polymorphisms in the ORF of the PRNP gene in a large sampling of outbred horse breeds, Jeju and Halla horses. In addition, we found significantly different genetic distributions of genotype, allele, and haplotype in three horse breeds in the PRNP gene and identified significantly weak LD between PRNP and PRND SNPs, unlike those of prion disease-susceptible animals. Furthermore, we evaluated the amyloid propensity of horse prion protein according to alleles of nonsynonymous SNPs. To the best of our knowledge, we analyzed potential impact of novel polymorphisms of the horse PRNP gene with the regard to susceptibility to prion disease. The weak LD results between horse PRNP and PRND SNPs correspond well with results from other prion disease resistant species. High amyloid propensity values do not align with prion disease susceptible species making the results generated for horse PrP difficult to interpret making further examination results.
Methods
Ethics statement
We extracted DNA from hair samples of 148 Jeju horses and 100 Halla horse horses in Jeju Island. All experimental procedures were approved by the Institute of Animal Care and Use Committee of Chonbuk National University (CBNU 2016-65). All experiments using horses were carried out following the Korea Experimental Animal Protection Act.
Genetic analysis
Genomic DNA was obtained from 10 hair bulbs using the HiYieldTM genomic DNA mini kit (Real Biotech Corporation, Taiwan) according to manufacturer’s recommendations. The horse PRNP and PRND were amplified from the genomic DNA using sense and antisense gene-specific primers. The DNA sequences of the primers were as follows: Horse PRNP-F (AGAAGTGCAGAGTGTGACATGC), Horse PRNP-R (CAAGCGTATTAGCCTACGGGTG), Horse PRND-F (GCCCGTTGCAGCTTCTTATCT) and Horse PRND-R (GCTGGAGGAGAGAAGTGGGAT). Polymerase chain reaction (PCR) was carried out using GoTaq® DNA Polymerase (Promega, Fitchburg, Wisconsin, USA) as described previously9. In brief, the PCR mixture consisted of 20 pmol of each primer, 5 μl of 10× Taq DNA polymerase buffer, 1 μl of 10 mM dNTP mixture and 2.5 units of Taq DNA polymerase. The PCR conditions were 94 °C for 2 min for denaturation; 35 cycles of 94 °C for 45 sec, 59 °C for 45 sec, and 72 °C for 1 min 30 sec; and then 1 cycle of 72 °C for 10 min to extend the reaction. PCR was performed using an S-1000 Thermal Cycler (Bio-Rad, Hercules, California, USA). The PCR products were purified by the PCR Purification Kit (Thermo Fisher Scientific, Bridgewater, New Jersey, USA) and 5 μl of purified PCR product (50 ng/μl) was sequenced with an ABI 3730 automatic sequencer (ABI, Foster City, California, USA) using 5 pmol of sense and antisense primers as described previously10. Sequencing results were analyzed by Finch TV software (Geospiza Inc., Seattle, USA).
Statistical analysis
Comparisons of genotype, allele and haplotype frequencies were performed by the chi-square test using SAS 9.4 Software (SAS Institute Inc., Cary, NC, USA) as described previously26. The HWE and haplotype analyses were performed using Haploview version 4.2 (Broad Institute, Cambridge, MA, USA). LD analysis was carried out between PRNP and PRND SNPs. LD score of the PRNP and PRND genes were calculated in 30 Jeju horses and 30 Halla horses using Haploview version 4.2 (Broad Institute, Cambridge, MA, USA) as described previously3.
Evaluation on potential impact of nonsynonymous SNP to horse prion protein
The biological impact of horse prion protein induced by the nonsynonymous SNPs was assessed by PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), PROVEAN (http://provean.jcvi.org/seq_submit.php) and PANTHER (http://www.pantherdb.org/) as described previously24. PolyPhen-2 uses a position-specific, independent count (PSIC) score (score ranges from 0.0 to 1.0). The prediction results can be presented as three types: ‘benign’, ‘possibly damaging’ and ‘probably damaging’21. PROVEAN is a program that measures the impact of nonsynonymous SNPs on protein function. If the final score is below −2.5, protein variants are predicted to be ‘neutral’, and if the final score is above −2.5, protein variants are predicted to be ‘deleterious’22. PANTHER utilizes a hidden Markov model (HMM) based on statistical modeling methods and multiple sequence alignments to perform evolutionary analysis of nonsynonymous SNPs. PANTHER predicts SNPs as two types: ‘deleterious’ or ‘neutral’23. Amyloid propensity of horse prion protein according to alleles was analyzed by AMYCO (http://bioinf.uab.cat/amyco) as described previously24. AMYCO utilizes an algorithm to predict amyloid fibril propensity from amino acid sequences19.
References
Prusiner, S. B. Prions. Proc. Natl Acad. Sci. USA 95, 13363–13383 (1998).
Prusiner, S. B. The prion diseases. Brain Pathol. 8, 499–513 (1998).
Kim, S. K., Kim, Y. C., Won, S. Y. & Jeong, B. H. Potential scrapie-associated polymorphisms of the prion protein gene (PRNP) in Korean native black goats. Sci. Rep. 9, 15293 (2019).
Kim, Y. C., Kim, S. K. & Jeong, B. H. Scrapie susceptibility-associated indel polymorphism of shadow of prion protein gene (SPRN) in Korean native black goats. Sci. Rep. 9, 15261 (2019).
Baylis, M. & Goldmann, W. The genetics of scrapie in sheep and goats. Curr. Mol. Med. 4, 385–396 (2004).
Aguilar-Calvo, P., Garcia, C., Espinosa, J. C., Andreoletti, O. & Torres, J. M. Prion and prion-like diseases in animals. Virus Res. 207, 82–93 (2015).
Jeong, B. H. & Kim, Y. S. Genetic studies in human prion diseases. J. Korean Med. Sci. 29, 623–632 (2014).
Kim, Y. C. & Jeong, B. H. Bovine spongiform encephalopathy (BSE) associated polymorphisms of the prion-like protein gene (PRND) in Korean dairy cattle and Hanwoo. J. Dairy. Res. 85, 7–11 (2018).
Jeong, M. J., Kim, Y. C. & Jeong, B. H. Prion-like protein gene (PRND) polymorphisms associated with scrapie susceptibility in Korean native black goats. PLoS One 13, e0206209 (2018).
Kim, Y. C., Jeong B. H. Lack of germline mutation at codon 211 of the prion protein gene (PRNP) in Korean native cattle - Short communication. Acta Vet Hung, 147-152 (2017).
Kim, Y. C. & Jeong, B. H. The first report of prion-related protein gene (PRNT) polymorphisms in goat. Acta Vet. Hung. 65, 291–300 (2017).
Kim, Y. C. & Jeong, B. H. First report of prion-related protein gene (PRNT) polymorphisms in cattle. Vet. Rec. 182, 717 (2018).
Perez, D. R., Damberger, F. F. & Wuthrich, K. Horse prion protein NMR structure and comparisons with related variants of the mouse prion protein. J. Mol. Biol. 400, 121–128 (2010).
Christen, B., Damberger, F. F., Perez, D. R., Hornemann, S. & Wuthrich, K. Structural plasticity of the cellular prion protein and implications in health and disease. Proc. Natl Acad. Sci. USA 110, 8549–8554 (2013).
Lloyd, S., Mead, S. & Collinge, J. Genetics of prion disease. Top. Curr. Chem. 305, 1–22 (2011).
Vaccari, G. et al. State-of-the-art review of goat TSE in the European Union, with special emphasis on PRNP genetics and epidemiology. Vet. Res. 40, 48 (2009).
Kim, Y. C., Jeong, M. J., Jeong, B. H. Strong association of regulatory single nucleotide polymorphisms (SNP) of the IFITM3 gene with influenza H1N1 2009 pandemic virus infection. Cell Mol Immunol, In press (2019).
Kim, Y. C. & Jeong, B. H. The first report of polymorphisms and genetic characteristics of the prion protein gene (PRNP) in horses. Prion 12, 245–252 (2018).
Iglesias, V., Conchillo-Sole, O., Batlle, C. & Ventura, S. AMYCO: evaluation of mutational impact on prion-like proteins aggregation propensity. BMC Bioinforma. 20, 24 (2019).
Tang, H. & Thomas, P. D. PANTHER-PSEP: predicting disease-causing genetic variants using position-specific evolutionary preservation. Bioinformatics 32, 2230–2232 (2016).
Adzhubei, I., Jordan, D. M., Sunyaev, S. R. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet Chapter 7, Unit7 20 (2013).
Choi, Y. & Chan, A. P. PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 31, 2745–2747 (2015).
Kim, Y. C., Jeong, M. J. & Jeong, B. H. The first report of genetic variations in the chicken prion protein gene. Prion 12, 197–203 (2018).
Kim, Y. C. & Jeong, B. H. In Silico Evaluation of Acetylation Mimics in the 27 Lysine Residues of Human Tau Protein. Curr. Alzheimer Res. 16, 379–387 (2019).
Mesquita, P. et al. The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep. Anim. Genet. 47, 128–132 (2016).
Won, S. Y., Kim, Y. C., Kim, K., Kim, A. D. & Jeong, B. H. The First Report of Polymorphisms and Genetic Features of the prion-like Protein Gene (PRND) in a Prion Disease-Resistant Animal, Dog. Int. J. Mol. Sci. 20, E1404 (2019).
Sanchez-Garcia, J. & Fernandez-Funez, P. D159 and S167 are protective residues in the prion protein from dog and horse, two prion-resistant animals. Neurobiol. Dis. 119, 1–12 (2018).
Zhang, J. The structural stability of wild-type horse prion protein. J. Biomol. Struct. Dyn. 29, 369–377 (2011).
Acknowledgements
This research was supported by the Basic Science Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2018R1D1A1B07048711). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1A6A1A03015876). Sae-Young Won was supported by the BK21 Plus Program in the Department of Bioactive Material Sciences. This work was supported by NRF (National Research Foundation of Korea) Grant funded by the Korean Government (NRF-2019-Fostering Core Leaders of the Future Basic Science Program/Global Ph.D. Fellowship Program).
Author information
Authors and Affiliations
Contributions
Y.C. Kim, S.Y. Won, K. Do and B.H. Jeong conceived and designed the experiment Y.C. Kim, S.Y. Won, K. Do performed the experiments. Y.C. Kim, S.Y. Won, K. Do and B.H. Jeong analyzed the data. Y.C. Kim, S.Y. Won, K. Do wrote the paper. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Kim, YC., Won, SY., Do, K. et al. Identification of the novel polymorphisms and potential genetic features of the prion protein gene (PRNP) in horses, a prion disease-resistant animal. Sci Rep 10, 8926 (2020). https://doi.org/10.1038/s41598-020-65731-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-020-65731-5
- Springer Nature Limited
This article is cited by
-
Identification of a novel risk factor for chronic wasting disease (CWD) in elk: S100G single nucleotide polymorphism (SNP) of the prion protein gene (PRNP)
Veterinary Research (2023)
-
Novel polymorphisms in the prion protein gene (PRNP) and stability of the resultant prion protein in different horse breeds
Veterinary Research (2023)
-
Detection of stage of lung changes in COVID-19 disease based on CT images: a radiomics approach
Physical and Engineering Sciences in Medicine (2022)