Novel Polymorphisms and Genetic Characteristics of the Prion Protein Gene (PRNP) in Dogs-A Resistant Animal of Prion Disease

Abstract

Transmissible spongiform encephalopathies (TSEs) have been reported in a wide range of species. However, TSE infection in natural cases has never been reported in dogs. Previous studies have reported that polymorphisms of the prion protein gene (PRNP) have a direct impact on the susceptibility of TSE. However, studies on polymorphisms of the canine PRNP gene are very rare in dogs. We examined the genotype, allele, and haplotype frequencies of canine PRNP in 204 dogs using direct sequencing and analyzed linkage disequilibrium (LD) using Haploview version 4.2. In addition, to evaluate the impact of nonsynonymous polymorphisms on the function of prion protein (PrP), we carried out in silico analysis using PolyPhen-2, PROVEAN, and PANTHER. Furthermore, we analyzed the structure of PrP and hydrogen bonds according to alleles of nonsynonymous single nucleotide polymorphisms (SNPs) using the Swiss-Pdb Viewer program. Finally, we predicted the impact of the polymorphisms on the aggregation propensity of dog PrP using AMYCO. We identified a total of eight polymorphisms, including five novel SNPs and one insertion/deletion polymorphism, and found strong LDs and six major haplotypes among eight polymorphisms. In addition, we identified significantly different distribution of haplotypes among eight dog breeds, however, the kinds of identified polymorphisms were different among each dog breed. We predicted that p.64_71del HGGGWGQP, Asp182Gly, and Asp182Glu polymorphisms can impact the function and/or structure of dog PrP. Furthermore, the number of hydrogen bonds of dog PrP with the Glu182 and Gly182 alleles were predicted to be less than those with the Asp182 allele. Finally, Asp163Glu and Asp182Gly showed more aggregation propensity than wild-type dog PrP. These results suggest that nonsynonymous SNPs, Asp182Glu and Asp182Gly, can influence the stability of dog PrP and confer the possibility of TSE infection in dogs.

Keywords: PRNP; SNP; dog; octapeptide; polymorphism; prion.

Figure 1

Gene map of polymorphisms identified in the canine prion protein (PRNP) gene on chromosome 24. The open reading frame (ORF) is indicated by a shaded block, and the 5′ and 3′ untranslated regions (UTRs) are indicated by white blocks. Arrows indicate the regions sequenced. The Y-shaped bar indicates the octapeptide deletion polymorphisms identified in the canine PRNP gene. Asterisks indicate the novel polymorphisms found in this study.

Figure 2

Electropherograms of 5 novel single nucleotide polymorphisms (SNPs) of the canine prion protein (PRNP) gene found in dogs. (A) Electropherograms showing the three genotypes at polymorphic codon 66 (Gly66Gly). Upper portion GGT/GGT, middle portion: GGT/GGC, and lower portion: GGC/GGC. (B) Electropherograms showing the two genotypes at polymorphic codon 124 (Ala124Ala). Upper portion: GCG/GCG and lower portion: GCG/GCA. (C) Electropherograms showing the two genotypes at polymorphic codon 182 (Asp182Glu). Upper portion: GAC/GAC and lower portion: GAC/GGC. (D) Electropherograms showing the three genotypes at polymorphic codon 182 (Asp182Gly). Upper portion: GAC/GAC, middle portion: GAC/GAA, and lower portion GAA/GAA. (E) Electropherograms showing the three genotypes at polymorphic codon 243 (Pro243Pro). Upper portion: CCT/CCT, middle portion: CCT/CCC, and lower portion: CCC/CCC.

Figure 3

Electropherograms of novel insertion/deletion polymorphism of the canine prion protein (PRNP) gene. (A) Electropherograms showing the polymorphism at the octapeptide repeat region. Upper portion: insertion/insertion homozygote of the canine PRNP and lower portion: insertion/deletion heterozygote of the canine PRNP. (B) The amino acid and nucleotide sequences of the repeat region of the canine PRNP gene. The box indicates the 64_71del HGGGWGQP polymorphism which was found between R2 and R3. R1: octapeptide repeat 1 (54Pro, 55Gln, 56Gly, 57Gly, 58Gly, 59Gly, 60Trp, 61Gly, 62Gln); R2: octapeptide repeat 2 (63Pro, 64His, 65Gly, 66Gly, 67Gly, 68Trp, 69Gly, 70Gln); R3: octapeptide repeat 3 (71Pro, 72His, 73Gly, 74Gly, 75Gly, 76Trp, 77Gly, 78Gln); R4: octapeptide repeat 4 (79Pro, 80His, 81Gly, 82Gly, 83Gly, 84Trp, 85Gly, 86Gln); and R5: octapeptide repeat 5 (87Pro, 88His, 89Gly, 90Gly, 91Gly, 92Gly, 93Trp, 94Gly, 95Gln).

Figure 4

Comparison of tandem repeat sequences in humans, sheep, water buffalos, goats, cattle, chickens, and dogs. Octapeptide repeat sequences were obtained from GenBank at the National Center for Biotechnology Information (NCBI), including human (Homo sapiens, BAG32277.1), sheep (Ovis aries, NP_001009481.1), goat (Capra hircus, NP_001301176.1), cattle (Bos taurus, NP_001258555.1), water buffalo (Bubalus bubalis, KC137622.1), chicken (Gallus gallus, NP_990796.2), and dog (Canis lupus familiaris, AGA63676.1). The arrow indicates the octapeptide repeat deletion polymorphism found in this study. R1 ~ R5 indicate octapeptide repeat regions of dog PrP. R1: octapeptide repeat 1 (54Pro, 55Gln, 56Gly, 57Gly, 58Gly, 59Gly, 60Trp, 61Gly, 62Gln); R2: octapeptide repeat 2 (63Pro, 64His, 65Gly, 66Gly, 67Gly, 68Trp, 69Gly, 70Gln); R3: octapeptide repeat 3 (71Pro, 72His, 73Gly, 74Gly, 75Gly, 76Trp, 77Gly, 78Gln); R4: octapeptide repeat 4 (79Pro, 80His, 81Gly, 82Gly, 83Gly, 84Trp, 85Gly, 86Gln); and R5: octapeptide repeat 5 (87Pro, 88His, 89Gly, 90Gly, 91Gly, 92Gly, 93Trp, 94Gly, 95Gln).

Figure 5

Comparison of the distribution of haplotypes according to dog breeds. (A) Comparison of the distribution of haplotype 1 among 8 dog breeds. (B) Comparison of the distribution of haplotype 2 between Maltese and other breeds. (C) Comparison of the distribution of haplotype 3 between Maltese and other breeds. (D) Comparison of the distribution of haplotype 4 between Maltese and other breeds. (E) Comparison of the distribution of haplotype 5 between Maltese and other breeds. (F) Comparison of the distribution of haplotype 6 between Maltese and other breeds. Parentheses indicate each number of dog breeds. * indicates p < 0.05. ** indicates p < 0.01.

Figure 6

Prediction of 3D structure and hydrogen bonds of dog prion protein (PrP). The white arrow indicates target amino acid residues. The red box indicates adjacent amino acid residues. The green dotted line indicates hydrogen bonds. The green numbers indicate the distance of the hydrogen bonds. (A) 3D structure of dog PrP with allele Asp163, (B) 3D structure of dog PrP with allele Glu163, (C) 3D structure of dog PrP with allele Asp182, (D) 3D structure of dog PrP with allele Glu182, and (E) 3D structure of dog PrP with allele Gly182.

Figure 7

Prediction of aggregation propensity according to alleles of dog prion protein (PrP) polymorphisms. The impact of polymorphisms on the aggregation propensity of dog PrP was evaluated as values from 0.0 to 1.0 by AMYCO analysis. AMYCO scores <0.45 and >0.78 indicate low and high aggregation propensities of the protein, respectively.