The First Report of Polymorphisms and Genetic Features of the prion-like Protein Gene (PRND) in a Prion Disease-Resistant Animal, Dog

Abstract

Prion disease has displayed large infection host ranges among several species; however, dogs have not been reported to be infected and are considered prion disease-resistant animals. Case-controlled studies in several species, including humans and cattle, indicated a potent association of prion protein gene (PRNP) polymorphisms in the progression of prion disease. Thus, because of the proximal location and similar structure of the PRNP gene among the prion gene family, the prion-like protein gene (PRND) was noted as a novel candidate gene that contributes to prion disease susceptibility. Several case-controlled studies have confirmed the relationship of the PRND gene with prion disease vulnerability, and strong genetic linkage disequilibrium blocks were identified in prion-susceptible species between the PRNP and PRND genes. However, to date, polymorphisms of the dog PRND gene have not been reported, and the genetic linkage between the PRNP and PRND genes has not been examined thus far. Here, we first investigated dog PRND polymorphisms in 207 dog DNA samples using direct DNA sequencing. A total of four novel single nucleotide polymorphisms (SNPs), including one nonsynonymous SNP (c.149G>A, R50H), were identified in this study. We also found two major haplotypes among the four novel SNPs. In addition, we compared the genotype and allele frequencies of the c.149G>A (R50H) SNP and found significantly different distributions among eight dog breeds. Furthermore, we annotated the c.149G>A (R50H) SNP of the dog PRND gene using in silico tools, PolyPhen-2, PROVEAN, and PANTHER. Finally, we examined linkage disequilibrium between the PRNP and PRND genes in dogs. Interestingly, we did not find a strong genetic linkage between these two genes. To the best of our knowledge, this was the first genetic study of the PRND gene in a prion disease-resistant animal, a dog.

Keywords: Doppel; PRND; dog; linkage disequilibrium; prion; prion-like protein gene.

Figure 1

Gene map and polymorphisms identified in the dog prion-like protein gene (PRND) on chromosome 24. (a) The open reading frame (ORF) is indicated by a shaded block, and the 5′ and 3′ untranslated regions (UTRs) are indicated by white blocks. Horizontal bars with edges indicate the regions sequenced. Arrows indicate the novel polymorphisms found in this study. The asterisk indicates the nonsynonymous single nucleotide polymorphism (SNP) of the dog PRND gene. (b) Electropherogram of four novel SNPs: c.149G>A (R50H), c.447T>C (F149F), c.465C>T (A155A) and c.556G>C, identified in this study. Four colors indicate individual bases of DNA sequence using an ABI 3730 automatic sequencer (blue: cytosine, red: thymine, black: guanine, green: adenine).

Figure 2

Comparisons of genotype and allele frequencies of c.149G>A (R50H) among eight dog breeds. Differences in the c.149G>A (R50H) genotype and allele frequencies among eight dog breeds (Maltese, Shih Tzu, Toy Poodle, Yorkshire Terrier, Pomeranian, Chihuahua, Cocker Spaniel, and Mixed dogs) were calculated by the chi-square test using Statistical Analysis Software (SAS) version 9.4. Parentheses indicate the number of dogs. Statistically significant differences are indicated below. * p value < 0.05, ** p value < 0.01, *** p value < 0.001.

Figure 3

The linkage disequilibrium (LD) scores between polymorphisms of the PRND gene and those of the PRNP gene. LD scores with r² values between PRND and PRNP polymorphisms in dogs. P1–P8 indicate PRNP polymorphisms as follows: P1, c.190in/del (codon 64); P2, c.198T>C (codon 66); P3, c.301A>G (codon 101); P4, c.372G>A (codon 124); P5, c.489C>G (codon 163); P6, c.545A>G (codon 182); P7, c.546C>A (codon 182); and P8, c.729T>C (codon 243).

Figure 4

Comparisons of amino acid sequences of prion-like protein (Doppel) in human, mouse, sheep, goat, rabbit, horse, and dog. Prion-like protein sequences were obtained from GenBank at the National Center for Biotechnology Information (NCBI), including those of human (Homo sapiens, AAQ89344.1), mouse (Mus musculus, AAH25140.1), sheep (Ovis aries, NP_001009261.1), goat (Capra hircus, AAO44923.1), rabbit (Oryctolagus cuniculus, XP_008254493.1), horse (Equus caballus, ABN79630.1), and dog (Canis lupus familiaris, XP_005634877.1). Doppel protein sequences were aligned among various species using ClustalW2. Colors symbolize the chemical properties of amino acids (blue: acidic; red: small and hydrophobic; magenta: basic; green: hydroxyl, sulfhydryl, amine and glycine). The arrow denotes the nonsynonymous single nucleotide polymorphism (SNP) (c.149G>A, R50H) found in this study. Asterisks indicate dog-specific residues.