Absence of Strong Genetic Linkage Disequilibrium between Single Nucleotide Polymorphisms (SNPs) in the Prion Protein Gene (PRNP) and the Prion-Like Protein Gene (PRND) in the Horse, a Prion-Resistant Species

Abstract

Prion disease is a fatal neurodegenerative disorder caused by a deleterious prion protein (PrP^Sc). However, prion disease has not been reported in horses during outbreaks of transmissible spongiform encephalopathies (TSEs) in various animals in the UK. In previous studies, single nucleotide polymorphisms (SNPs) in the prion protein gene (PRNP) have been significantly associated with susceptibility to prion disease, and strong linkage disequilibrium (LD) between PRNP and prion-like protein gene (PRND) SNPs has been identified in prion disease-susceptible species. On the other hand, weak LD values have been reported in dogs, a prion disease-resistant species. In this study, we investigated SNPs in the PRND gene and measured the LD values between the PRNP and PRND SNPs and the impact of a nonsynonymous SNP found in the horse PRND gene. To identify SNPs in the PRND gene, we performed direct sequencing of the PRND gene. In addition, to assess whether the weak LD value between the PRNP and PRND SNPs is a characteristic of prion disease-resistant animals, we measured the LD value between the PRNP and PRND SNPs using D’ and r² values. Furthermore, we evaluated the impact of a nonsynonymous SNP in the Doppel protein with PolyPhen-2, PROVEAN, and PANTHER. We observed two novel SNPs, c.331G > A (A111T) and c.411G > C. The genotype and allele frequencies of the c.331G > A (A111T) and c.411G > C SNPs were significantly different between Jeju, Halla, and Thoroughbred horses. In addition, we found a total of three haplotypes: GG, AG, and GC. The GG haplotype was the most frequently observed in Jeju and Halla horses. Furthermore, the impact of A111T on the Doppel protein was predicted to be benign by PolyPhen-2, PROVEAN, and PANTHER. Interestingly, a weak LD value between the PRNP and PRND SNPs was found in the horse, a prion disease-resistant animal. To the best of our knowledge, these results suggest that a weak LD value could be one feature of prion disease-resistant animals.

1. Introduction

Prion diseases are fatal neurodegenerative disorders that produce an abnormal deleterious prion protein (PrP^Sc) from a normal prion protein (PrP^C) [1,2,3,4]. The prion protein (PrP) is encoded by the prion protein gene (PRNP), and polymorphisms in the PRNP gene affect the susceptibility of prion diseases in humans and ruminants [5,6,7,8,9,10,11]. In humans, susceptibility to sporadic and variant Creutzfeldt–Jakob disease (CJD) is related to PRNP codon 129 methionine homozygosity and/or 219 glutamic acid homozygosity [12,13]. In addition, the vulnerability of sheep scrapie is associated with valine at codon 136, arginine at codon 154 and glutamine at codon 171 (VRQ) and Alanine at codon 136, Arginine at codon 154 and glutamine at codon 171 (ARQ) haplotypes at PRNP codons 136, 154, and 171 [14,15,16]. Over 40 polymorphisms in the PRNP gene have been reported in goats and sheep. Among them, 7 polymorphisms, namely, 142, 143, 146, 154, 171, 211, and 222, are related to prion vulnerability [17,18,19]. In contrast, recent studies have reported that prion disease-resistant species, such as horses in perissodactyls and dogs in carnivores, have distinct genetic features of the PRNP gene [20,21]. Compared with prion disease-susceptible species, dogs have a unique dog-specific amino acid, aspartic acid (D) located at PRNP codon 163, which affects prion resistance [22]. In addition, horses have a distinct structure of the PrP, β2-α2 loop, which contributes to the structural stability of horse PrP [23]. In addition, replacing the dog-specific amino acid D159 and horse-specific amino acid S167 showed toxicity in a transgenic model of dog and horse PrP, respectively [24]. Furthermore, a recent study found that four salt bridges in horse PrP also contribute to its structural stability [25]. However, spontaneous prion aggregation has been observed in mice with a specific amino acid of the horse [26]. Thus, the hypothesis, which can explain prion disease with horse PrP alone, is complicated. Therefore, we investigated the prion-like protein gene (PRND), which is related to the PRNP gene.

Recent studies have reported a strong genetic linkage of single nucleotide polymorphisms (SNPs) between the PRNP gene and the PRND gene in prion disease-susceptible species. Strong linkage disequilibrium (LD) between PRNP codons 136, 154, and 171 and PRND codon 26 was first reported in Portuguese sheep. The alanine at codon 136, arginine at codon 154 and arginine at codon 171 (ARR) haplotype of the PRNP gene is associated with resistance to prion infection and significantly linked with the G allele at PRND codon 26. In contrast, the ARQ haplotype is associated with susceptibility to prion infection and linked with the A allele at codon 26 of the PRND gene [27]. In addition, in prion disease-susceptible animals, goats also have strong LD between codon 143 of the PRNP gene and codons 28, 151, and 385 of the PRND gene and between codon 102 of the PRNP gene and codon 99 of the PRND gene, respectively [28]. However, in prion disease-resistant species such as dogs, all SNPs in the PRND gene show weak LD with all SNPs in the PRNP gene (r² value: below 0.3) [29]. However, it is elusive whether the weak LD value between the PRNP gene and the PRND gene in dogs is characteristic of prion disease-resistant species.

In our previous study, we did not discover SNPs in the horse PRND gene in the Thoroughbred breed [30]. Because the Thoroughbred breed is known to strictly regulate mating, the genetic distribution of the PRND gene may be biased. Thus, we investigated outbred horses, Jeju and Halla horses, to validate the absence of polymorphisms in the open reading frame (ORF) of the PRND gene in horses. The Jeju horse is a Korean native horse that may have originated from Mongolian or independent lineage [31]. Halla horses are a crossbreed of the Jeju and Thoroughbred horses with the purpose of horse racing [32].

In this study, we investigated SNPs using direct sequencing of the horse PRND gene in Jeju and Halla horses. We also analyzed the genotype, allele, and haplotype frequencies of horse PRND SNPs and compared their distributions between Thoroughbred, Jeju, and Halla horses. In addition, we evaluated the influence of the identified nonsynonymous SNPs in the horse PRND gene using PolyPhen-2, PROVEAN, and PANTHER [33,34,35,36]. Furthermore, we measured the LD value between the PRNP and PRND SNPs in horses to assess whether strong genetic linkage is a characteristic of prion disease-resistant animals.

2. Materials and Methods

2.1. Ethical Statement

The hair samples of 148 Jeju and 100 Halla horses were collected from Jeju Island. All experimental procedures were approved by the Institute of Animal Care and Use Committee of Jeonbuk National University (JBNU 2016-65). All experiments using horses were carried out following the Korea Experimental Animal Protection Act.

2.2. Genetic Analysis

The genomic DNA of Halla and Jeju horses was isolated from 10 hair bulbs using a HiYield™ Genomic DNA Mini Kit (Real Biotech Corporation, Banqiao city, Taiwan) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) on the horse PRND coding region located at exon 3 was performed with sense and antisense primers as follows: horse PRND forward 5′-GCCCGTTGCAGCTTCTTATCT-3′ and horse PRND reverse 5′-GCTGGAGGAGAGAAGTGGGAT-3′. PCR was performed in a final volume of 25 µL, including 1 µL of genomic DNA, 10 pmol of each primer, 2.5 µL of 10X Taq DNA polymerase buffer, 0.5 µL of a 0.2 µM dNTP mixture, 2.5 µL of 5X band helper, and 0.2 µL Taq DNA polymerase. The PCR conditions were as follows: denaturing at 95 °C for 2 min, 33 cycles of 95 °C for 20 s, 61 °C for 40 s, 72 °C for 1 min 30 s, and 1 cycle 72 °C for 5 min. The purification of PCR products was performed using a FavorPrep GEL/PCR Purification Mini Kit (FAVORGEN, Pingtung city, Taiwan). The PCR products were directly sequenced using an ABI 3730 sequencer (ABI, Foster City, CA, USA), and sequencing results were analyzed using Finch TV software (Geospiza, Inc., Seattle, WA, USA).

2.3. Statistical Analysis

The genotype, allele, and haplotype frequencies of the horse PRND gene were compared between 3 horse breeds by the chi-square test using SAS 9.4 software. In addition, we examined LD calculated by Lewontin’s D’ (|D’|), and pairwise linkage disequilibrium (r²) was examined using Haploview version 4.2 (Broad Institute, Cambridge, MA, USA). The Hardy–Weinberg equilibrium (HWE) test and haplotype analysis were carried out using Haploview version 4.2 (Broad Institute, Cambridge, MA, USA).

2.4. Analysis of Genetic Linkage between SNPs in the PRNP and PRND Genes

We performed LD analysis between PRNP and PRND SNPs in 135 Jeju and 72 Halla horses. In addition, we examined LD calculated by Lewontin’s D’ (|D’|), and pairwise linkage disequilibrium (r²) was examined using Haploview version 4.2 (Broad Institute, Cambridge, MA, USA).

2.5. Measurement of Protein Functional Alterations Induced by Nonsynonymous SNPs

We predicted the damaging effect of amino acid substitution induced by nonsynonymous SNPs using the PolyPhen-2 v.2.2.2, PROVEAN v.1.1.3, and PANTHER 15.0 programs. PolyPhen-2 evaluates the effect of amino acid changes from an independent count (PSIC), represented as a score ranging from 0 (tolerated) to 1 (deleterious). The prediction range of 0.0 to 0.15 indicates ‘benign’, 0.15 to 1.0 indicates “possibly damaging”, and 0.85 to 1.0 indicates “damaging”. PROVEAN estimates nonsynonymous or indel variants; a score below −2.5 is considered “deleterious”, and a score above −2.5 is considered “neutral”. PANTHER classifies amino acids into groups with similar molecular biology. In PANTHER, a score below −3 indicates “deleterious” and a score above −3 indicates “neutral”.

2.6. Physical Map Distance between the PRNP and PRND Genes among Several Species

Physical map distances between the PRNP and PRND gene sequences were investigated in humans (Homo sapiens, PRNP gene [Gene ID: 5621], PRND gene [Gene ID: 23627]), sheep (Ovis aries, PRNP gene [Gene ID: 493887], PRND gene [Gene ID: 443194]), goats (Capra hircus, PRNP gene [Gene ID: 102169975], PRND gene [Gene ID: 102170246]), dogs (Canis lupus familiaris, PRNP gene [Gene ID: 485783], PRND gene [Gene ID: 485782]), and horses (Equus caballus, PRNP gene [Gene ID: 100065904], PRND gene [Gene ID: 100048937]). We obtained sequence information from GenBank at the National Center for Biotechnology Information (NCBI).

3. Results

3.1. Investigation of Polymorphisms in the Horse PRND Gene in 140 Jeju and 80 Halla Horses

The horse PRND gene consists of two exons, and exon 2 contains the ORF. We investigated polymorphisms in exon 2 of the PRND gene in two horse breeds, Jeju and Halla. The PRND gene sequences of Halla and Jeju horses are identical to that of Equus caballus registered in GenBank (Gene ID: 100048937). We discovered a total of two novel SNPs, c.331G > A and c.411G > C. c.331G > A (A111T) is a nonsynonymous SNP, and c.411G > C is a synonymous SNP (Figure 1a,b). The genotype and allele frequencies of the PRND SNPs are described in

Table 1. Genotype and allele frequencies of the PRND c.331G > A polymorphism in Jeju, Halla, and Thoroughbred horses.

Breed	Total, n	Genotype Frequency, n (%)			p-Value	Allele Frequency, n (%)		p-Value	HWE	Ref
		GG	GA	AA		G	A
Jeju	140	83 (59.3)	52 (37.1)	5 (3.6)	-	218 (77.9)	62 (22.1)	-	0.360	Current study
Halla	80	78 (97.5)	2 (2.5)	0 (0)	<0.0001	158 (98.8)	2 (1.2)	<0.0001	0.909	Current study
Thoroughbred	242	242 (100)	0 (0)	0 (0)	<0.0001	484 (100)	0 (0)	<0.0001	NA *	[30]

* NA, Not available.

and

Table 2. Genotype and allele frequencies of the PRND c.411G > C polymorphism in Jeju, Halla, and Thoroughbred horses.

Breed	Total, n	Genotype Frequency, n (%)			p-Value	Allele Frequency, n (%)		p-Value	HWE	Ref
		GG	GC	CC		G	C
Jeju	140	130 (92.9)	10 (7.1)	0 (0)	-	270 (96.4)	10 (3.6)	-	0.661	Current study
Halla	80	79 (98.8)	1 (1.2)	0 (0)	0.06	159 (99.4)	1 (0.6)	0.0631	0.955	Current study
Thoroughbred	242	242 (100)	0 (0)	0 (0)	<0.0001	484 (100)	0 (0)	0.0022	NA	[30]

. Both SNPs were in HWE.

Next, we compared the genotype and allele frequencies of the c.331G > A (A111T) and c.411G > C SNPs in the horse PRND gene in Jeju, Halla, and Thoroughbred horses. Regarding the c.331G > A (A111T) SNP, the genotype and allele frequencies in the Jeju horse were significantly different from those in the Halla (p < 0.0001) and Thoroughbred (p < 0.0001) horses. However, regarding the c.411G > C SNP, the genotype distribution in the Jeju horse showed no significant difference with that in the Halla horse (p = 0.06) but showed a significant difference with that in the Thoroughbred horse (p < 0.0001). In addition, the allele frequency in the Jeju horse showed no significant difference with that in the Halla horse (p = 0.0631) but showed a significant difference with that in the Thoroughbred horse (p = 0.0022) (Figure 2, Table 1 and Table 2).

We performed a haplotype analysis of the two PRND SNPs in Jeju and Halla horses. A total of three haplotypes, GG, AG, and GC, were observed in these two horses. The GG haplotype was the most frequently observed in the Jeju (74.3%) and Halla (98.1%) horses. The haplotype frequencies between Jeju and Halla horses were significantly different (p < 0.0001) (

Table 3. Haplotype frequency of two PRND polymorphisms in Jeju and Halla horses.

Haplotype	Frequency		p-Value
	Jeju	Halla
GG	208 (0.743)	157 (0.981)
AG	62 (0.221)	2 (0.013)	<0.0001
GC	10 (0.036)	1 (0.006)

). We also estimated the biological impact of the nonsynonymous SNP c.331G > A (A111T) using PolyPhen-2, PROVEAN, and PANTHER. A111T was predicted to be “benign”, with a score of 0.006 by PolyPhen-2, “neutral”, with a score of −0.969 by PROVEAN, and “probably benign”, with a score of 176 by PANTHER (

Table 4. Prediction of the deleterious effect of the nonsynonymous polymorphism in the horse PRND gene by PolyPhen-2, PROVEAN, and PANTHER.

Variation	Method	Score	Prediction
c.331G > A (A111T)	PolyPhen-2	0.006	Benign
	PROVEAN	−0.969	Neutral
	PANTHER	176	Probably benign

).

Next, we measured LD of the two horse PRND SNPs by (|D’|) and r² values. Both SNPs were strongly linked, with a D’ value of 1.0 according to the (|D’|) value in Jeju and Halla horses. However, the r² value showed weak LD, with values below 0.02 (

Table 5. Linkage disequilibrium (LD) between two polymorphisms in the PRND gene in Jeju and Halla horses.

	Jeju		Halla
	c.331G > A (Codon 111)	c.411G > C (Codon 137)	c.331G > A (Codon 111)	c.411G > C (Codon 137)
c.331G > A (codon 111)	-	1.0	-	1.0	D’
c.411G > C (codon 137)	0.011	-	0	-	r2

).

3.2. Analysis of Genetic Linkage between SNPs in the PRNP and PRND Genes

To examine whether horse PRND SNPs have genetic linkage with the PRNP gene, we measured the LD values between SNPs in the PRNP and PRND genes. All PRND SNPs showed weak LD with the PRNP SNPs (r² value: below 0.1) (Figure 3). The detailed r² values are described in

Table 6. Linkage disequilibrium (LD) between single nucleotide polymorphisms (SNPs) in the PRNP and PRND genes with r² values in Jeju and Halla horses.

PRNP		PRND
	Jeju		Halla
	c.331G > A (Codon 111)	c.411G > C (Codon 137)	c.331G > A (Codon 111)	c.411G > C (Codon 137)
c.-3A > G	0.005	0.002	0.025	0.001
c.301T > A (codon 101)	0.0	0.0	0.0	0.0
c.525C > A (codon 175)	0.089	0.003	0.01	0.005
c.570G > A (codon 190)	0.002	0.002	0.003	0.002

.

3.3. Measurement of the Physical Map Distance between the PRNP and PRND Genes among Several Species

We measured the physical map distance between the PRNP and PRND genes in prion disease-susceptible species, including humans, sheep, and goats, and prion disease-resistant species, including dogs and horses. Notably, prion disease-resistant species, including dogs (17 kb) and horses (16 kb), showed relatively short physical map distances between the PRNP and PRND genes compared to those of prion disease-susceptible animals, including humans (20 kb), sheep (25 kb), and goats (25 kb) (Figure 4).

3.4. Genetic Linkage between PRNP and PRND Genes among Several Species

Lastly, we summarized the genetic linkage between PRNP and PRND genes in prion disease-susceptible species, including humans, sheep, and goats, and prion disease-resistant species, including dogs and horses. Notably, prion disease-susceptible animals showed strong genetic linkage between the PRNP and PRND genes compared to prion disease-resistant species (

Table 7. Genetic linkage between PRNP and PRND genes in several animals.

Common Name	Scientific Name	Prion Disease Susceptibility	Method	Genetic Linkage	References
Human	Homo sapiens	Susceptible animal	Pairwise disequilibrium	Strong linkage	[37]
Sheep	Ovis aries	Susceptible animal	Haplotype analysis	Strong linkage	[27]
Goat	Capra hircus	Susceptible animal	Pairwise disequilibrium	Strong linkage	[28]
Dog	Canis lupus familiaris	Resistant animal	Pairwise disequilibrium	Weak linkage	[29]
Horse	Equs canbalus	Resistant animal	Pairwise disequilibrium	Weak linkage	Current study

).

4. Discussion

A previous study surveyed PRND gene polymorphisms in the Thoroughbred horse. However, we did not find polymorphisms in the PRND gene [30]. Because mating in the Thoroughbred horse is strictly regulated, it may cause nonpolymorphic genetic features of the PRND gene. Therefore, we investigated polymorphisms in the horse PRND gene in outbred breeds such as Jeju and Halla.

The Jeju horse is a Korean native horse and is registered and protected as Natural Monument No. 347. According to molecular genetic studies of Korean native Jeju horses, some of the ancestors of Jeju are believed to be of Mongolian origin [31], and some Jeju horses are estimated to have an independent maternal lineage. Halla horses are a crossbreed of the Jeju and Thoroughbred horses, which has been deemed a better breed of horses by the National Institute of Animal Science for horse racing. The lineage of Jeju horses is managed by the Jeju Horse Database System [32], operated by the Livestock Promotion Agency of Jeju Special Self-Governing Province. According to the Livestock Promotion Agency, 5223 Jeju horses were registered and are being raised in 613 breeding farms so far. In addition, 5201 Halla horses were registered and are being raised in 662 breeding farms. The habitat of Jeju and Halla horses is Jeju Island, which is a small island with an area of 1845 km². Interestingly, we found a total of two novel SNPs, including one nonsynonymous SNP (c.331G > A, A111T) and one synonymous SNP (c.411G > C), in the ORF of the horse PRND gene. Unlike Thoroughbred horses, which are distributed in a wide range of habitats, SNPs were first discovered in Jeju and Halla horses despite their limited range of habitats. It can be assumed that the Jeju horse may have evolved longer than the Thoroughbred horse or maintained by free crossing.

According to a previous study, prion disease-susceptible species, such as sheep and goats, show strong LD between SNPs in the PRNP and PRND genes [27,28]. In contrast, prion disease-resistant species, such as dogs, show low LD values between these two genes. In this study, we first discovered a weak LD value between horse PRNP and PRND SNPs. A weak LD value may be regarded as a common feature of prion disease-resistant species (e.g., dogs and horses) [29]. However, the study is limited to a few breeds of only one alleged resistant species and should be extended to more breeds and/or other resistant species. Thus, careful validation is needed in future study.

In addition, prion disease-resistant species showed weak LD values despite the short physical map distance between the PRNP gene and the PRND gene (humans: 20 kb; sheep: 25 kb; goats: 25 kb; dogs: 17 kb; horses: 16 kb) (Figure 4). Interestingly, although there was a short physical map distance between the PRNP and PRND genes, weak LD between these two genes was identified in prion disease-resistant animals (Table 7). Strong genetic linkage between PRNP and PRND genes in prion disease-susceptible animals indicated that prion disease-susceptible animals were selected with prion disease-contributable alleles of PRNP and PRND genes. Thus, evaluation of susceptibility to prion disease according to alleles of polymorphisms in the PRNP and PRND genes will be highly needed in the future.

5. Conclusions

In conclusion, we found a total of two novel SNPs, one nonsynonymous SNP (c.331G > A, A111T) and one synonymous SNP (c.411G > C), in the PRND gene in Jeju and Halla horses. The genotype and allele distributions of horse PRND SNPs were significantly different between Jeju, Halla, and Thoroughbred horses. In addition, we measured LD between SNPs in the PRNP and PRND genes and found a weak LD value. Furthermore, we investigated the physical map distance between the PRNP and PRND genes in prion disease-resistant and -susceptible species. Notably, although there was a short physical map distance between the PRNP and PRND genes, weak LD between these two genes was identified in prion disease-resistant animals. To the best of our knowledge, we provide evidence that a weak LD value could be a feature of prion disease-resistant animals.