Polymorphisms and variants in the prion protein sequence of European moose (Alces alces), reindeer (Rangifer tarandus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Scandinavia

Abstract

The prion protein (PrP) sequence of European moose, reindeer, roe deer and fallow deer in Scandinavia has high homology to the PrP sequence of North American cervids. Variants in the European moose PrP sequence were found at amino acid position 109 as K or Q. The 109Q variant is unique in the PrP sequence of vertebrates. During the 1980s a wasting syndrome in Swedish moose, Moose Wasting Syndrome (MWS), was described. SNP analysis demonstrated a difference in the observed genotype proportions of the heterozygous Q/K and homozygous Q/Q variants in the MWS animals compared with the healthy animals. In MWS moose the allele frequencies for 109K and 109Q were 0.73 and 0.27, respectively, and for healthy animals 0.69 and 0.31. Both alleles were seen as heterozygotes and homozygotes. In reindeer, PrP sequence variation was demonstrated at codon 176 as D or N and codon 225 as S or Y. The PrP sequences in roe deer and fallow deer were identical with published GenBank sequences.

Introduction

The prion protein (PrP) encoded by the prion protein gene (PRNP) is highly conserved among mammals pointing to an essential function for the organism. However, the normal function of cellular PrP is not fully known despite extensive investigations mainly driven by its association with transmissible spongiform encephalopathies (TSE) or prion diseases.¹

Minor variations in the amino acid sequence at critical positions in PrP have profound influence on prion disease susceptibility, incubation time, pathology and the possibility to transmit the disease from one species to another.^2-8

Chronic wasting disease (CWD) is an emerging prion disease in North America⁹ affecting white-tailed deer, mule deer, rocky mountain elk and American moose^10-14and amino acid polymorphisms associated with susceptibility to CWD have been shown.^15-23 In white-tailed deer (Odocoileus virginianus) amino acid variants have been identified in codons 95, 96, 116, 226 and 230.^15,18,21 A comparison of CWD-positive and CWD-negative white-tailed deer suggested that a glutamine (Q) to histidine (H) polymorphism at position 95 (Q95H) and a glycine (G) to serine (S) polymorphism at position 96 (G96S) were underrepresented in CWD-positive populations.²¹ Also, 96S deer PrP transgenic mice showed no evidence of disease after infection.¹⁹ In mule deer (Odocoileus hemionus), polymorphisms in the PrP sequence have been described at codon 20 and 225^15,22where animals heterozygous for serine (S) and phenylalanine (F) at position 225 (225SF) were associated with longer incubation periods in an experimental CWD infection compared with animals homozygous for serine (225SS). In Rocky Mountain elk (Cervus canadensis nelsoni), homozygosity for methionine (M) at PrP position 132 (132MM), corresponding to the human position 129,⁵ were over-represented in CWD-affected elk.¹⁸ Studies in transgenic mice expressing elk PrP sequence variants at amino acid 129 suggest that this polymorphism controls susceptibility and influences the host range of CWD.^16,23 However, a study of the natural variation demonstrated that all three genotypes (132MM, 132ML and 132LL) showed equal susceptibility to infection.¹⁷ The indication of a latent infection²³ and the suggestion of more than one CWD strain^20,24add further complications. Both Shiras moose (Alces alces shirasi) and Alaskan moose (Alces alces gigas) carry a polymorphic PrP sequence variation at codon 209^14,25and natural cases of CWD in Shiras moose have been described.¹⁴ Alaskan Caribou (Rangifer tarandus grantii) have polymorphic positions at amino acids 2, 129, 138 and 169.²⁶

The Cervidae family can be divided into 16 different genera.²⁷ Roe deer is classified into the genus Capreolus, reindeer into Rangifer and fallow deer into the genus Dama. The European moose (Alces alces alces) falls into the genus Alces together with American moose (Alces alces americana). The Alces genus evolved in Eurasia and entered North America through the Bering land bridge.²⁸ Glaciating periods then allowed the American Alces population to disperse into four subspecies, A. a. gigas, A. a. americana, A. a. andersonii and A. a. shirasi.

Recently, the European food Safety Authority presented a survey aimed at detecting the possible presence of CWD in wild and farmed cervids in the EU and Norway. From 2006 to 2010 around 13,000 brain stem samples from cervids were tested and no TSE positive results were found.²⁹ In the survey, a recommendation was made to investigate the PRNP genetic diversity of European cervids to compare with the variation described in the North American cervid population. An initial study of PRNP polymorphisms in free ranging European red deer and roe deer from Scotland and Italy confirmed that the red deer carries polymorphisms that are compatible with a susceptibility to CWD.³⁰ No other investigations have been made in European ruminants to analyze PRNP variability.

This study was undertaken to examine the presence of sequence variants in the PrP from European moose, reindeer, roe deer and fallow deer from Scandinavia to compare with PrP sequence variants found in North American cervids.

Results and Discussion

Although a limited number of animals were analyzed, unique amino acid variants in the PrP sequence (i.e., genotypes) were found (Table 1). European moose (A. a. alces) PrP codon 109 encoded lysine (K) or (Q) and individuals were either homozygous (KK or QQ) or heterozygous (KQ). The 109K/Q and 109Q/Q variants are unique for the European moose since Alaskan moose (A. a. gigas) and other cervids studied here are homozygous for 109K/K.²⁵ This variation at position 109 is situated in a charged cluster³¹ of around 10 amino acids directly on the N-terminal side of the C1 cleavage site.³² The change from a lysine to an uncharged glutamine will influence the charge and polarity of the sequence. The region is highly conserved in vertebrates³³ and the only known natural variant is the European moose K109Q found in this study. Human PrP sequence variants in this charged cluster are associated with a Gerstmann–Sträussler–Scheinker syndrome (GSS) phenotype³⁴ and transgenic mice carrying mutations corresponding to moose K109I/H110I develop neurodegenerative disease spontaneously.³⁵

Table 1. Polymorphisms in the PrP sequence of European moose, reindeer, roe deer and fallow deer in Scandinavia. The results were obtained by sequencing the respective ORF of the PRNP.

Species	Codon
	36	109a	176	209	225	226
European moose (n = 15)	T/T	K/K (n = 6)	N/N	M/M	S/S	Q/Q
		K/Q (n = 6)b
		Q/Q (n = 3)b
Reindeer (n = 9)	T/T	K/K	N/N (n = 8)	M/M	S/S (n = 5)	Q/Q
			D/D (n = 1)		S/Y (n = 2)
					Y/Y (n = 2)
Roe deer (n = 11)	T/T	K/K	N/N	M/M	S/S	Q/Q
Fallow deer (n = 11)	T/T	K/K	N/N	M/M	S/S	E/E
Canadian moose c (n = 7)	T/T (n = 5)	K/K	N/N	M/M (n = 3)	S/S	Q/Q
	T/N (n = 2)			M/I (n = 2)
				I/I (n = 2)

^aVariant codon in bold; ^b This variant has until now only been found in European moose; ^cSamples from Canada as sequencing control.

During the 1980s a complex wasting syndrome in Swedish moose, Moose Wasting Syndrome (MWS), was described.^36-38 The diseased animals showed signs of central nervous disturbances, lesions in mucosal membranes and intestines and atrophied lymphoid organs. Unusual behaviors like circling, no fear of man and anorexia were shown. Pathological investigations indicated no association with a spongiform encephalopathy.³⁹

In order to investigate a possible link between MWS and the variant K109Q, a single-nucleotide polymorphism (SNP) analysis of historical DNA samples collected during the outbreak of MWS in the years 1991 to 1993 was performed. European moose diagnosed with MWS (n = 54) and time matched healthy animals (n = 86) were included in the analysis. The observed genotype proportions of the variants 109K/K, 109K/Q and 109Q/Q corresponding to the nucleotides A/A, A/C and C/C, respectively, are shown in Figure 1. A higher proportion of the heterozygous K/Q variant was seen among the MWS animals compared with the non-diseased population (0.46 and 0.35 respectively). The homozygous Q/Q variant is less common and differs between the animals diagnosed with MWS and the non-diseased animals (0.04 and 0.14, respectively). These data could suggest a possible association between MWS and the K109Q polymorphism. The observed genotype proportions for the homozygous KK variant were 0.49 and 0.50, respectively, for the MWS animals and the healthy animals.

Figure 1. Observed genotype proportions obtained by SNP analysis of European moose diagnosed with MWS (n = 54) and healthy European moose (n = 86) and being homozygous KK, heterozygous KQ or homozygous QQ at codon 109 in the PrP sequence. Samples were collected during the outbreak of MWS in the years 1991 to 1993.

The allelic frequencies for the 109K- and the 109Q codon were for the MWS animals 0.73 and 0.27, respectively and for the healthy animals 0.69 and 0.31, respectively. The genotype frequencies for animals diagnosed with MWS were A/A 0.53, A/C 0.39 and C/C 0.07 and for healthy animals A/A 0.47, A/C 0.43 and C/C 0.10. The genotype frequencies for both groups were not significantly different from Hardy-Weinberg equilibrium. When comparing the proportion of the genotypes A/C to C/C, a significantly greater proportion of A/C was found in the MWS animals than in the healthy animals, 0.93 and 0.71 respectively, (Fischer’s exact test; 2-tail: p = 0.037).

Further, European moose is homozygous for 209M compared with Alaskan moose and Shiras moose that show variation at amino acid position 209 as 209M or 209I.^25,40 Experimental CWD transmission to captive Shiras moose homozygous for MM at position 209 has been demonstrated.40 The ORF of the PRNP from seven samples of Canadian moose were analyzed as a comparison. Individuals from this group were either homozygous MM or II, or heterozygous MI at position 209. A polymorphism was also found in the PrP sequence of Canadian moose (Alces alces andersonii) at amino acid position 36 with T or N as either homozygous 36TT or heterozygous 36TN animals. Silent substitutions were found at codons 63 and 246 in both European moose and Canadian moose.

The PrP sequence from reindeer (Rangifer tarandus tarandus) showed variation at codon 225 with heterozygous SY and homozygous YY and SS animals (Table 1). The position 225 in mule deer is also polymorphic but with 225S or 225F where the heterozygotic 225SF variant has been associated with longer incubation periods of CWD infection compared with the 225SS animals.^22,41 In reindeer a further variant at position 176 was found as either aspartic acid (176D) or asparagine (176N). The176DD variant is unique for reindeer.

Studies of the PrP sequence in Alaskan Caribou²⁶ detected polymorphisms in codons 129 (G or S), 138 (S or N) and 169 (V or M). The corresponding sequence variants in reindeer are G129, S138, and V169.

The PrP sequence from roe deer (Capreolus capreolus capreolus) studied here, were identical to the sequence deposited in GenBank (accession no AY639096). However, two individuals revealed synonymous nucleotide substitutions at codon 24.

In fallow deer (Dama dama dama) all samples analyzed here showed identical PrP sequences (Table 1) and were identical to the PrP sequences deposited in GenBank (accession nos. AY286007, EF139175 and EF165089).

All species in this study were homozygous for methionine at position 132 (132MM), corresponding to the polymorphic position 129 in humans.⁵ In Rocky Mountain elk, variability has been found at amino acid 132 with either M or L¹⁸ where the 132MM individuals were over-represented among CWD-positive animals.

In white-tailed deer, the amino acid at position 95 exists as two allelic variants with Q or H and position 96 shows the two variants G and S. Here, the European moose, roe deer, fallow deer and reindeer were all found to be homozygous QQ at position 95 and homozygous GG at position 96.

The PrP genotypes presented in Scandinavian cervids are similar to genotypes connected with variation in CWD susceptibility in certain North American cervid species. This could warrant a more active investigation of the genetic diversity and further surveillance⁴² in Scandinavia since sheep, reindeer and wild ruminants graze in overlapping areas. This would be in line with recommendations given in the recent EU survey for CWD in cervids.²⁹

Materials and Methods

The present study was performed on recently collected (2006 to 2011) liver samples from hunter killed European moose (Alces alces alces, n = 15, eight from southern, six from middle and one from the northern part of Sweden, National Veterinary Institute, SVA). Historical liver samples from European moose collected between 1991 -1993 and diagnosed with MWS (n = 54, from southern and the middle part of Sweden) and from healthy European moose collected during the same time span (n = 86). European moose diagnosed with MWS were given a combined score of 1–7 according to clinical and pathological findings, where “1” represented severe findings like circling, weakness, emaciation, lesions in the intestines and mucous membranes and a thin spleen, and “7” a healthy moose.³⁶ Of the 54 European moose diagnosed with MWS analyzed in this study, 33 were graded as severely affected (score 1 and 2), 16 with movement disturbances and intestinal lesions (score 3 to 5) and five animals had not been scored.³⁷ Samples of reindeer (R. t. tarandus, n = 9, seven half-domesticated from Finnmark, Norway, one wild reindeer from Hardangervidda, Norway and one wild reindeer from Svalbard), roe deer (C. c. capreolus, n = 11, three from northern, four from middle and four from the southern part of Sweden) and fallow deer (D. d. dama, n = 11 from farms in the south part of Sweden) were all collected from the repository at Animal Genetics Laboratory, Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences. Canadian moose (A. a. andersonii, n = 7) liver samples were kindly provided by C. Strobeck, University of Alberta, Edmonton, Canada to S. Mikko. DNA was prepared from frozen liver samples according to standard methods. The PrP open reading frame was amplified using the following primers; 5′-GCT GAC ACC CTC TTT ATT TTG C-3′ and 5′-GCA AGA AAT GAG ACA CCA CCA C-3′. After purification (QIAquick PCR purification kit, Qiagen) PCR products were sequenced at Uppsala Genome Center, Uppsala, Sweden, (http://www.rudbeck.uu.se/node15). Sequence data were analyzed using DNAStar SeqMan version 9.1 (DNAStar Inc.). For further verification, amplification products from selected animal samples were cloned and both strands of independently isolated PrP clones were sequenced. Sequences obtained in this study have been reported to GenBank (Alces alces alces 109K allele AY6390957, Alces alces alces 109Q allele JQ290077, Capreolus capreolus capreolus AY639096, Rangifer tarandus tarandus 225S allele AY639093, Rangifer tarandus tarandus 225Y allele JQ290076, Rangifer tarandus tarandus 176D allele JQ290075, Dama dama dama AY639094).

All moose samples were genotyped for the A/C SNP using Taqman chemistry on the StepOnePlus™ Real-Time PCR System (Applied Biosystems). The assay consisted of a forward primer: 5′-GTA CCC ACA GTC AGT GGA ACA AG-3′, reverse primer: 5′-GCT CCT GCC ACA TGC TTC A-3′ and two allele-specific fluorescent dye labeled probes VIC- 5′-CAG TAA ACC CAA AAC CA-3′ and FAM- 5′-CAG TAA ACC CCA AAC CA-3′.