Genetic Polymorphism at 15 Codons of the Prion Protein Gene in 156 Goats from Romania

Background: The variability of prion protein gene (PRNP) codons and the frequency of alleles (K222, D146, and S146) that appear to confer genetic resistance to classical scrapie are still unknown in several goat populations/breeds prevalent in Romania. This work aims to assess the genetic polymorphism at 15 PRNP codons in Romanian goat populations to inform the development of goat breeding programs for scrapie resistance. Methods: Whole blood and hair follicles from Carpathian (50), French Alpine (53), and Banat’s White (53) breed goats were sampled to extract genomic DNA for genetic analyses and Sanger sequencing. In the targeted goat groups, one classical scrapie-positive Banat’s White goat was included. Results: The codons without polymorphisms were G37G, W102W, N146N, R151R, S173S, and I218I. The following non-synonymous polymorphisms of PRNP were recorded: P110P, P110S, P110T, T110T, G127G, G127S, I142I, I142M, T142I, H143H, P143P, R143R, R154R, H154R, P168P, Q168Q, Q211Q, Q211R, Q222Q, H222Q, K222K, S240S, P240P, P240S, and S240P. Conclusions: PRNP polymorphism was recorded in 60% (9/15) of codons. The scrapie-positive Banat’s White goat had G37G, W102W, T110T, G127G, I142I, H143H, N146N, R151R, R154R, P168P, S173S, R211R, I218I, Q222Q, and S240S. The K222 allele had a frequency of 6% (3/50) in Carpathian, 9.43% (5/53) in Banat’s White, and 15.09% (8/53) in French Alpine. Therefore, the polymorphisms detected in this sample of Romanian goat breeds are too rare to design a breeding program at the current time.


Introduction
The genotypes of the prion protein gene (PRNP) play an important role in the susceptibility of sheep and goats to scrapie, a naturally occurring transmissible spongiform encephalopathy (TSE) produced by prions [1]. While in sheep, numerous studies led to the development of national genetic selection plans in agreement with Regulation (EC) 999/2001 [2][3][4][5][6][7][8][9], there are insufficient studies involving local goat populations/breeds to validate Romanian control strategies. In the ovine selection programs for scrapie, the haplotype alanine/arginine/arginine (ARR) at codons 136, 154, and 171 is associated with decreased susceptibility or resistance to scrapie, whereas the haplotypes valine/arginine/glutamine (VRQ) and alanine/arginine/glutamine (ARQ) are linked with high susceptibility to scrapie [10].
The importance of active surveillance and control of TSEs has been generally accepted all over the world. European Union (EU) regulations set out general provisions for TSE prevention (monitoring systems, breeding programmes, prohibitions concerning animal feeding, specified risk material, and education programmes), control, and eradication measures (notification, measures with respect to suspect animals, measures following confirmation of the presence of a TSE, and contingency plans) [3]. The use of genetic screening programs in sheep increases the chances of eradicating scrapie in sheep. A similar approach should be applied to goats; however, this requires the accumulation of data concerning PRNP polymorphism in several local goat populations and breeds.
The highly protective effect of the K222 allele against classical scrapie has been demonstrated in several studies [29][30][31][32][33][34][35][36]. However, the European Food Safety Authority (EFSA) noted a very low frequency and heterogeneous distribution of the K222 allele in different goat breeds and EU Member States, which could lead to adverse effects on genetic diversity if a scrapie-resistance breeding programme were developed at the EU level [37]. Before developing a multinational resistance programme for scrapie in goats, each EU member state should assess the appropriateness of implementing national breeding programmes adapted to the genetic characteristics of the local goat population/breeds.
In addition, the PRNP polymorphisms at codons 21,23,37,49,102,110,127,133,137,139,142,142,143,146,146,151,154,168,173,211,218, 220, and 240 have been evaluated for scrapie genetic resistance in goats [15][16][17]22,26,[30][31][32][33]. However, the EFSA summary results of goat genetic surveys in the EU emphasizes only codons 142, 146, 154, and 211-222 as codons with alleles of interest in breeding programmes to promote resistance to classical scrapie in goats. Insufficient data prevented a ranking of susceptibility at genotype level; however, the EFSA used a combination of the "weight of evidence" and the "strength of resistance" to rank the K222 allele with the highest resistance to classical scrapie, followed by D146, S146, Q211, H154, and M142 [37].
Considering the diseases produced by prions, the EFSA monitors the surveillance activities on TSE in cattle, sheep, goats, cervids, and other species by all Member States and some non-EU countries (the United Kingdom, Bosnia and Herzegovina, Iceland, Montenegro, North Macedonia, Norway, Serbia, and Switzerland). Every year, more than 300,000 sheep and 120,000 goats are tested in the EU [38][39][40]. In 2018, classical scrapie was confirmed in 1338 small ruminants (of which 38.69% were goats) and atypical scrapie in 119 small ruminants (of which 5.04% were goats) [38]. In 2019, classical scrapie was recorded in 1290 small ruminants (of which 29.38% were goats) and atypical scrapie in 97 small ruminants (of which 11.34% were goats) [39]. In 2020, there were 908 small ruminants (of which 35.13% were goats), while there were 107 small ruminants (of which 8.41% were goats) with atypical scrapie [40]. Up until 2017, 824 classical and zero atypical scrapie cases in sheep and goats were reported in Romania. Between 2018 and 2020, Romania was one of the most affected EU states by scrapie, with 206 cases in 2018 (out of 523 goats in the EU), 141 cases in 2019 (out of 390 goats in the EU), and 57 cases in 2020 (out of 328 goats in the EU) [38][39][40]. In Romania, only one atypical scrapie case was reported in 2020 (out of 26 goats with atypical scrapie in the EU) [40].
EU policy is designed to eradicate classical scrapie, which poses a higher risk of transmission between animals than atypical scrapie. The scientific report on atypical scrapie monitoring in 22 countries, published in 2021 by the EFSA, concluded that "there is no new evidence that atypical scrapie can be transmitted between animals under natural conditions, and it is considered more likely (subjective probability range 50-66%) that atypical scrapie is a non-contagious, rather than a contagious disease" [41].
This work aims to assess the genetic polymorphism at 15 codons (37,102,110,127,142,143,146,151,154,168,173,211,218,222, and 240) of the PRNP in two local goat breeds (Carpathian and Banat's White) and one cosmopolitan breed (French Alpine) reared in eastern Romania. Our research was justified by the lack of data on the susceptibility of Romanian goat populations to scrapie and whether the alleles K222, D146, and S146 that seem to confer genetic resistance to classical scrapie strains are present in Romanian goat populations. Furthermore, we aimed the amino acid polymorphism analysis at the same codons for a scrapie-positive Banat's White goat. Goats from six farms located in eastern and south-eastern Romania were selected to meet the study's objectives (Table 1). In Romania, goats are reared mainly in extensive lowinput production systems that are oriented primarily towards milk production [42]. The 156 goats targeted in this study were from the main breeds grown in Romania: Carpathian (50), Banat's White (53), and French Alpine (53). Goat ages were between 2 and 15 years. The Romanian breed structure of goats is dominated by the indigenous breeds Carpathian and Banat's White, which represent over 90% of the Romanian goats [42]. Carpathian goats seem to have a high degree of heterogeneity in meat production in relation to the rearing system [43] and the high quality of the meat in comparison to other breeds [44]. In addition, Carpathians have a remarkable organic resistance and adaptation to climatic conditions [42], milk yields between 220 and 350 kg/lactation, litter size between 130 and 160%, and growth rates in kids between 90 and 110 g/day [45,46]. These characteristics have led to the spread of the breed in Eastern European countries and to our interest in including it in this research. Banat's White is currently listed as endangered and included in a genetic conservation program [42]. Banat's White has a milk yield between 370 and 400 kg/lactation and a litter size between 200 and 225% [47]. French Alpine is one of the most popular exotic breeds in Romania, with a milk yield between 525 and 585 kg/lactation [48] and an average litter size of 115% [49].

Samples Collection
Whole blood and hair follicles from each goat were sampled to extract genomic DNA using a protocol described by Gurău et al. (2021) [50]. Gurău et al. (2021) established that the hair follicles of goats can be used as a non-invasive method of sampling biological material for genomic DNA isolation (in whole blood and hair follicle samples, the DNA concentrations are enough for the specific amplification of 1200 bp DNA fragments). Whole blood was collected in a 5 mL sterile tube with K3-EDTA and the hair follicles in a 50 mL sterile transport tube with a screwcap (30-40 hair follicles/sample). The samples were transported in refrigerated conditions (+1-3 • C) within 24 h of sampling on the farm to the laboratory where they were stored at −20 • C until DNA extraction.

Genomic DNA Extraction
Genomic DNA extraction was conducted using PureLink Genomic DNA Kits (Invitrogen, Waltham, MA, USA) by using previously described protocols [50,51]. Briefly, 200 µL of whole blood collected on an EDTA sample and 30-40 hair follicles crushed with a sterile scalpel on a Petri dish were used for each animal to extract DNA in two different protocols described by the manufacturer.

Total DNA Quantification
The amount of total DNA extracted was quantified with a fluorometer (Qubit 4 Fluorometer, Thermo Fisher Scientific Inc., Waltham, MA, USA) using Qubit dsDNA HS Assay Kits [52,53]. The protocol of quantification was previously described by Gurău et al. (2021) [50]. Briefly, the method measures the intensity of the signal from fluorescent dyes which are bound to the DNA molecule and the results are expressed in ng/µL.

Statistical Analysis
Genotypic and allelic frequencies regarding codons 37,102,110,127,142,143,146,151,154,168,173,211,218,222, and 240 were calculated for Carpathian, Banat's White, and French Alpine breed goats. In this study, we were not focused on evaluating the possible inbreeding and transmission of unfavourable linked traits; the Hardy-Weinberg equilibrium state was examined in order to assess the genetic variability conservation.

Results
In all the goats tested, the total DNA amounts extracted from 200 mL of whole blood and 30-40 hair follicles were enough for the specific amplification of targeted DNA fragments and DNA Sanger sequencing of the PRNP gene. This provided high-quality sequences from the two types of biological samples that were easily analysed with BioEdit Sequence Alignment Editor, ver. 7.2.5 (Tom Hall, Ibis biosciences, Carlsbad, CA, USA). PRNP polymorphisms were evaluated for 15 codons in 50 Carpathian, 53 Banat's White, and 53 French Alpine breed goats (Table 4).  [56]. The number of animals is reported in brackets. n/N = the number of animals detected in the population with the specified amino acid/the total number of analysed animals in each breed. -: not detected in breed.

Discussion
Initially validated by Gurău et al. (2021), genomic DNA isolation from hair follicles proved to be, in the case of the 156 goats included in this study, a method with performance similar to the method in which the necessary DNA isolation is made from whole blood samples collected on K3-EDTA. Consequently, the recommendation of this sampling method in selective goat rearing programs to increase scrapie resistance should be considered. Moreover, hair follicles can be sampled from goats by trained farm workers and the materials required for individual sampling and packaging of the sample are inexpensive.
The scientific knowledge concerning genetic resistance to classical scrapie and polymorphisms of the PRNP gene in European goat breeds/populations is sufficient to consider the development of guidance on how to increase goat genetic resistance to classical scrapie by disseminating resistant alleles in goat breeds [15,19,26,28,35,37,44,48,49]; our data give particular information for a future Romanian guide of breeding goats resistant to scrapie. Moreover, our research covered two local breeds (a total of 103 GenBank IDs of Carpathian and Banat's White were obtained) and one of the most common European goat breeds (French Alpine); it also generated data for future metanalyses.
In 2017, the EFSA Panel on Biological Hazards concluded that "the field and experimental data available for the K222, the D146 and the S146 alleles are greater than those available in the public domain for the ARR allele in sheep when the 2002 SSC opinion on safe sourcing of small ruminant materials was produced" [37].
The D146 and the S146 alleles were not detected in the analysed goats. Unfortunately, the K222 allele was rare in all the analysed Romanian herds. Only 16 homozygote K222K goats were detected in our study, which included: two females and one male in Carpathian goats; three females and two males in Banat's White goats; and seven females and one male in French Alpine goats. In our study, 89.10% (139/156) of the goats were homozygote Q222Q. One female of the French Alpine breed was heterozygote H222Q.
In Romania, selection plans for scrapie resistance in goats have not been developed due to the lack of information on the genetic resistance of goats to TSEs. Therefore, the PRNP polymorphisms, alleles, and haplotypes identified and analysed in our study could contribute to the development of breeding programs to reduce the possible risk of TSEs in goats. Extending the survey to more goats and encouraging genotyping by providing subsidies to farmers may be a future direction for stakeholders. The best start for a future breeding program can be the use of homozygous K222 male goats.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes13081316/s1, Figure S1: Electropherograms showing the polymorphism of the prion protein gene (PRNP) identified in Romanian goats by using BioEdit Sequence Alignment Editor (ver. 7.2.5); Figure S2: Electropherograms of three goats with silent mutations; and Table S1: Goats with silent mutations and homo-or heterozygous polymorphisms at 15 codons of the PRNP gene.