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PRNP polymorphisms in Tunisian sheep breeds

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PRNP polymorphisms in Tunisian sheep breeds
S. Kdidi
a,b,
n
, M.H. Yahyaoui
a
, M. Conte
c
, B. Chiappini
c
, G. Zaccaria
c
,
M. Ben Sassi
d
, A. Ben Ammar El Gaaied
b
, T. Khorchani
a
, G. Vaccari
c
a
Livestock and Wildlife Laboratory, Arid Lands Institute, Rte. El Djorf, Km 22.5, 4119 Medenine, Tunisia
b
Laboratory of Genetics, Immunology and Human Pathology, Faculty of Sciences, Tunis-El Manar University, Tunis 2092, Tunisia
c
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
d
Office de lElevage et des Pâturages, 1002 Tunis, Tunisia
article info
Article history:
Received 11 January 2014
Received in revised form
6May2014
Accepted 9 May 2014
Keywords:
PRNP
Sheep
Polymorphism
Tunisia
abstract
In this study, genetic variation of ovine prion protein in Tunisian sheep breeds was
analysed. Sequencing of the entire coding sequence of prion protein gene (PRNP)was
performed in a total of 201 samples belonging to four breeds (Barbarin, Western Thin Tail,
Sicilo Sarde and Black Thibar). Five haplotypes (ARQ, ARR, ARH, AHQ and VRQ) and 10
genotypes were observed based on codons 136, 154 and 171, with different frequencies
among the investigated breeds. The ARQ, ARR and ARH haplotypes were present in all
breeds, the VRQ haplotype was observed at low frequencies in Barbarin and Western Thin
Tail breeds. The ARQ and ARR haplotypes were the most common with frequencies
ranging from 33.4% to 47.8% and from 26.5% to 46.5% respectively, in the different breeds.
Moreover, 12 additional non-synonymous (Q101R, M112T, G127S/V, M137T, L141F, H143R,
N146S, R151G, Y172D, N176K and H180Y) as well as 2 synonymous polymorphisms at
codons 231 and 237 were found in the PRNP gene. Among them, the R151G polymorphism
has never been described in sheep. Moreover an insertion of an octarepeat in the ARQ
haplotype has been observed. These results represent the first survey on PRNP variability
in Tunisian sheep and may serve as basis for the development of breeding programme to
increase scrapie resistance in national sheep breeds.
&2014 Elsevier B.V. All rights reserved.
1. Introduction
Transmissible spongiform encephalopathies are a group
of fatal neurodegenerative disorders that can occur in sheep
and goat (scrapie), cattle (bovine spongiform encephalo-
pathy), or humans (CreutzfeldtJakob disease). The causa-
tive agent is a pathological isoform (PrP
Sc
)ofthenormal
cellular prion protein (PrP
C
) that accumulates in brain and
lymphoid tissues of the infected individuals (Prusiner,
1998). In sheep, susceptibility to scrapie is influenced by
polymorphisms in the amino acid sequence of the prion
protein gene (PRNP)(Goldmann, 2008). The most studied
polymorphisms at codons 136 (A/V), 154 (R/H) and 171
(Q/R/H) of the protein are combined in five main haplotypes
(expressed in single-letter amino acid code at positions 136,
154, 171): ARQ, VRQ, AHQ, ARH and ARR. The ARR haplo-
type has been associated with the highest level of protec-
tion from classical scrapie, whereas VRQ, ARQ, AHQ and
ARH with different degrees of susceptibility (Baylis et al.,
2004). Additional non-synonymous mutations have been
reported in several breeds, (Goldmann, 2008)mainlyasso-
ciated with the ARQ haplotype giving rise to at least 43
haplotypes. Nor98 or atypical scrapie, first detected in
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/livsci
Livestock Science
http://dx.doi.org/10.1016/j.livsci.2014.05.005
1871-1413/&2014 Elsevier B.V. All rights reserved.
n
Corresponding author at: Livestock and Wildlife Laboratory, Arid
Lands Institute, Rte. El Djorf, Km 22.5, 4119 Medenine, Tunisia.
Tel.: þ216 97 911 581; fax: þ216 75 633 006.
E-mail address: kdidi_samia@yahoo.fr (S. Kdidi).
Livestock Science 167 (2014) 100103
Norway in 1998, is a prion disease that showed distinct
phenotypic characteristics compared with classical scrapie
(Benestad et al., 2008). Nor98 has been identified in most
European countries, in North America (Mitchell et al., 2010;
Loiacono et al., 2009) and New Zealand (Kittelberger et al.,
2010) with sporadic distribution. The susceptibility of sheep
to this apparently spontaneous disease is also under the
control of the PrP gene. Indeed the AHQ and AF
141
RQ
haplotypes are associated with the occurrence of the disease.
In Tunisia no scrapie cases have been so far detected.
It should be mentioned that although scrapie is named
within the list of communicable infectious disease of
animals it is not considered a priority (Dr. H. Kilani
Deguiche, personal communication). Therefore, there is
not a specific surveillance programme implemented in
Tunisia and no central register of tested animals. These
make impracticable to draw any consideration about the
presence of the disease in the country.
Although scrapie has not been detected in Tunisia, it
would be of interest to establish the frequencies of haplo-
types that may render animals resistant to the disease.
There are 7.2 million heads of sheep in Tunisia (ONAGRI,
2010) belonging to four different breeds: Barbarin (60.3%),
Western Thin Tail (34.6%), Black Thibar (2.1%) and Sicilo
Sarde (0.7%). The Barbarin and Western Thin Tail are
common breeds found in Tunisia and Algeria (Iniguez,
2006). Barbarin breed originates from the Asiatic steppes
(Khaldi, 1989). The Black Thibar breed resulted from cross-
breeding the native Western Thin Tail and the French
Merinos dArles breeds (Chalh et al., 2007). The Sicilo-
Sarde breed derived from a cross between the Italian Sarda
and the Comisana breeds realized in late 19th century
(Djemali, 2000). The Sicilo Sarde breed is the only dairy
breed in the North of Africa, while the other three breeds
are used for meat production contributing for more than
40% of the total red meat production (OEP, 2011).
2. Materials and methods
In the present work, we analysed the genetic poly-
morphism of PRNP in these breeds. A total of 201 blood
samples were collected from Barbarin (n¼63), Western
Thin Tail (n¼51), Black Thibar (n¼46) and Sicilo Sarde
(n¼41). Sampling was carried out in 2011, and was
obtained from different flocks located in the north, centre
and south, and representing all geographic regions of the
country. A total of 23 and 17 flocks were sampled from the
northern departments of the country for the Black
Thibar and Sicilo Sarde breeds, respectively. Barbarin and
Western Thin Tail are reared throughout the whole coun-
try, and then, 49 and 29 flocks were sampled, respectively.
A maximum of three samples from unrelated animals were
taken per flock. Genomic DNA was extracted according to
the standard protocol of phenol chloroform, and the entire
PRNP coding sequence was amplified using standard con-
ditions and F1 (5
0
-CAT TTA TGA CCT AGA ATG TTT ATA GCT
GAT GCC A-3
0
) and R1 (5
0
-TTG AAT GAA TAT TAT GTG GCC
TCC TTC CAG AC-3
0
) primers. Sequencing reactions were
performed with primers T3 (5
0
-TTT ACG TGG GCA TTT GAT
GC-3
0
) and T4 (5
0
-GGC TGC AGG TAG ACA CTC C-3
0
) using
Big Dye Terminator Cycle sequencing Kit v1.1 and an ABI
PRISM 3130 apparatus (Applied Biosystems).
Deviations from HardyWeinberg equilibrium were
evaluated using Genepop software version 4 (Raymond
and Rousset, 1995) and chi-square test.
3. Results
The PRNP genotypes, considering the amino acids at
positions 136, 154 and 171, of the four studied sheep
breeds are shown on Table 1. From the fifteen genotypes
commonly found in sheep, only 10 were detected in this
study, ranging between 5 in Black Thibar to 8 in Sicilo
Sarde. The ARR/ARQ was the most frequent genotype in
Barbarin and in Black Thibar while in Western Thin Tail the
common genotype was ARQ/ARQ. Two genotypes, ARQ/
ARQ and ARR/ARQ showed the highest proportions (36.6%)
in Sicilo Sarde breed. The second preponderant genotype
was the ARQ/ARQ in both Barbarin and Black Thibar, the
ARQ/ARR in Western Thin Tail breed and ARQ/AHQ in the
Sicilo Sarde breed.
Among genotypes with the VRQ haplotype, the ARQ/
VRQ was observed only in the Western Thin Tail with low
frequency (2%) whereas the ARR/VRQ in the Barbarin
(1.6%), and it was absent in the other studied breeds. The
ARR/ARR genotype, which provides a high resistance to
classical scrapie, was present in all breeds, the frequencies
ranged between 7.32% (Sicilo Sarde) and 23.9% (Black
Thibar). Based on Fisher's exact test, the four breeds were
in HardyWeinberg equilibrium (P¼0.819) and no devia-
tion was detected (P40.05).
Three haplotypes (ARR, ARQ and ARH) have been
detected in all the studied sheep breeds (Table 2) being
ARQ the most frequent in three breeds. Indeed the ARR
frequency was higher than that of ARQ (46.4% and 33.4%,
respectively) only in the Black Thibar breed.
Twelve non-synonymous polymorphisms have been
detected (Q101R, M112T, G127S/V, M137T, L141F, H143R,
N146S, R151G, Y172D, N176K and H180Y) in addition to
two silent nucleotide substitutions (691a-c and 711c-g)
and the insertion of an octarepeat (OR). Interestingly the
R151G (cgt/ggt) polymorphism is reported here for the
first time (Accession number KF830261). This codon has
Table 1
PRNP genotype frequencies (in %) of Tunisian sheep breeds.
PrP
genotypes
Breeds
Barbarin
(n¼63)
Western Thin
Tail(n¼51)
Black Thibar
(n¼46)
Sicilo Sarde
(n¼41)
ARQ/ARQ 30.2 43.1 28.3 36.6
ARQ/AHQ 2 9.8
ARQ/ARH 7.95 9.8 2.15 2.42
ARQ/VRQ 2
ARR/ARQ 42.8 33.3 43.5 36.6
ARH/ARH 2.42
ARR/AHQ 2.15 2.42
ARR/ARH 7.95 2.42
ARR/ARR 9.5 9.8 23.9 7.32
ARR/VRQ 1.6
n¼number of animals.
S. Kdidi et al. / Livestock Science 167 (2014) 100103 10 1
already been described as polymorphic: R/C (Tranulis et al.,
1999)orR/H(Acin et al., 2004) however to our knowledge
the G variant has never been reported. An insertion of 24 bp
(GGTGGCTGGGGTCAGCCCCATGGA) in the octarepeat region
between nucleotides 186 and 187 of the open reading frame
has been observed in one animal of the Western Thin Tail
breed. This insertion in the ARQ haplotype generates a new
haplotype designed ARQ
6OR
(Accession number KF830262)
consisting of an additional octarepeat (PHGGGWGQ), similar
tothe6ORhaplotypeobservedinbovinespecie(McKenzie
et al., 1992;Hunter et al., 1994). Although variation on the
number of the octarepeats has been already observed in goat
(Goldmann et al., 1998), it has never been described in sheep.
The V
127
ARQ was showed in the Barbarin and Black Thibar
breeds, while AS
127
RQ, AT
137
RQ, AG
151
RQ, ARQD
172
and
ARQY
180
haplotypes occurred only in one of the four breeds.
4. Discussion
The VRQ and the AF
141
RQ have been associated with
high susceptibility to classical scrapie and Nor98 (Moum
et al., 2005). These haplotypes were observed at low
frequencies, the VRQ only in Barbarin and Western Thin
Tail (0.7% and 1% respectively) while the AF
141
RQ in all the
four breeds (with frequencies lower than 1.5%). The AHQ
haplotype, also associated with atypical scrapie suscept-
ibility, was found in Western Thin Tail, Black Thibar and
Sicilo Sarde.
The frequency of the ARR haplotype, associated with
resistance to scrapie, varied between 26.5% in Western Thin
Tail and 46.5% in Black Thibar. Another haplotype that has
been associated with scrapie resistance (Vaccari et al., 2007,
2009a), the AT
137
RQ was observed only in Sicilo Sarde
(3.9%). Interestingly this haplotype has been observed in
several European breeds among which the Sarda breed in
Italy (Vaccari et al., 2001) from which this breed derives.
The AS
146
RQ haplotype is the homologous of one of the
haplotypes associated to scrapie resistance in goats (for
review see Vaccari et al., 2009b) and has been already
observed in Asian sheep (Ün et al., 2008;Alvarez et al.,
2011;Karami et al., 2011;Meydan et al., 2013). Interest-
ingly, this haplotype has been observed in the Barbarin
and Western Thin Tail, probably reflecting their breed
origin. The ARQK
176
, haplotype has been also associated
with scrapie resistance (Vaccari et al., 2007,2009a), and it
was observed in all breeds with a frequency ranging from
3.0% to 11. 7%.
This work represents the first report on Tunisian sheep
PrP gene variability. Our results showed the presence of
relatively high frequencies of the ARR haplotype but also of
other haplotypes associated with scrapie resistance such as
ARQK
176
. The VRQ haplotype, associated with higher sus-
ceptibility to scrapie, was observed in only two breeds with
very low frequencies. Overall, our results indicated that the
ovine population in Tunisia could be susceptible to both
classical and atypical scrapie. These results will eventually
help the development of breeding programs in Tunisia to
render sheep resistant to scrapie.
Conict of interest statement
The authors declare that there are no conflicts of
interest.
Acknowledgements
The authors thank sheep owners and OEP (central
office and regional directions) in Tunisia for providing
blood samples and Dr Hajer Kilani Deguiche (Ministry of
Agriculture, DGSV) for providing information about scrapie
in Tunisia. Kdidi S. was supported by a scholarship from
the Tunisian Ministry of Higher Education.
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PRNP haplotype frequencies (in %) of Tunisian sheep breeds.
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Tail(n¼51)
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Sarde
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ARQ 43.5 41.3 33.4 47.8
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101
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AT
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RQ 0.7 2 1.9 0
AS
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S. Kdidi et al. / Livestock Science 167 (2014) 100103 10 3
... An earlier study linked AC 151 RQ genotype to prolonged incubation period after scrapie exposure [7]. Similarly, several studies reported variants such as G126A, G126G, G127G, G127V, G127A, and S138S [10][11][12] in sheep prion protein with or without direct effect to scrapie susceptibility. ...
... As in previous studies, non synonymous and synonymous substitutions i.e. G127G, S138S, R231R, L237L, G126A, 126GA, 127GV, 127GA, 142IT, N146S, N146NS were also identified in the population under study [10][11][12]. In the present study, the variant at codon 127 in particular was highly polymorphic. ...
... Studies from Tunisia and Algeria reported ARQ, ARR, AHQ, ARH, and VRQ as major alleles. In the same studies, additional polymorphisms which were also previously reported in Spanish and Italian sheep breeds were identified [12,[35][36][37]. ARQ was recorded at a significantly higher frequency in scrapie-affected Spanish sheep. ...
Article
Full-text available
Background: Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene (PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at codon 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent's sheep breeds. Therefore, genotyping native Ethiopian sheep breed's PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results: Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-- > CAA), H99L (CAC-- > CTA), A116E (GCA-- > GAA), A116T (GCA-- > ACA), and one synonymous N103 N (AAC-- > AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231, and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion: Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141 L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie.
... An earlier study linked AC 151 RQ genotype to prolonged incubation period after scrapie exposure [7]. Similarly, several studies reported variants such as G126A, G126G, G127G, G127V, G127A, and S138S [10][11][12] in sheep prion protein with or without direct effect to scrapie susceptibility. ...
... As in previous studies, non synonymous and synonymous substitutions i.e. G127G, S138S, R231R, L237L, G126A, 126GA, 127GV, 127GA, 142IT, N146S, N146NS were also identified in the population under study [10][11][12]. In the present study, the variant at codon 127 in particular was highly polymorphic. ...
... Studies from Tunisia and Algeria reported ARQ, ARR, AHQ, ARH, and VRQ as major alleles. In the same studies, additional polymorphisms which were also previously reported in Spanish and Italian sheep breeds were identified [12,[36][37][38]. ARQ was recorded at a significantly higher frequency in scrapie-affected Spanish sheep. ...
Preprint
Full-text available
Background Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene (PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at codon 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA), and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231, and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie.
... An earlier study linked AC 151 RQ genotype to prolonged incubation period after scrapie exposure [7]. Similarly, several studies reported variants such as G126A, G126G, G127G, G127V, G127A, and S138S [10][11][12] in sheep prion protein with or without direct effect to scrapie susceptibility. ...
... As in previous studies, non synonymous and synonymous substitutions i.e. G127G, S138S, R231R, L237L, G126A, 126GA, 127GV, 127GA, 142IT, N146S, N146NS were also identified in the population under study [10][11][12]. In the present study, the variant at codon 127 in particular was highly polymorphic. ...
... Studies from Tunisia and Algeria reported ARQ, ARR, AHQ, ARH, and VRQ as major alleles. In the same studies, additional polymorphisms which were also previously reported in Spanish and Italian sheep breeds were identified [12,[36][37][38]. ARQ was recorded at a significantly higher frequency in scrapie-affected Spanish sheep. ...
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Full-text available
Background: Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene (PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at position 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results : Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA), and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231, and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion: Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie.
... An earlier study linked the AC 151 RQ genotype to a prolonged incubation period after scrapie exposure [7]. Similarly, several studies reported variants such as G126A, G126G, G127G, G127V, G127A and S138S [10][11][12] in sheep prion protein with or without direct effect to scrapie susceptibility. ...
... As in previous studies, non synonymous and synonymous substitutions i.e. G127G, S138S, R231R, L237L, G126A, 126GA, 127GV, 127GA, 142IT, N146S and N146NS were also identified in the population under study [10][11][12]. In the present study, the variant at codon 127 in particular was highly polymorphic. ...
... Studies from Tunisia and Algeria reported ARQ, ARR, AHQ, ARH, and VRQ as major alleles. In the same studies, additional polymorphisms which were also previously reported in Spanish and Italian sheep breeds were identified [12,36]. ...
Preprint
Full-text available
Background Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene ( PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at position 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA) and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231 and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie. Keywords: Ethiopian sheep; Novel Variations; Polymorphism; Prion gene; Scrapie Susceptibility
... PCR amplification and sequencing were carried out as previously reported [41]. ...
... PCR amplification and sequencing were carried out as previously reported [41]. Briefly, primers were designed over the available sequence (GenBank accession number EU032305.1) ...
Article
Full-text available
Scrapie is a fatal prion disease. It belongs to transmissible spongiform encephalopathies (TSEs), and occurs in sheep and goats. Similarly, to ovine species, the prion protein gene (PRNP) plays a major role in conferring resistance or susceptibility to TSE in goats. This study assesses the variability of PRNP in native and crossed-breed goat populations raised in the Southeast of Tunisia and provides information on the distribution of PRNP haplotypes and genotypes in these goat populations. A total of 116 unrelated goats including 82 native and 34 crossed-breed goats were screened for PRNP polymorphisms using Sanger sequencing. Sequence analysis revealed 10 non-synonymous polymorphisms (G37V, M137I, R139S, I142M, H143R, N146D, R154H, R211Q, Q222K, and S240P), giving rise to 12 haplotypes and 23 genotypes. Moreover, four silent mutations were detected at codons 30, 42, 138, and 179; the former was reported for the first time in goat (nucleotide 60 c→t). Interestingly, the PrP variants associated with resistance (D146 and K222) or with a prolonged incubation time of goat to scrapie (M142, R143, H154, Q211) were absent or detected with low frequencies except for H154 variant, which is present with high frequency (1%, 1%, 4%, 0%, 88%, and 6%, respectively, for native goats, and 0%, 1%, 0%, 1%, 78%, and 1%, respectively, for crossed goats). The analysis of PRNP polymorphisms of goats raised in other regions of the country will be useful in getting a global view of PRNP genetic variability and the feasibility of goat breeding programs in Tunisia.
... accessed on 27 January 2021) and classified as "Deleterious" and "Neutral". All the missense mutations were gathered from Ensembl (EN-SOART00020017839.1) and through database searching [13][14][15][16][17][18]. The alignment was performed using T-Coffee program [9] and edited using Genedoc software [10]. ...
... accessed on 27 January 2021) and classified as "Deleterious" and "Neutral". All the missense mutations were gathered from Ensembl (ENSOART00020017839.1) and through database searching [13][14][15][16][17][18]. [30] Camel Prion Disease Dromedary camel 134E to be determined [31] To be determined [32] Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 33 Figure 3. Missense mutations of prion protein (PrP) in Ovis aries. ...
Article
Full-text available
Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are a fatal group of infectious, inherited and spontaneous neurodegenerative diseases affecting human and animals. They are caused by the conversion of cellular prion protein (PrPC) into a misfolded pathological isoform (PrPSc or prion- proteinaceous infectious particle) that self-propagates by conformational conversion of PrPC. Yet by an unknown mechanism, PrPC can fold into different PrPSc conformers that may result in different prion strains that display specific disease phenotype (incubation time, clinical signs and lesion profile). Although the pathways for neurodegeneration as well as the involvement of brain inflammation in these diseases are not well understood, the spongiform changes, neuronal loss, gliosis and accumulation of PrPSc are the characteristic neuropathological lesions. Scrapie affecting small ruminants was the first identified TSE and has been considered the archetype of prion diseases, though atypical and new animal prion diseases continue to emerge highlighting the importance to investigate the lesion profile in naturally affected animals. In this report, we review the neuropathology and the neuroinflammation of animal prion diseases in natural hosts from scrapie, going through the zoonotic bovine spongiform encephalopathy (BSE), the chronic wasting disease (CWD) to the newly identified camel prion disease (CPD). CITATION: Orge, L.; Lima, C.; Machado, C.; Tavares, P.; Mendonça, P.; Carvalho, P.; Silva, J.; Pinto, M.d.L.; Bastos, E.; Pereira, J.C.; Gonçalves-Anjo, N.; Gama, A.; Esteves, A.; Alves, A.; Matos, A.C.; Seixas, F.; Silva, F.; Pires, I.; Figueira, L.; Vieira-Pinto, M.; Sargo, R.; Pires, M.d.A. Neuropathology of Animal Prion Diseases. Biomolecules 2021, 11, 466. https://doi.org/10.3390/biom11030466
... accessed on 27 January 2021) and classified as "Deleterious" and "Neutral". All the missense mutations were gathered from Ensembl (EN-SOART00020017839.1) and through database searching [13][14][15][16][17][18]. The alignment was performed using T-Coffee program [9] and edited using Genedoc software [10]. ...
... accessed on 27 January 2021) and classified as "Deleterious" and "Neutral". All the missense mutations were gathered from Ensembl (ENSOART00020017839.1) and through database searching [13][14][15][16][17][18]. [30] Camel Prion Disease Dromedary camel 134E to be determined [31] To be determined [32] Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 33 Figure 3. Missense mutations of prion protein (PrP) in Ovis aries. ...
Article
Full-text available
Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are a fatal group of infectious, inherited and spontaneous neurodegenerative diseases affecting human and animals. They are caused by the conversion of cellular prion protein (PrPC) into a misfolded pathological isoform (PrPSc or prion- proteinaceous infectious particle) that self-propagates by conformational conversion of PrPC. Yet by an unknown mechanism, PrPC can fold into different PrPSc conformers that may result in different prion strains that display specific disease phenotype (incubation time, clinical signs and lesion profile). Although the pathways for neurodegeneration as well as the involvement of brain inflammation in these diseases are not well understood, the spongiform changes, neuronal loss, gliosis and accumulation of PrPSc are the characteristic neuropathological lesions. Scrapie affecting small ruminants was the first identified TSE and has been considered the archetype of prion diseases, though atypical and new animal prion diseases continue to emerge highlighting the importance to investigate the lesion profile in naturally affected animals. In this report, we review the neuropathology and the neuroinflammation of animal prion diseases in natural hosts from scrapie, going through the zoonotic bovine spongiform encephalopathy (BSE), the chronic wasting disease (CWD) to the newly identified camel prion disease (CPD).
... An earlier study linked AC 151 RQ genotype to prolonged incubation period after scrapie exposure [7]. Similarly, several studies reported variants such as G126A, G126G, G127G, G127V, G127A, and S138S [10][11][12] in sheep prion protein with or without direct effect to scrapie susceptibility. ...
... As in previous studies, non synonymous and synonymous substitutions i.e. G127G, S138S, R231R, L237L, G126A, 126GA, 127GV, 127GA, 142IT, N146S, N146NS were also identi ed in the population under study [10][11][12]. In the present study, the variant at codon 127 in particular was highly polymorphic. ...
Preprint
Full-text available
Background Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene (PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at codon 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA), and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231, and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie.
... Genetic diversity of Tunisian sheep breeds was previously investigated through morphologic and morphometric descriptors (Khaldi et al., 2011). Moreover, molecular markers were used to study the genetic structure of Tunisian sheep populations, such as RAPD-PCR (Khaldi et al., 2010;Hentati et al., 2012), microsatellites markers (Ben Sassi-Zaidy et al., 2014; Kdidi et al., 2015a), and sequence polymorphisms at the Prion protein (PRNP) gene (Kdidi et al., 2014) showing the presence of relatively high frequencies of the ARR haplotype associated to scrapie resistance. Analysis of genome-wide SNP markers has been shown to provide an unprecedented resolution in reconstructing genetic relationships, inferring genetic structure, and assessing breed distinctiveness and conservation status in sheep (Kijas et al., 2009;Zhao et al., 2017). ...
Article
Assessing the status of genetic variability of native sheep breeds could provide important clues for research and policy makers to devise better strategies for the conservation and management of genetic resources. In this study, a genetic investigation of Tunisian sheep breeds using a genome-wide scan of approximately 50,000 SNPs was performed. To reconstruct genetic structure and relationships among four sheep breeds, 40 samples belonging to fat-tailed Barbarine, Queue Fine de l’Ouest, Noire de Thibar and D’Man breeds were genotyped using Illumina Ovine SNP50 BeadChip. Tunisian breeds averaged 96% polymorphic loci with an expected heterozygosity (He = 0.36). Genetic analysis of relationship between breeds using Bayesian clustering, MDS and Neighbor-Network analysis, and estimation of FST genetic structure, highlighted the genetic differentiation of Noire de Thibar breed from the other local breeds, reflecting the effect of past events of introgression of European gene pool. The Queue Fine de l’Ouest breed showed a genetic heterogeneity and was close to Barbarine and D'Man breeds, as evidenced by MDS and the lowest level of differentiation with Barbarine breed (FST = 1.8%). The D'Man breed shared a considerable gene flow with the thin-tailed Queue Fine de l'Ouest breed. Possible factors explaining the genetic patterns observed, such as considerable gene flow probably due to anthropogenic activities in the light of population management and conservation programs.
Article
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Classical scrapie is a contagious prion disease of sheep and goats. It is endemic in many countries in Europe, North America, and Asia. In Africa, imported scrapie cases have been described in South Africa and Kenia in the past. More recently, several cases have been reported from different regions of Libya, based on clinical signs and histological lesions. Here, we report the results of thorough investigations carried out on a suspect case of scrapie in a 6-year-old Barbarine sheep, born, and bred in Tunisia, showing behavioral changes, weight loss, itching, skin lesions, wool loss, and motor incoordination. Histopathology and immunohistochemistry revealed spongiform change in several brain areas with associated pathological prion protein deposition. Western blotting confirmed the diagnosis and showed a classical scrapie-like molecular pattern of PrPres, different from atypical scrapie and bovine spongiform encephalopathy (BSE) in small ruminants. Sequence analysis of the prion protein gene showed that the animal carried the ARQ/ARQ genotype, one of the most susceptible to classical scrapie. The inoculation of sheep brain homogenate in a susceptible rodent model proved the experimental transmissibility of the disease. These results demonstrate the circulation of classical scrapie in Tunisia and confirm its presence in North Africa, indicating the need to improve epidemiological surveillance and diagnostic capacity for prion diseases in the region.
Article
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Several PrP gene polymorphisms modulate sheep scrapie susceptibility. Recently, an increase of scrapie outbreaks has been reported in Italy. A vaccine containing sheep brain homogenate was used in most of the outbreaks. We investigated PrP gene polymorphisms in scrapie-affected and clinically healthy Sarda breed sheep from a flock exposed to the aforementioned vaccine, and in affected Sarda sheep from unexposed flocks. All affected animals were (Gln/Gln)171 homozygous. Moreover, we observed no variation for Ala136 and a new polymorphism (Lys to Asn) at codon 176. Our findings confirm the correlation between scrapie and (Gln/Gln)171 in breeds with no variation for Ala136.
Article
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The aim of this study was to identify the PRNP polymorphisms outside the standard codons 136, 154 and 171 in 1110 sheep with no clinical sign of scrapie from all 18 Turkish native sheep breeds and compare our results with published data on ovine PRNP polymorphism from other regions of the world. Among the 22 amino acid polymorphisms and three silent mutations, 10 were novel for ovine PRNP: p.Gly94Gly, p.Leu128Ile, p.Met132Leu, p.Ser135Arg, p.Met137Val, p.Asn146Lys, p.Arg159Arg, p.Tyr160Asn, p.Gln163His and p.Thr193Ser. These data reveal that sheep breeds close to the historic center of small ruminant domestication have remained highly diverse in the prion gene locus, with distinctive genetic similarities to both Asian and European sheep breeds.
Article
Ovine scrapie is a neurodegenerative disease caused by polymorphisms of the prion protein gene (Prnp); especially the amino acid residue alterations at codons 136, 154, and 174, in sheep have been found to be associated with susceptibility to scrapie disease. We studied Prnp polymorphisms in local sheep of Khuzestan, Iran. In this study, a total of 250 healthy and randomly chosen sheep were investigated. Information from the breeder was considered in order to avoid family connections. The genetic DNA of blood samples was extracted, amplified and sequenced. At codon 136, are two different amino acid residues A and V with frequencies of 98.8 and 1.2%, respectively. In our study, the amino acid residue at 154 in all the prion protein (PrPs) was A. At codon 171, are three different amino acid residues Q, R and H with frequencies of 68.4, 21.8 and 9.8%, respectively. The frequency of the amino acid residues ARQ/ARQ at codons 136, 154, and 171, respectively, which is associated with medium-high susceptibility to scrapie, was 43.2%. The frequencies of genotypes ARQ/ARR, ARQ/ARH, ARQ/VRQ, ARR/ARH and ARH/ARH were 38, 10, 1.2, 5.2 and 2.4, respectively. The scrapie-resistant genotype ARR/ARR was not found. Also, the highly susceptible genotype VRQ/VRQ at these codons were not detected in the tested sheep. In addition, five polymorphism were identified ( G127V, N146S, Y172D, S173N, and V179E ) at different codons of PrP gene. Key words : Prion protein gene (Prnp), polymorphisms, susceptibility, scrapie.
Article
Ovine susceptibility to scrapie is largely controlled by polymorphisms in the PRNP gene. Beginning in 2003, breeding programmes based on the known association between this gene and the susceptibility/resistance status of this disease have been implemented in many European countries. This is not the case in Turkey, where the PRNP gene was not genotyped in native sheep breeds until recently. We sequenced the complete open reading frame of the PRNP gene in 100 sheep belonging to five native Turkish sheep breeds (Akkaraman=21; Morkaraman=20; Tuj=17; Hemsin=23; Karayaka=19). Based on the variability found at codons 136, 154 and 171 (also referred to as standard codons), we determined six alleles VRQ, TRQ, ARR, ARH, ARK and ARQ. The archetype, ARQ, was the most frequent variant in each of the five breeds (across-breed frequency=0.710), while the second most frequent was the resistance-associated ARR allele (0.140). The susceptible VRQ allele exhibited the lowest frequency (0.015). The AHQ allele was not found in any of the analysed breeds. Beyond variability in the standard codons, we found ten additional amino acid variants (M112T, G127S, G127V, M137T, L141F, H143R, N146S, Y172D, Q189L and V213E), one of which, V213E, has not been previously described to our knowledge. Overall, this report may serve as a complement of previous studies on the genetic background of native Turkish sheep regarding the resistance/susceptibility status to classical and atypical scrapie.
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Scrapie is an infectious fatal disease of sheep that affects the central nervous system. Polymorphisms in sheep PrP gene are known to be related to scrapie susceptibility. Selection programmes for scrapie based on PrP genotyping data are available for many of the European sheep breeds. So far comparable data for local Turkish sheep breeds are not available. The aim of this study was genotyping of PrP gene in Turkish native sheep to find out their risk groups. Therefore, in this study 109 native Turkish sheep belonging to Kivircik, Sakiz and Imroz breeds were genotyped by means of PCR and direct sequencing. The polymorphism detected in the prion gene was made up six alleles ARR, ARQ, AHQ, VRQ, TRQ, ARH and 12 genotypes, ARR/ARR, ARR/TRQ, ARR/ARQ, ARQ/AHQ, ARH/TRQ, TRQ/TRQ, ARQ/TRQ, ARQ/ARQ, ARQ/ARH, ARH/ARH, ARR/VRQ and ARQ/VRQ. In addition, eight polymorphisms were identified (M112T, A116E, R138M, N146S, Y172D, S173N, V179E and R231R) at different codons of PrP gene. The data have shown that PrP genes of the analysed sheep are highly variable and the most of the genotypes belong to risk groups 1 and 2.
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Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt–Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high β-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.
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The primary amino acid sequence of the prion protein (PrP) has previously been correlated with changes in the incubation period of subacute spongiform encephalopathies. We have analyzed the PrP gene from 65 different cattle representing 14 breeds by polymerase chain reaction and restriction enzyme analysis. Two distinct PrP alleles differing in the number of octapeptide repeats are present. The predominant genotype is homozygous for 6 octapeptide repeats. Few individuals (8) were found to be heterozygous for these repeats and only 1 animal was homozygous for the 5 octapeptide repeat allele.