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Short communication
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 l’Elevage 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 (Creutzfeldt–Jakob 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) 100–103
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 d’Arles 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 Hardy–Weinberg 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 Hardy–Weinberg 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) 100–103 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.
Conflict 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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