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transcript
Sheep genotyping for PrP gene polymorphisms in rare Greek breeds
Loukia V. Ekateriniadou, Cynthia H. Panagiotidis, Anastasios Terzis, Konstantoula
Ploumi, Alexandros Triantafyllidis, Panagiotis Deligiannidis, Costas Triantaphyllidis,
Theodoros Sklaviadis
L. V. Ekateriniadou, PhD,
National Agricultural Research Foundation - Veterinary Research Institute,
570 08 Ionia, Thessaloniki, Greece
C. H. Panagiotidis, PhD,
A. Terzis, BA,
T. Sklaviadis, PhD
Prion Disease Research Group,
School of Pharmacy, Aristotle University of Thessaloniki
54 124 Thessaloniki, Greece
A. Triantafyllidis, PhD,
P. Deligiannidis, MS,
C. Triantaphyllidis, PhD,
School of Biology, Aristotle University of Thessaloniki
54 124 Thessaloniki, Greece
K. Ploumi, DVM,
National Agricultural Research Foundation, Agricultural Research Station of
Chalkidiki, 63200 Agios Mamas, Greece
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Summary Current data indicate that at least 1-1.5% of Greek sheep flocks are affected by
scrapie (Greek Ministry of Rural Development and Food). The aim of the present
work was to determine the PrP genotype profile of rare Greek sheep breeds, in order
to find breeding animals resistant to scrapie. Thus, the PRNP genes of 388 sheep
from 13 rare breeds were analysed for polymorphisms at codons 136, 154, 171 and a
total of five haplotypes and eleven different genotypes were found. Genotypes of low
risk Group R1 (ARR/ARR) and Group R2 (ARR/ARQ, ARR/ARH, and ARR/AHQ)
appeared at high frequencies, i.e. 14.43% and 44.4%, respectively. As the frequency
of the VRQ haplotype, known to be associated with a high risk for scrapie, is very
low (0.4%), these breeds cannot be considered valine breeds. The highest frequency
of scrapie resistance genotypes was observed in the Kimi and Kalaritiko breeds. A
pilot-breeding programme to increase the frequency of scrapie resistant genotypes
will be established soon in Greece. The high frequency of scrapie-resistant genotypes
in the country’s rare breeds (a total of 58.83%) and the availability of recently
genotyped low-risk Class R1 and R2 individuals should greatly facilitate and expedite
such a breeding programme.
KEY WORDS: scrapie resistance, rare Greek sheep breeds, allelic (haplotype) frequency,
PCR, RFLP.
2
Introduction
Scrapie is a slowly progressive infectious disease of sheep and goats that causes
degeneration of the central nervous system. The main constituent of the infectious
agent is an aberrant isoform (PrPSc) of the normal cellular (PrPC) prion protein (PrP),
which is a cell–surface glycoprotein (Madec and others 1997, Prusiner 1998). In
sheep, polymorphisms at codons 136, 154 and 171 of the host gene that encodes PrP
are known to be closely linked to susceptibility to natural and experimental scrapie
(Laplanche and others 1993, Westaway and others 1994, Belt and others 1995, Smits
1995, Bossers and others 1996, Hunter and others 1996, Dawson and others 1998,
Baylis and others 2002).
Breed differences in both the frequencies of different PrP alleles and the correlation of
the alleles with the disease are known; however, some clear genetic rules have
emerged. According to the British National Scrapie Plan (Dawson 2003), a
classification system of five risk groups (R1-R5) has been established with R1
indicating a very low risk of scrapie and R5 indicating genotypes associated with the
highest susceptibility.
The PrP genotypes of a majority of the North-Central European, as well as some of
the Asian sheep breeds, have been studied and the polymorphisms characteristic of
each breed are known (Hunter and Cairns 1998, Bossers and others 1999,
Thorgeirsdottir and others 1999, Tranulis and others 1999, Vaccari and others 2001,
Siros and others 2002, Gombojav and others 2003). Breeds can be characterized as
either “simple” or “complex” types (Ikeda and others 1995, Hosie and Dawson 1996,
O’ Doherty and others 2001), based on plurality of their PrP allele variants and
resultant geneotypes.
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In Greece, where the sheep population stands at about 9.5 million, there are twenty-
six native breeds (Rogdakis 2002), nine of which represent significant populations. As
of 2004, scrapie has been diagnosed in 70 Greek flocks, with all the scrapie-affected
sheep being crossbred individuals. As scrapie has not yet been found in any of the rare
Greek breeds, it is of great interest to determine if their seeming resistance can be
correlated to any of the known scrapie resistance-associated genotypes.
The Chios breed, a well-known Greek breed of sheep and one of the world’s best milk
producers, is found both in Northern Greece (Chios Mainland) and on the Aegean
island of Chios (Chios island). The Chios Mainland breed is the result of
crossbreeding between local sheep of the Chios Island breed and breeds from
Anatolia.
In addition to the Chios breed, Northern Greece is also home to the breeds of Thrace,
Katsika, Kozani, Florina and Sarakatsaniko. The Florina breed is raised in the
lowlands of Northern Greece and in some regions of Former Yugoslavian Republic of
Macedonia. The mountainous breeds of Thrace and Sarakatsaniko have many
similarities and probably share a similar origin (Kivircik). Finally, the Katsika breed
originated from the Zackel breed, as did the Kozani breed. It is worth noting that the
latter breed is particularly well-adapted to the hostile environmental conditions of
Greece.
Central Greece has four main native breeds of sheep, the Kalaritiko, Pilio,
Karamaniko and Argos breeds. The mountainous Kalaritiko breed originated from an
ancient local breed and the Italian breed, Comisana. The lowland Karamaniko breed
and another mountainous breed, that of Pilio, both originated from the breed of
Zackel. Argos sheep are a lowland breed of Central Greece.
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In addition to the previously mentioned breed of Chios island, the Skopelos, Kimi and
Zakynthos breeds are also located mainly on islands. The breed of Skopelos, one of
the best known old Greek breeds, is found on the Sporades islands of Skopelos and
Skiathos. It is a crossbreed between local sheep and breeds from Anatolia. Kimi
sheep live in the mountains of Evia, an island just northeast of Athens and the
Zakynthos breed, having many similarities to the Italian breed Berkamasca, is found
on the Ionian island of Zakynthos. These insular island breeds share not only
exceptional breed characteristics such as high milk yield and excellent prolificacy, but
also the unfortunate designation of being in serious danger of extinction (Boyazoglu,
1991).
In response to European Union regulations requiring member countries to institute
scrapie resistance breeding schemes, a pilot-breeding programme to increase the
frequency of scrapie resistant genotypes will be established soon in Greece. To
facilitate and expedite such a breeding programme, the present study to determine the
PrP genotype profile of rare Greek sheep breeds and to identify “low-risk”
individuals suitable for such a programme was undertaken.
Materials and Methods
Sheep
Blood samples were obtained from a total of 388 healthy sheep, from 47 farms and 13
rare Greek breeds raised in three geographic areas of Greece: Northern Greece
[Thrace (THR), Chios Mainland (CMD), Katsika (KAT), Kozani (KOZ), Florina
(FLO) and Sarakatsaniko (SAR) a total of 176 sheep], Central Greece [Karamaniko
(KAR), Argos (ARG), Kalaritiko (KAL) and Pilio (PIL) a total of 117 sheep] and
some Greek islands (Chios (CIL), Kimi (KIM), Skopelos (SKO) and Zakynthos
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(ZAK) a total of 95 sheep]. All these breeds have been characterized by the Greek
Ministry of Agriculture as rare Greek breeds.
DNA extraction
DNA was isolated from EDTA-treated blood by a standard phenol-chloroform
extraction protocol (Sambrook and others, 1989).
DNA amplification and Genotype analysis
Two pairs of primers (V1 and V2) designed by Yuzbasiyan-Gurkan and others (1999)
were used for the amplification of the PrP gene as described by Yuzbasiyan-Gurkan
and others (1999).
Detection of polymorphisms at codons 136 and 154. Polymorphisms at codons 136
and 154 were detected by RFLP analysis as described by Hunter and others (1993)
with one modification: for the DNA amplification, the V2 primers of Yuzbasiyan-
Gurkan and others (1999) were used. One hundred of the samples were checked by
both methods to verify our modified protocol.
Detection of polymorphisms at codon 171. The three common codon 171 alleles were
detected using two sets of primers (V1 and V2) by a series of two digestions. After
the DNA amplification with the V1 primers or the V2 primers, the PCR products
were treated with the enzyme BslI to detect the R allele and with the enzyme AccI to
detect the H allele, respectively. In both tests, the allele refractory to digestion was the
Q allele (Yuzbasiyan-Gurkan and others 1999).
Twenty-two samples (two from each genotype found) were confirmed with a capillary
electrophoresis method (Zslonai and others 2003).
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Statistical Analysis
Genetic heterogeneity among the entire set of samples, pairwise differentiation among
population samples and deviations from Hardy-Weinberg equilibrium for each locus
were tested using probability tests or, where possible, exact tests. The program
GENEPOP 3.3 (Raymond and Rousset 1995) was used to perform the above analyses.
RESULTS
PrP allelic (haplotype) variants
Dimorphisms A/V and R/H in codons 136 and 154, as well as the trimorphism Q/R/H
in codon 171, of the gene encoding PrP were determined for 388 individual healthy
animals. Samples were obtained from 47 farms and 13 different breeds located in
three different geographical regions (Northern Greece, Central Greece and Greek
Islands). Five different PrP haplotype were observed, resulting in eleven separate
genotypes. The haplotype frequency distribution for each breed is shown in Table 1.
In general, most of the breeds examined possess three to four different haplotypes.
Two haplotypes, ARQ and ARR, are present at high mean frequencies overall (50.5%
and 36.7% respectively) in the population tested. The breeds with the highest
frequencies (>45%) of the scrapie-resistance associated ARR haplotype are Kimi,
Kalaratiko and Katsika.
With regard to the AHQ haplotype, it is the dominant haplotype (44.7%) in one breed
examined, that of Skopelos. In all other breeds tested, however, the AHQ haplotype
frequency is lower than 15%, and it is absent in the CIL, KAL and ARG breeds (in
the KOZ breed the AHQ allele is also absent but possibly due to the low sample size).
These latter breeds, i.e. CIL, KAL and ARG all have a significant (>10%) frequency
of the ARH polymorphism. Most importantly, perhaps, is the finding that the VRQ
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haplotypes is present in only two breeds, FLO and SKO, and at a low frequency
(5.5%) in those.
The breeds of KAR, PIL, ZAK and THR have many similarities. As noted above,
none of them have the ARH haplotype but all do have a significant AHQ component.
Moreover, the ARR, ARQ and AHQ haplotype frequency profiles are very similar for
these three breeds. Consistent with these similarities, no pairwise differentiation test
among these breeds was statistically significant (p> 0.05).
Another similarity in breed frequency profiles was observed with the ARR, ARH and
ARQ haplotypes in the CMD and SAR breeds of Northern Greece, with the pairwise
test between these breeds revealing no statistical significance (p> 0.05). It is
interesting to note that the pairwise test between the CMD breed and the CIL breed,
from which it originated, is highly significant (p<0.001), indicating a substantial
genetic drift of the CMD breed.
PrP genotypes
As mentioned above, the present study of thirteen rare Greek sheep breeds detected
five PrP haplotype variants giving rise to eleven different genotypes. Table 2 shows
the obtained total results per breed, per geographical area along with the frequency of
each genotype. All breeds were in Hardy Weinberg equilibrium (P>0.05) except for
the Florina breed (P<0.001), due possibly to the complete absence of ARR/ARQ
heterozygotes in the population tested despite the high frequencies of the
corresponding homozygotes. Four genotypes (ARR/VRQ, AHQ/ARH, AHQ/AHQ
and AHQ/VRQ) were detected for the first time in Greece (Table 2). No VRQ/ VRQ
sheep were found in any of the breeds tested and, in fact, genotypes with valine at
codon 136 appeared only in the breeds of FLO and SKO.
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In general, breeds of Northern Greece have a large variety of PrP genotypes (Table 2),
while those of Central Greece display a lesser variety. For purposes of scrapie-
resistance breeding, the most interesting breed of Northern Greece is that is that of
KAT which has a high frequency of ARR/ARR genotype (25.4%), a very good
proportion (46.1%) of genotypes with at least one ARR allele and a low frequency of
genotypes belonging to risk Group R3. Similarly, in Central Greece, the KAL breed
shows a high frequency (90.4%) of scrapie resistant genotypes (risk Group R1 and
R2). Of the Greek island breeds sampled, the best, in terms of its overall PrP
genotype profile, seems to be KIM, with a 40.0% frequency of ARR/ARR and
53.35% frequency of ARR/ARQ and ARR/AHQ genotypes (Table 2).
Table 2 also displays the general distribution of PrP genotypes in the 388 samples.
More than half of the individuals sampled (58.83%) are genotypes of the risk Groups
R1 and R2. Polymorphisms with valine in codon 136 appeared only at a frequency of
0.77% for the overall population tested. The comparison of the frequency of the
various genotypes in the three different regions is shown also in Table 2.
DISCUSSION
The results of the present study reveal the frequencies of the haplotypes, as well as
those of the genotypes, for the three scrapie susceptibility-associated PrP codons (136,
154 and 171) in 13 different rare Greek breeds, from three different geographical
areas. It is the first report of PrP genotype frequencies in these breeds. The analysis
revealed five different PrP allelic variants resulting in eleven different genotypes. The
VRQ haplotype, which is linked to the highest risk for scrapie, was present only in
two breeds (FLO and SKO, Table 1) and with an average frequency of 0.4% for the
total population tested. This is far lower than the VRQ frequencies found in other
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European breeds (Belt and others 1995, Hunter and others 1996, Bossers and others
1999, Tranulis and others 1999). From these results it seems that Greek rare breeds
belong mainly to the group of non-valine breeds.
Of the 13 breeds examined, the lowest number of genotype variants per breed was
four, and that was seen with only four breeds (THR, KAR, PIL and KIM). All other
breeds have five or more (5 to 9) genotypes, indicating that they are rather complex.
With regard to phylogenetic evolution, the ARQ haplotype is the oldest PrP haplotype
in the ovis species (Thorgeisdottir and others 1999). High frequencies of this
haplotype were found in the breeds of KOZ, CMD, SAR and THR (75%, 67.8%,
67.2% and 64.6%, respectively). Despite the high frequency of the ARQ/ARQ
genotype in the breeds of CMD, THR, and KOZ (50.8%, 50%, and 50%, respectively)
no scrapie cases have been reported in purebred flocks of these breeds. In Greece, the
only cases of sheep affected by scrapie are those of crossbred flocks, mainly from
crosses with the breed of CMD (unpublished data).
The AHQ haplotype was present in the majority of breeds tested in this study. It was
not found, however, in the breeds of KOZ, ARG, KAL and CIL. This haplotype
frequency is highest, at 44.7%, in the breed of SKO. Interestingly, while the CMD
breed carries the AHQ allele (6.9%), the CIL breed, from which it originated, does
not.
It is difficult to hypothesize why there are such differences in the PrP haplotype
frequencies among the Greek breeds. Apart from possible selective advantages of
special alleles that have already been pointed out, one must bear in mind the effect of
the small populations of many of these breeds. Genetic drift phenomena are quite
possible, a fact that has also been suggested based on previous examination of these
Greek breeds by isozyme analyses (Rogdakis 2002, Koutsouli and Rogdakis 2002)
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and Restriction Fragment Length Polymorphism analysis of mitochondrial DNA
(Katana 2002).
Importantly, four genotypes (ARR/VRQ, AHQ/ARH, AHQ/AHQ and AHQ/VRQ)
were detected for the first time in Greece (Table 2). Moreover, sheep with the
VRQ/VRQ genotype were not found in the present study of purebred breeds.
In accordance with the Greek Surveillance Programme for TSE’s Control (2004),
scrapie positive farms in Greece will be checked and all the animals that do not carry
at least one ARR allele will be stamped out. ARR/ARR rams, and ewes carrying at
least one ARR allele, would be kept, or introduced from an available breeding
population (once established), to such scrapie positive farms. It is worth noting, in this
regard, that in a recent survey of more than two hundred crossbred sheep from 16
scrapie-affected flocks across Greece (Billinis and others 2004), the frequency of
individuals carrying at least one ARR allele was reported to be less than 15 percent.
To support our Surveillance Programme for TSEs, a pilot-breeding programme for
increasing the frequency of the scrapie resistant genotypes will begin soon, based on
ARR/ARR rams and ewes carrying at least one ARR allele. Our current aim is to
identify and to provide “scrapie resistant” animals for breeding. The high frequency
of scrapie-resistant genotypes (58.83%) in Greek breeds presents a strong possibility
for selecting low-risk, recently PrP-genotyped animals for reproduction.
In parallel studies aimed at developing potential targets for scrapie therapy, all
haplotypes described in this work are currently being cloned and expressed to
generate a new battery of molecules to study the molecular mechanisms of self-
aggregation due to the differential conformations of the PrP variants.
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ACKNOWLEDGEMENTS The authors thank the staff at CISA – INIA, Madrid, Spain who performed the
capillary electrophoresis experiments. The authors also thank the European Union for
partial funding of this study through QLRT-2001-00959 Small ruminant TSE
epidemiology pathology and diagnostic tests (SRTSENETWORK).
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Table 1. Frequency distribution of PrP haplotypes (n – number of chromosomes)*
Breed Abr n PrP haplotype ARR ARH ARQ AHQ VRQ
Kozani KOZ 8 25.0% - 75.0% - -
Katsika KAT 78 48.7% 15.4% 28.2% 7.7% -
Thrace THR 48 31.2% - 64.6% 4.2% -
Florina FLO 30 40.0% - 43.3% 13.35% 3.35%
Chios, Mainland CMD 130 23.8% 1.5% 67.8% 6.9% -
Sarakatsaniko SAR 58 19.0% 6.9% 67.2% 6.9% -
Karamaniko KAR 24 37.5% - 58.3% 4.2% -
Argos ARG 62 30.6% 11.3% 58.1% - -
Kalaritiko KAL 84 54.8% 13.1% 32.1% - -
Pilio PIL 64 42.2% - 54.7% 3.1% -
Chios Island CIL 78 35.9% 11.5% 52.6% - -
Kimi KIM 30 66.7% - 23.3% 10.0% -
Skopelos SKO 38 26.3% - 23.7% 44.7% 5.3%
Zakynthos ZAK 44 38.7% - 54.5% 6.8% -
All breeds 776 36.7% 5.8% 50.5% 6.6% 0.4%
* The highest ARR haplotype frequencies, the lowest ARQ haplotype frequencies and the unusual high frequency of the AHQ haplotype are shown in bold.
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Table 2. Comparison of the genotypes frequencies (%) of the thirteen different breeds in the three regions (North Greece, Central Greece, Islands)
GENOTYPES NORTHERN GREECE CENTRAL GREECE ISLANDS THR CMD KAT KOZ FLO SAR KAR KAL ARG PIL CIL KIM SKO ZAK
ARR/ARR 12.5 10.8 25.6 33.3 8.35 19.0 9.7 21.9 5.15 40.0 10.5 9.1
R1 Total 12.5 10.8 25.6 33.3 8.35 19.0 9.7 21.9 5.15 40.0 10.5 9.1 ARR/ARQ 29.2 23.2 20.5 50.0 34.6 50.0 50.0 25.8 40.6 43.6 46.6 15.8 50.0
ARR/ARH 1.5 23.1 3.4 21.4 16.1 17.9
ARR/AHQ 8.3 1.5 2.6 6.7 8.35 6.7 15.8 9.1
R2 Total 37.5 26.2 46.2 50.0 6.7 38.0 58.35 71.4 41.9 40.6 61.5 53.3 31.6 59.1ARQ/ARQ 50.0 50.8 12.8 50.0 33.3 37.9 33.3 4.8 41.9 31.3 28.2 5.3 27.3
ARQ/ARH 1.5 5.1 10.3 4.8 6.5 5.15
ARQ/AHQ 9.2 5.1 20.0 13.8 6.2 21.05 4.5
AHQ/ARH 2.6
AHQ/AHQ 1.5 2.6 6.7 21.05
R3 Total 50.0 63.0 28.2 50.0 53.3 62.0 33.3 9.6 48.4 37.5 33.35 6.7 47.4 831.ARR/VRQ 6.7
R4 Total .7 6AHQ/VRQ 10.5
R5 Total 10.5
17
18