POLYMORPHISM OF GENE ENCODING C-REACTIVE PROTEIN IN SUCKLING
PIGLETS.
Chandra S. Pareek and Krystyna Życzko
Department of Animal Genetics, University of Warmia and Mazury, Olsztyn, Poland.
Abstract
An investigation aimed to perform on one polymorphic SNP (G > A) in the 3’-UTR at the position of 1271bp of
porcine CRP gene (GenBank accession no. AY714055) in 172 suckling piglets originating from cross between
Polish Large White x Polish Landrace sows x Duroc boar. The HinfI PCR-RFLP results revealed a 454bp undigested
amplicon as CRPG allele and digested product with 379bp and 75bp as CRPA allele. The progenies originating from
boar GG crossed with GG and AG sows were in accordance to the expected genotypic frequencies, while progenies
originating from boar AG crossed with GG and AG sows, showed the deficit of genotype AG. Genotype GG was
observed as the most frequent, covering about 72.7% of analysed population, whereas genotype frequencies of AG
and AA were 25.6% and 1.7%, respectively. The allele frequency of CRPA and CRPG was 0.145 and 0.855,
respectively. Obtained results revealed the elimination of AA and AG genotypes in the suckling piglets aged 19-25
days. The observed highly significant differences of allele A frequency in the younger and older aged suckling
piglets did not depend on the state of health. The rear allele A frequency in suckling piglets aged 12-18 days and 1525 days were 0.195 (healthy), 0.175 (piglets with diarrhoea) and 0.058 (healthy), 0.096 (piglets with diarrhoea),
respectively.
Keywords: CRP, SNP, Polymorphism, suckling piglets, diarrhoea.
The earliest host response to tissue injury, infection, inflammation or trauma is the acute phase
proteins called as C-reactive proteins (CRP). CRP has not only to be considered as a marker, but
also as a potential participant in the pathogenesis of diseases such as various roles in cellular
activation and in inflammatory processes (Pepys et al. 2003). In animals, CRP is an acute-phase
inflammatory serum protein marker and a mediator of innate and adaptive immunity (Szalai
2002). In human, the baseline levels of plasma CRP show a clear heritability (h2) of 40% and
35% in family studies ((Pankow et al. 2001, Vickers et al. 2002). The human CRP gene lies on
chromosome 1q23, within a conserved genetic region that encodes for proteins critical to the
immune system and to intercellular communication (Watson et al. 1990). In pig, CRP encoding
gene is physically allocated to Sscr 4q13 (Pinton et al. 2000). The porcine CRP gene is comprised
of 1762bp (accession no. AB005545). Two SNPs in the 3’-UTR region at position of 788bp
(CRP_SNP-1) and at 1271bp (CRP_SNP-2) of the cDNA sequence (GenBank accession no.
AY714055) were detected (Chomdej et al. 2004). In presented paper, we studied the CRP
polymorphism in the healthy and diarrhoea affected piglets (PLW x PL x Duroc) at two different
stages of early physiological life.
Material and methods
The material was comprised of 172 suckling piglets originating from cross between Polish Large
White (PLW) x Polish Landrace (PL) sows and Duroc boars. The experimental design was
constructed according to health status (healthy and diarrhoea affected) and different ages (12-18
days and 19-28 days) of suckling piglets. The DNA was isolated from whole blood using
MasterPureTM DNA purification kit (Epicentre, USA). The HinfI PCR-RFLP for CRP gene were
performed according to Chomdej et al. (2004). The obtained HinfI PCR-RFLP result revealed
one undigested fragment of 454 bp and two DNA fragments of 379bp and 75bp of size. The
uncut amplicon of 454bp represented the CRP G allele, while cut amplicons of 379bp and 75bp
represented the CRP A allele (Figure 1). A 2 test was employed to find out the distribution and
effect of CRP polymorphism towards the health status of suckling piglets at different
physiological stage of life.
1353
1078
872
603
454 bp
379bp
310
287
234
M
GG AG GG AG
AG GG
Figure 1: HinfI PCR-RFLP genotyping of porcine CRP gene. M denotes as Phix174/HaeIII.
Results
The distribution of CRP genotype frequencies revealed a non significant effect of CRP genotypes
on healthy and infected piglets in both age groups (Table-1). The frequencies of genotype AA
was observed as less frequent, while frequencies of genotype AG as intermediate and frequencies
of genotype GG as highest in the all analysed piglets. However, The distribution of CRP allele
showed highly significant relationship (p  0.01) between CRP alleles and piglets aged 12-18
days and 19-25 days irrespective to the piglets’ health status.
Table 1: Distribution of genotypes and allele frequencies of porcine CRP gene in piglets.
Piglets at the age of 12-18 days
Piglets at the age of 19-23 days
Healthy
Diarrhoea
Together
Healthy
Diarrhoea
Together
n
(%)
n
(%)
n
%
n
%
n
%
n
%
AA
1
1,56
2
3,57
3
2,50
–
–
–
–
–
–
AG
23
35,94
13
23,21
36 30.00
3
11,54
5
19,23
8
15,38
GG
40
62,50
41
73,22
81 67,50
23
88,46
21
80,77
44
84,62
Together
64
100
56
100
120 100
26
100
26
100
52
100
CRPA
0,195
0,152
0,175a
0,058
0,096
0,077b
CRPB
0,805
0,848
0,825a
0,942
0,904
0,923b
a,
b
Assigned letters and means allele frequencies differ highly significantly at different age groups at p  0.01.
Genotypes
/ alleles
The segregation pattern of CRP alleles in examined piglets revealed no discrimination of CRP
alleles in examined healthy and diarrhoea affected piglets, and a state of genetic equilibrium was
observed (Table 2).
Table 2: State of genetic equilibrium of porcine CRP gene according to health status of piglets.
Assigned letters ns means non-significant.
The distribution of CRP genotypes in progenies from various parental mating combinations
presented in Table 3. Obtained results showed the progenies originating from parental mating of
CRP
genotypes
AA
Healthy piglets
observed (%)
expected (%)
1,1
2,45
Piglets with diarrhoea
observed (%)
expected (%)
2,4
1,80
AG
28,9
26,33
22,0
23,2
GG
70,0
71,22
75,6
75,0
CRP alleles
A
G
2
Healthy piglets
0,156
0,844
0.897ns
Piglets with diarrhoea
0,134
0,866
0,241ns
GG x GG and GG x AG were in accordance to the expected genotypic frequencies, while
progenies originating from parental mating of AG x GG and AG x AG, showed a highly
significant (p  0.01) deficit of genotype AG (Table 3).
Table 3: Distribution of porcine CRP genotypes in progenies from various parental mating.
**: highly significant at p 0.01.
Discussion
Type of parental mating Progenies
number
Boars
Sows
n
GG
GG
62
GG
AG
78
Together GGxAG, GG
140
AG
GG
18
AG
AG
14
Together AGxAG, GG
32
Distribution of CRP genotypes (n) in progenies
AA
AG
GG
observed expected observed expected observed expected
62
62
38
39
40
39
38
39
102
101
4
9
14
9
3
3,5
2
7
9
3,5
3
3,5
6
16
23
12,5
2
0,105
15,14**
Recently published CRP polymorphism (Chomdej et al. 2004) was studied in the progenies
originating from PLW x PL x Duroc crossbred suckling piglets. Although, highly significant
effect of CRPA allele was recorded in examined progenies, this polymorphism did not show any
influence on the state of health of suckling piglets. The segregation of CRP alleles among the
examined progenies was in a state of genetic equilibrium. However, this segregation pattern in
examined PLW x PL x Duroc suckling piglets was observed quite different then in the
commercial purebred pig population. In the Large White, Pietrain and German Landrace
population frequencies of CRP allele A were reported as high (0.70), intermediate (0.35), and
very low (0.04) (Chomdej et al. 2004) respectively. With the advancement of age, the tendency of
existence of alleleA become lower and resulted in the elimination of AA and AG genotypes. The
study confirm the genetic variability of porcine CRP gene in the examined crossbred population,
however, further investigation are necessary in context to validate the porcine CRP gene as a
favourable candidate gene (Szalai et al. 2001) for complement activity and host defence in the
pig.
References
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Chandra S. Pareek and Krystyna Życzko
POLIMORFIZM GENU KODUJĄCEGO BIAŁKO C-REAKTYWNE U SSĄCYCH
PROSIĄT
STRESZCZENIE
Celem badań było określenie występowania mutacji punktowej (G > A) w obrębie obszaru 3’UTR w pozycji 1271pz
genu CRP (GenBank numer acc: AY714055). Badaniami objęto 172 ssące prosięta pochodzące z krzyżowanie świń
rasy Wielka Biała Polska x Polska Biała Zwisłoucha z knurami rasy Duroc. Metodą PCR-RFLP przy użyciu enzymu
restrykcyjnego HinfI, identyfikowano allele od 454pz – allel G i dwa fragmenty o długości 379pz i 75pz – allel A.
Determinowały one genotypy: AA, AG i GG. Wykazano, że po knurach o genotypie GG kojarzonych z lochami GG
i AG, obserwowany rozkład genotypów w ich potomstwie był zgodny z oczekiwanym. Natomiast wśród prosiąt
pochodzących po knurach o genotypie AG, kojarzonych z lochami AG i GG, stwierdzono statystycznie wysoko
istotny niedobór heterozygot AG. Wśród potomstwa najczęściej występował genotyp GG. Stanowił on 72.7% ogółu
prosiąt,. Udział prosiąt AG wynosił 25.6% zaś AA zaledwie 1.7%. Frekwencja allelu CRPA wynosiła 0.145 zaś allelu
CRPG- 0.855. Wykazano, że wraz z wiekiem prosiąt, następowała eliminacja genotypu AA i AG. Wysoko istotne
różnice we frekwencji CRPA między młodszymi i starszymi prosiętami nie zależały jednak od ich stanu zdrowia.
Frekwencja allelu CRPA u młodszych prosiąt wynosiła 0.195 (zdrowe prosięta) i 0.175 (prosięta z biegunką) zaś u
starszych, odpowiednio 0.058 i 0.096.