Vortragstagung der DGfZ und GfT am 6./7. September 2006 in Hannover
Genetic analyses of porcine MBL genes and their association with immunological traits
in pigs
C. Phatsara1, D. Jennen
1, S. Ponsuksili
2, E. Murani
3, D. Tesfaye
1,
K. Schellander1, K. Wimmers
3
1Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn,
Endenicher Allee 15, 53115 Bonn; 2Research Institute for the Biology of Farm Animals
(FBN), Research Group Functional Genomics, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf,
Germany, 3Research Institute for the Biology of Farm Animals, Molecular Biology Research
Division, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf
Introduction
Mannose-binding lectin (MBL) or mannose-binding protein (MBP), a member of the collectin
family of proteins, is an important factor in the lectin pathway of the complement cascade.
Binding of MBL leads to opsonization through complement activation and deposition of C3.
Its association to MBL-associated serine protease (MASP) initiates the lectin pathway. Two
types of MBL (MBL-A and MBL-C), were characterized in rat (Mizuno et al., 1981), mouse
(Hansen et al., 2000), and bovine (Kawai et al., 1997). While only one MBL was identified in
human and chickens (Laursen & Nielsen, 2000), two forms of MBL were characterized in pig.
A porcine liver MBL cDNA of 723-bp was characterized (Agah et al., 2001) and showed a
higher identity to rat MBL-C than MBL-A proteins. Another MBL were characterized and
proposed as the porcine MBL1 gene, which is homologous to the rodent MBL1 gene and
MBL1P1 pseudogene of humans and chimpanzees (Lillie et al., 2006). In this study,
chromosomal assignment, expression in different tissues, and association with complement
activity of the porcine MBL genes, were analyzed.
Materials and methods
Standard PCR was performed in 20 µl reaction volume. Thermocycling program was initial
denaturation at 95 °C for 5 min, followed by 35 cycles at 94 °C for 30 seconds, 59 °C for 30
seconds, 72 °C for 1 min, and final extension at 72 °C for 10 min. Additionally, touchdown
PCR was used to amplify MBL1. Gene-specific primers used in this study are described in
Table 1. Semiquantitative RT-PCR was employed in order to survey expression of the porcine
MBL genes in muscle, heart, spleen, tonsil, lymph node, lung, liver, kidney, testis, and brain
from adult animals. First-strand cDNA was synthesized from 1 µg of total RNA using random
hexamer primers and oligo (dT)12N with superscript II (Invitrogen, Karlsruhe, Germany) as
reverse transcriptase enzyme. Standard PCR using MBL1-a, and MBL2-a primer sets were
done to detect MBL1 and MBL2 transcripts. The 18S gene was used as an internal control.
Mapping of MBL genes on chromosome was performed by radiation hybrid panel. Moreover,
genetic mapping was performed by two-point linkage analysis using CRIMAP program.
Partial porcine MBL2 gene of 15 pigs were amplified and sequenced using CEQ™ 8000
Genetic Analysis System (Beckman Coulter, Krefeld, Germany) to screen for sequence
variants. PCR Restriction Fragment Length Polymorphism (PCR-RFLP) was performed using
HinfI and AdeI digestive enzymes for MBL1 and MBL2 polymorphic sites. For the MBL1
gene, animals were genotyped at a previously identified C to T substitution on position 328 of
the sequence (AF208528) reported by Marklund et al. (2000). For the MBL2 gene, animals
were genotyped at the G to A transition, which is found in this study, on position 645 of
sequence (NM 214125). Digested PCR products were visualized on 2.5% agarose gel to
determine the genotype distribution. Analysis of variance was performed using the procedure
‘mixed’ and the ‘repeated measure’ statement of the SAS software package. A model was
fitted in order to identify other significant environmental and genetic effects apart from the
MBL1 and MBL2 genotypes and its interaction by stepwise elimination of non-significant
effects. Animal effect was the subject specified in the repeated statement.
Tab. 1 Gene-specific primers (5´-3´) used for porcine MBL genes amplification in this study
Primer set Sequence Annealing Temp.
(°C)
Product size
(bp)
MBL1-a CCCCAATATTTCCTGGAGGT
TCCTCCTTCTGTGTGTGGTG 59 222
MBL2-a GGGAGAAAAGGGAGAACCAG
CACACAGAGCCTTCACTCCA 59 278
MBL1-b AAGGGAGAACCAGGTATAGG
TGAACCCTGGCCCTGTTG 62-66 702
MBL2-b CTTCGCTCAGGGAAAACAAG
GTCATTCCACTTGCCATCCT 59 319
Results and discussion
Semiquantitative RT-PCR of ten tissues showed differential expression of porcine MBL
genes. Both MBL genes were highly expressed in liver. However, low MBL1 expression was
also found in lung, testis and brain, while expression of MBL2 was low in testis and kidney.
Porcine MBL1 expression pattern in this study is similar to the results reported by Lillie et al.,
(2006) showing high expression of MBL1 in liver as well. Differential expression of MBL1
and MBL2 in murine tissues was reported. RT-PCR study revealed that the liver is the major
site of expression for both MBL genes. Also low expression was found in kidney, brain,
spleen and muscle, but only murine MBL1 is expressed in testis (Wagner et al., 2003). An
SNP was found at the position 645 (G to A) of porcine MBL2 cDNA (NM_214125) in F2
DUMI pigs, but did not effect amino acid composition in the translated protein. This SNP
affecting an AdeI restriction site was found to be segregating in the F2 DUMI pigs. The AdeI
PCR-RFLP generates fragments of 319 bp (allele A), 286 bp and 33 bp (allele G). Genotyping
F2 DUMI pigs revealed frequencies of MBL1 alleles C and T of 0.67 and 0.33, respectively.
Frequencies of genotypes C/C, C/T, and T/T were 0.48, 0.38 and 0.14, respectively. For
MBL2, genotyping showed allele frequencies for allele G and A to be 0.41 and 0.59,
respectively. The distributions of gene frequencies were 0.21, 0.40 and 0.39 for G/G, G/A, and
A/A genotypes, respectively. The results of RH mapping assigned both porcine MBL genes to
chromosome 14 with retention frequencies of 16% for both genes. The most significantly
linked markers (two-point analysis) for porcine MBL1 and MBL2 were SW210 (89 cR; LOD
= 3.32) and SW1552 (35 cR; LOD =10.66), respectively. The closest marker on the linkage
map was S0007 with recombination frequencies and two-point LOD scores of 0.32, 3.34 and
0.23, 8.26, for porcine MBL1 and MBL2, respectively. MBL2 chromosomal location
established by RH mapping in our study is confirmed by gene assignment of porcine MBL2 to
position 3226.0 cR of SSC14 with nearest gene and markers DKK1 and SW1552, as reported
by Meyers et al. (2005) and Yasue et al. (2006). Comparative mapping data indicate that
porcine MBL1 might be located between SFPTA and SFPTD genes on SSC14. The
association analysis using the MBL1 and MBL2 and their interactions with time point revealed
no effect on haemolytic complement activity in classical (CH50) and alternative (AH50)
pathways of the SNPs that were analysed (Table 2). The C3c protein level, which reflects in
vivo complement activity, tended to be higher in MBL1 heterozygous genotypes (C/T) than in
the homozygous genotypes (C/C and T/T) (p=0.067). There was a highly significant effect of
time of measurement (P<0.001). Interactions of time and MBL genotypes in the repeated
measures models reflect the dependency of the profile of complement activity along the
experiment on the genotype. For complement activity, a slight MBL1 genotype dependent
deviation of the profiles of C3c concentration over time was found (p=0.056). This deviation
is most prominent late after Mycoplasma vaccination. No significant effect of interaction
between genotypes and time point on haemolytic complement activity assayed in the
alternative and classical pathway was found. Both parameters of haemolytic complement
activity, CH50 and AH50 do not directly involve MBL1 or MBL2 depending on sequences of
the complement cascade. In contrast, C3c serum concentration reflects complement activation
after vaccination that may act on the lectin pathway controlled by MBL.
Tab. 2: Least square means of haemolytic complement activity traits (AH50, CH50) and C3c
serum concentration for the effect of MBL1 and MBL2 genotype in DUMI resource
population
Genotype AH50 CH50 C3c
MBL1
C/C 56.29 ± 2.47 68.12 ± 3.31 0.190 ± 0.004
C/T 58.13 ± 2.60 65.25 ± 3.55 0.198 ± 0.005
T/T 54.27 ± 3.40 62.90 ± 4.31 0.192 ± 0.005
Effect (P):
MBL1 0.355 0.313 0.067
Time < 0.001 < 0.001 < 0.001
MBL1*Time 0.690 0.479 0.056
MBL2
G/G 58.24 ± 3.14 67.59 ± 4.36 0.201 ± 0.006
G/A 60.58 ± 2.60 68.89 ± 3.50 0.201 ± 0.005
A/A 56.69 ± 2.63 65.54 ± 3.61 0.194 ± 0.005
Effect (P):
MBL2 0.141 0.499 0.136
Time < 0.001 < 0.001 < 0.001
MBL2*Time 0.664 0.723 0.967
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