Transcript Document

Research Experience in Molecular Biotechnology & Genomics
Summer 2007
Center for Integrated Animal Genomics
Associations of Single Nucleotide Polymorphisms in bovine
Toll-like receptor 4 with incidence of Infectious Bovine Keratoconjunctivitis in American Angus calves
Manuel A. Ortega1, Ranjit S. Kataria2, Dinesh Kumar2, Richard G. Tait Jr3., and James M. Reecy3
1Industrial Biotechnology, University of Puerto Rico- Mayaguez,
2National Bureau of Animal Genetic Resources, Karnal, India-132 001
3Department of Animal Science, Iowa Sate University
Abstract
Results
Toll-like receptor 4 (TLR4) is a receptor protein. Its main function is to activate
immune responses. Because of its function in identifying pathogenic molecules, it has
been implicated in resistance to diseases. To investigate its possible role in resistance
to IBK, we evaluated the extent to which Single Nucleotide Polymorphism were
associated with pinkeye incidence. We PCR amplified the TLR4 gene of 382
American Angus calves in order to genotype two SNP. One in Int1-Ex 2 (-26)
position (G/A) and Ex 3 (1678) position (C/T). Pinkeye phenotypic information was
available for two time point – August and October. At the August time point, the Ex 2
SNP could account for 2.2% of the phenotypic variation in incidence of pinkeye. A/A
animals exhibited more infection than other G/G animal (65% vs. 28% infection
rate). While the Ex 3 SNP could account for .09% of the phenotypic variation , there
was no statistical difference between genotypes of ex 3. At the October time point,
the Ex 2 SNP followed the same pattern as August, it accounted for 3.1% of the
phenotypic variation in pinkeye infection. A/A animals exhibited more infection G/G
animals. As for Ex 3, it accounted for only .008% of the phenotypic variation, and
again, neither of the three genotypes showed any difference at all regarding pinkeye
incidence. These data indicate that there is a close relationship between Ex 2
genotype and incidence of pinkeye .
SNP
SNP
SNP
Figure.1 Chromatogram of TLR4 Ex 2 sequence. SNP
G/A found on Int1-Ex 2 (-26) position.
Figure. 2 Chromatogram of TLR4 Ex 3 sequence. SNP
C/T found on Ex3 (1678) position.
Table 1. Exon 2 or exon 3 as sole sources associated
with August incidence of pinkeye
(Data analyzed
separately).
Table 2. Exon 2 or exon 3 as sole sources associated
with October incidence of pinkeye (Data analyzed
separately).
Source
R-Square
F value
Pr > F
Source
R-Square
F value
Pr > F
Exon 2
0.021923
4.23
0.0153
Exon 2
0.030771
5.84
0.0032
Exon 3
0.000861
0.16
0.8501
Exon 3
0.000075
0.01
0.9863
Materials and Methods
Purebred American Angus calves were used to evaluate the extent to which
variation in the TLR4 locus was associated with pink eye.
Estimation of severity of pinkeye infections were obtained in either August (n =
382) or October (n = 371).
100
90
80
70
60
50
40
30
20
10
0
5. Selected samples were sequenced to identify single nucleotide polymorphism
(SNP).
6. Restriction Fragments Length Polymorphism assays were designed to genotype
each SNP location. The restriction enzyme used for the Exon 2 SNP was Aci I and
ApeK I for the exon 3 SNP.
7. Genotype results were statistically analyzed for associations with pinkeye using the
GLM procedures of SAS. In addition to pinkeye, we analyzed each genotype with
respect to weight, age of dam, and early weaning of the individual.
August measurements
October measurements
B
A/A
B
G/A
100
90
80
70
60
50
40
30
20
10
0
G/G
B
C/C
Exon 2 Genotype
B
August measurements
October measurements
C/T
B
T/T
Exon 3 Genotype
Figure 3. Infection rate percentage of pinkeye
on exon 2 genotypes.
Figure 4. Infection rate percentage of pinkeye
on exon 3 genotypes.
Conclusions
Two SNP were found on bovine TLR4 gene, one in Int1-Ex 2 (-26) position and in Ex 3 (1678) position.
The genotype of Ex 2 SNP was G/A and C/T for Ex 3 SNP.
For August and October measurements, Ex 2 was identified as a significant source on pinkeye incidence. (Pr < .01)
According to the Pr values, Ex 3 did not show any relevance on pink eye incidence.
Between the three genotypes A/A, G/A and G/G on Ex 2 SNP, A/A showed a higher infection rate
(65% for August, 82% for October).
Further genotyping is needed to determined in fact that Ex 2 cause an impact in pinkeye incidence.
References
1. DNA was extracted as described in “Current Protocols in Human Genetics”
(Dracopoli et al., 2000).
4. DNA was amplified in a 15μL PCR reaction that contained 50 ng genomic DNA,
5.5 μL of distilled water, 7.5 μL of of GoTaQ Master Mix (Promega) and 0.5 μL of
both forward and reverse primers. The PCR program consisted of 2 min at 94˚C,
followed by 35 cycles of 94˚C, 56˚C and 70˚C for 30 sec. each and a final 5-min
extension at 72˚C.
A
Infection rate (%)
Infectious Bovine Keratoconjunctivitis or pinkeye is an inflammatory eye disease
caused by Moraxella bovis. Some of the effects in cattle are excessive tearing, and
inflammation of the conjunctiva. There exist many treatments for this disease, but none
of them have a genetic approach. Toll-like receptor 4 has been identified as a protein
that belongs to a group of receptors called Pattern Recognition Receptors. Its function
is to recognize lipopolysaccharides and lipoteichoic acid, serving as one of the first
immune response of animals. Because of its function in the immune system, TLR4 can
be considered as a candidate gene for resistance to a large number of diseases (White et
al. 2003). To determine TLR4 as a candidate, Single Nucleotide Polymorphisms were
screened on the TLR4 gene. SNP will be used as markers for the identification of a
specific genotype on an individual showing any type of resistance to this disease.
Infection rate (%)
(%)
Introduction
(%)
Association of the different genotypes with incidence of pinkeye
2. DNA quantification
1. DNA extraction
from White
Blood Cells
5. Sequence DNA
to identify Single
Nucleotide Polymorphism
Dracopoli, N.C., Haines, J.L., Korf, B.R., Morton, C.C., Seidman, C.C., Seidman
J.G., Smith, D.R., and Boyle A.L. 2000. Current Protocols in Human Genetics, 3,
John Wiley & Sons Inc, New York, pp. A.3B.3, A.3C.2
3. Dilution of DNA samples
(50 ng/uL)
White S.N., Taylor K.H., Abbey C.A., Gill C.A., Womack J.E. 2003. Haplotype
variation in bovine Toll-like receptor 4 and computational prediction of a
positively selected ligand-binding domain. Proc Natl Acad Sci U S A 100: 103649
4. Amplification of TLR4 DNA
(PCR Technique)
Special Thanks to…
7. Data Analysis
6. Restriction Enzyme
Digestion of PCR products
Jim Reecy for giving me the opportunity to work in his lab. Dinesh Kumar and Ranjit
Kataria for all their mentoring and support. Mary Sue Mayes for all her help in the
laboratory. J.R. Tait for all his statistical guidance. Jennifer Young, James Koltes, and
Jessica Frerichs for editing purposes. And everyone else who contributed to my
experience at Iowa State.
Funding provided by
USDA-CSREES &
National Beef Cattle Evaluation Consortium
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371