Genetic polymorphism in TLR2 genes of HF cross bred cattle through PCR SSCP

Toll like receptors (TLRs) are molecules found to play a pivotal role in innate as well as acquired immunity (Akira et al., 2001). These are cell-surface receptors which recognize a broad class of pathogen-associated molecular patterns (PAMPs) that activates innate and adaptive immune responses, through activating various cell signaling molecules and induce the expression of inflammatory factors like Interleukin (IL)-1, IL-6, and IL-8, which participate in innate immune responses and then confer disease resistance (Shizuo et al., 2001). Furthermore, TLR-mediated activation of innate immunity has been reported to be mandatory for the development of antigen-specific adaptive immunity (Iwasaki et al., 2004).
       
Thorough perusal of literature suggests a possible role of TLR2 gene in resistance to infection mostly due to gram positive bacteria (Mariotti et al., 2009). Bovine TLR2 was assigned to Bos taurus (BTA) chromosome 17 using radiation hybrid mapping as reported by McGuire et al., (2006). It spans over 13.2 kb which includes 2 exons (Zhang et al., 2009). The first exon is of 179bp while the second one is 3333bp interspersed by an intronic region of 9714bp.
       
In all species, the protein shared common domain architecture: an extra-cellular domain containing 20 leucine rich repeats (LRR) between AA54 and AA584, a transmembrane domain at AA585– AA607 and an intracellular TIR domain at AA633–AA783. In some domains of TLR2, sequence variation is high, particularly in the region between AA200 and AA310, comprising LRR 7–10. Most of the other domains are more conserved, particularly LRR 12 and LRR 13 (AA340–AA390) and the cytoplasmic domain (AA610-end). Keeping in view the vital role of TLR2 gene in host defense mechanism, present study was planned to explore genetic variability at molecular level and their distribution in TLR2 gene in HF crossbred cattle of Bangalore and Kolar districts of Karnataka.

Animals
 
Blood samples were collected aseptically in vacutainer tubes containing 0.5% EDTA from 214 HF crossbred cattle. Genomic DNA was isolated by modified high salt method as described by Miller et al. (1988). After evaluation of purity, quality and quantity the genomic DNA was used for PCR.
 
Primers and PCR amplification
 
Primers reported by Zhang et al., 2009 for exon 1 and exon 2 of TLR2 (Table 1) gene were used to amplify the region of interest. The polymerase chain reactions (PCR) were carried out in a total volume of 25 µl solution containing 100 ng template DNA, 1X buffer (Tris-HCl 100 mmol/l, pH 8.3; KCl 500 mmol/l), 0.5 mmol/l primers, 2.0 mmol/l MgCl2, 0.25 mmol/l dNTPs, and 0.5U Taq DNA polymerase (Amnion Biosciences, Bangalore, India) with the thermal cycler. Thermal cycling conditions have been exhibited in Table 2. PCR products were confirmed through electrophoresis with standard marker on 1.5% agarose gels.
 

 

 
PCR-SSCP analysis
 
PCR Single-Strand Conformation Polymorphism (PCR-SSCP) was used to detect SNPs in TLR2 gene. Aliquots of 10 µl PCR products were mixed with 10 µl denaturing solution (95% formamide, 25mM EDTA, 0.025% xylene cyanole, and 0.025% bromophenol blue), heated for 10 min at 98°C and chilled on ice for 5 min. The samples were separated by an electrophoresis on a 10% neutral polyacrylamide gel (acrylamide:bisacrylamide= 29:1) at 140 volt for 10–12 hours. The gels were stained with Ethidium Bromide (10 mg/ml) to identify SSCP patterns.
 
Sequence analysis
 
Representative PCR products of each electrophoresis patterns were sequenced by Xcleris Labs Ltd., Ahmadabad. Chromatogram drawn by data collection software was used to extract the sequence data. BLAST analysis was performed to confirm gene identity. Sequence alignment was carried out by Bioedit software.
 
Population genetic analysis
The genotypic patterns obtained for TLR2 gene in HF  crossbred population under present study were determined by manual segregation. The degree of heterozygosities, c² (Chi-square) test for Hardy Weinberg equilibrium and F statistics for the population were performed using population genetic analysis software POPGENE version 3.2 (Yeh et. al., 2006).

Allelic and genotypic frequencies
 
In present study exon 2 was found to be polymorphic with six SNPs viz., A827G, C1088G, C2155A, G2281C, G2410A and C2600T identified. Allelic and genotypic frequencies of all these segments of SNPs of TLR2 gene are indicated in Table 3.
 

 
Genotypic frequencies of AA, AG and GG in A827G of TLR2 gene were 0.66, 0.04 and 0.30 respectively. Bai et al., (2011), assigned the genotypes based on segments of exon indicating them as 2.1, 2.2, 2.3, 2.4, 2.5 and 2.6. The genotype frequencies of AA, AB and BB genotypes for exon 2.2 were 0.88, 0.11 and 0.01 respectively, and for the exon 2.3 were 0.93, 0.07 and 0 for the same genotypes for HF breed in China. In the present study three genotypes CC, CG and GG were found for SNP C1088G region with frequencies of 0.74, 0.02 and 0.24 and with allele frequencies as 0.2523 and 0.0187 for allele C and G respectively. For the SNPs C2155A and G2281C allelic frequencies observed were 0.2804 for allele A and G while for C it was 0.7196.
       
At SNP C2115A region three genotypes AA,CA and CC  were observed with the frequencies of 0.15, 0.27 and 0.58 where as at SNP C2155A portion also revealed three genotypes GG,GCand CC with their corresponding frequencies were 0.15, 0.27 and 0.58. Zhang et al., (2009) reported that the frequency of A and B were 0.03 and 0.97 respectively for exon 2.5 of TLR2 gene in HF cattle with genotype frequencies 0, 0.055 and 0.945 for genotypes AA, AB and BB respectively. It was observed that the allele A maintained in the population with low frequency.
       
Genetic analysis of SNP G2410A of TLR2 gene indicated that the genotypic frequencies of GG, GA and AA were 0.26, 0.22 and 0.52 respectively showing a higher frequency of the AA genotypes. The allelic frequencies were 0.3692 for G and 0.6308 for A, indicating the allele A was more frequent. Similar trend was observed for SNP C2600T where T was more frequent in the population with a frequency of 0.6308. However for the same locus in HF cattle in China (Bai et. al., 2011), it was reported that there were three genotypes BB, AA and AB with frequencies of zero, 0.94 and 0.06 respectively suggesting a deviation from HW equilibrium. There were no individuals with genotype AA which is not the case in our study, though the frequency of one genotype was lesser than that of the other.
 
Level of heterozygosityies and effective number of alleles (n_e)
 
The distribution of genotypic frequencies of SNP A827G of TLR2 exon 2 in HF crossbred population revealed that the observed heterozygosity value of 0.0374, which was considerably lower in comparison to the expected heterozygosity value of 0.4380. Since the observed heterozygosity values were found to be lower than the expected heterozygosity values, there was a high degree of homozygosity at SNP A827G of TLR2 exon 2.
       
Genotypic frequencies of C1088G SNP showed an observed heterozygosity value of 0.0187 and it was lower than the expected heterozygosity value (0.3782). Similarly, the observed heterozygosity values for C2155A and G2281C in exon 2.5 and G2410A and C2600T in exon 2.6 were 0.2710 and 0.2150 respectively, which were lower than the expected heterozygosity values of 0.4045 and 0.4669 respectively for these SNPs. Thus, the results revealed that there was a high degree of homozygosity for TLR2 gene genotypes in the studied HF crossbred population. However, the degree of homozygosity was lower for C2155A, G2281C, G2410A and C2600T than both the SNPs viz., A827G and C1088G since the difference between the observed and expected heterozygosity values for former are less as compared to the later.
       
The effective number of alleles (n_e) was estimated for A827G, C1088G, C2155A, G2281C, G2410A and C2600T as indicated by Kimura and Crow (1964). The n_e and observed and the expected heterozygosity values along with the effective number of alleles are presented in Table 4.
 

 
c² test for Hardy Weinberg equilibrium
 
The c² test was applied to test whether the TLR2 genotypes in A827G, C1088G, C2155A, G2281C, G2410A and C2600T loci were in accordance to Hardy Weinberg equilibrium. The POPGENE analysis revealed that the estimated c² value was 180.11 at 1 degree of freedom, with probability value of < 0.001 for C1088G. Similarly, it was observed that the estimated c² values were 194.87, 23.45, 23.45, 62.63 and 62.63 for loci C1088G, C2155A, G2281C, G2410A and C2600T respectively at 1 df, with probability value of < 0.001.
       
Thus genotypic frequencies showed a significant deviation from HW equilibrium probabilities in this study. However, similar studies in HF breed in China (Bai et al., 2011), revealed the calculated c² values in the range of 0.15 to 6.34 and were all non-significant also the genotype frequencies of 15 SNPs fitted with the Hardy-Weinberg equilibrium (P > 0.05).
 
F statistics and Shannon’s Information index
 
The FIS (inbreeding coefficient) values for A827G, C1088G, C2155A, G2281C, G2410A and C2600T loci were 0.9144, 0.9505, 0.3284, 0.3284, 0.5385 and 0.5385 respectively. The results revealed a high degree of genetic differentiation in the population. The Shannon index estimated for each of the loci using POPGENE software analysis by formula indicated by Lewontin (1972). The Shannon index values were 0.6287, 0.5649, 0.5933, 0.5933, 0.6585 and 0.6585 respectively for the A827G, C1088G, C2155A, G2281C, G2410A and C2600T loci. These values represent genetic variation within the population. Thus, the genetic variation within the population was higher for G2410A and C2600T locus followed by A827G, G2281C, G2410A and C1088G respectively.

Molecular characterization of TLR2 gene was carried out by PCR- SSCP analysis. Exon 1 was amplified as a whole fragment while exon 2 of TLR2 gene was amplified in overlapping fragments covering the whole exon 2. Six SNPs were found in the HF crossbred population studied viz., A827G (exon 2.2), C1088G (exon 2.3), C2155A (exon 2.5), G2281C (exon 2.5), G2410A (exon 2.6) and C2600T (exon 2.6). Gene and genotypic frequencies indicated that some of the alleles were more frequently distributed in the population than the other. This was evident for SNP G2410A and SNP C2600T. The results also suggested that there was a high degree of homozygosity for TLR2 gene genotypes. The study also revealed that there was a difference between the observed and expected heterozygosity values for all SNPs studied. The genotypic frequencies showed a significant deviation from HW equilibrium. Values of inbreeding coefficient indicated high degree of genetic differentiation in the population. It was also seen that the genetic variation within the population was higher for G2410A and C2600T locus followed by A827G, G2281C, G2410A and C1088G respectively (as indicated by values of Shannon’s index).