Single Nucleotide Polymorphism in the Promoter Region of Bovine Interleukin 8 Gene and its Association with Milk Production Traits and Somatic Cell Score of Holstein Cattle in Iran

1. Background
Mastitis is a polygenic trait that is caused predominantly by bacterial infection. During mastitis, it is common to observe an increased number of somatic cells (macrophages, neutrophils, and lymphocytes) in milk. These somatic cells play a crucial role in host resistance. In dairy cattle, due to positive genetic correlation between Somatic Cell Count (SCC) and clinical mastitis, most genetic studies focused on milk SCC and clinical mastitis as phenotypic measures to predict the bacterial status of udders (7, 19). Somatic cells migration from the bloodstream to mammary gland tissue occurs as a response to pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b) and IL8. There is compelling evidence that suggests IL8 is a more powerful neutrophil chemotactic cytokine than the other cytokines (1). Therefore, IL8 is a potential candidate gene for mastitis resistance.    The gene encoding bovine IL8 cytokine is mapped on chromosome 6 (6) and contains four exons and three introns, typical of the gene organization of CXC subfamily of chemokines (14). The gene encodes a protein that acts as a potent chemokine for neutrophils when ligating with IL8RA receptor expressed on the surface of neutrophils (21). Identification of SNP in the promoter region of cytokine genes is fundamental for understanding the cytokine gene expression in response to infection diseases. Several studies have confirmed associations between SNPs in the promoter region of different immune response genes and performance traits in dairy cattle (9, 15, 18).

2. Objectives

The objective of this study was to assess the association between -180 SNPs in the bovine IL8 promoter region with milk production traits and SCS in Holstein cattle of Iran.

3. Materials and Methods

Blood samples were collected from 601 multiprous Holstein dairy cows from one farm at Esfahan province of Iran. Genomic DNA was extracted from EDTA anticoagulated blood using the AccuPrep® (BiONEER, South Korea) genomic DNA extraction kit, according to the manufacturer’s instructions.
    The 260bp of promoter region (-216 to +44) of the bovine IL8 gene was amplified by polymerase chain reaction (PCR) using the following conditions: Initial denaturing cycle (2 min at 95°C); the amplification step composed of 30 cycles of 95 °C for 30 s, 63°C for 30 s and 72°C for 25 s, and the last step of 72°C for 7 min for final extension. Forward (5’-cagatgactcagatgtgctctca-3’) and reverse (5’-caggaaaagctgccaagaga-3’) primers were designed by Primer3 software (http://frodo.wi.mit.edu/primer3/), according to the promoter region of bovine IL8 sequence (GenBank accession no. AY627308.1). The polymerase chain reaction (PCR) was carried out using a PCR kit (BiONEER, South Korea) with the lyophilized components.
     For single-strand conformation polymorphism (SSCP) analyses 2 mL of the PCR product was mixed with 6 mL SSCP gel loading dye (95% formamide, 20 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue). Then samples were heated for 5 min at 95ºC and immediately cooled on ice. The total volume was loaded onto a 10% polyacrylamide gel (37.5:1acrylamide/ bisacrylamide).
     Electrophoresis was carried out at room temperature in 0.5×TBE buffer for 20 h. The gels were subsequently fixed in 10% acetic acid, stained with 0.15% AgNO3 and revealed with 1.5% Na2CO3.
    For sequence analysis, the PCR products (2 samples) were purified using the QIA quick PCR purification kit (Qiagen, Iran) and bidirectional sequenced by forward and reverse primers. Multiple alignments of the nucleotide sequences for different SSCP patterns were carried out using the CLUSTALW (http:// workbench.sdsc.edu). To evaluate the effect of IL8-180 SNP on transcription factor binding sites within amplified fragment of IL8 promoter region, In silico analysis was performed using TFSEARCH v1.3 (www.cbrc.jp/ researh/db/ TFSEARCH. html) software.

Statistical Analysis
   Test-day records of milk yield, fat percentage, protein percentage and SCC were obtained from the routine milk recoding scheme in the center of animal breeding at Isfahan province. The distribution frequency of SCC values was highly skewed. Therefore, the somatic cell counts were transformed to a log scale and converted to SCS (shooke, 1982).
    Frequencies of genotypes, alleles and Hardy-Weinberg test were computed using TFPGA version 1.3. For association studies, the data regarding the milk production traits and SCS were analyzed with the mixed procedure of the SAS 9.1 program according to the following statistical model:

yijkl = m+Gi+Sj+Lk+Sil+eijkl
Where:
 
yijkl: phenotypic value of interest traits;  m: mean of the traits; Gi: fixed effect of the IL8 genotype;  Sj: fixed effect of season; Lk: fixed effect of lactation; Sil:  random effect of sire and eijkl: random error.

4. Results 

In this study, the amplified fragment of IL8 promoter region revealed three distinct SSCP patterns which were highly reproducible. Different banding patterns were observed, which are indicated by A, B or C band patterns observed were denominated from A to C, as shown in Figure 1.
     Sequence analysis of the region revealed a substitution of G to A at position -180 relative to the initiation codon site. Since the forward primer was too close to the -180 SNP, sequencing withthis primer could not distinguishdifferent geno-types of G to Aat IL8-180. Therefore, thesequencing was performed using reverse primer (Figure 2). According to sequence analysis, theSSCPpatterns of A, B and C is equal to heterozy-gous (G/A), homozygous (A/A) and homozygous (G/G), respectively for -180 mutation. Theobtained sequences for promoter region of thebovine IL8 gene were submitted to NCBI(KF551877, KF551878).
     The genotype frequencies of AA, GA and GG for IL-8-180 were 0.270, 0.545 and 0.185 respectively. The estimated frequencies of A and G alleles were 0.54 and 0.46, respectively. The individual frequencies of the genotypes deviated from the Hardy-Weinberg equilibrium for identified SNP by c2 test (P= 0.014).
    Following a search for possible transcription factor binding sites using TFSEARCH, the amplified sequence of the IL8 promoter region revealed several potential transcription factor binding sites, including AP-1, C/EBPb, NF-kap, c-Rel, GATA-1, Nkx-2 and CdxA. A potential TATA box was also located -114 from start codon site of bovine IL8 gene. On the other hand, in silico analysis of the consequences of identified SNP on potential cis-acting elements predicted that the G to A transition at position -180 does not disrupt any consensus sequences for known transcription factors, but creates a putative binding site for Oct-1 transcription factor (Table 1) . 
     The associations between IL8-180 genotypes with milk production traits and SCS are listed in Table 3. Results indicated that different genotypes in this fragment had a significant association with average daily milk yield (P < 0.05). Cows with GG genotype had significantly increased milk yield relative to the GA and AA genotypes. There was a high tendency to associate between detected genotypes and SCS (P = 0.06), so that the AA genotype had lower SCS rather than AG and GG genotypes. No other significant associations were observed between any of the genotypes at -180 positions for fat percentage and protein percentage (Table 2).

5. Discussion

The amplified fragment of bovine IL8 promoter region revealed 3 SSCP genotypes and sequence analysis of them showed one SNP (G/A) at position -180. The genotypes of IL8 gene significantly deviated from the Hardy-Weinberg proportions (P=0.014), indicating that IL8 gene is under directional selection. The high frequency of heterozygotes (an observed and expected count 327 and 298, respectively) caused this deviation from Hardy-Weinberg equilibrium.
     Transcription factors play important roles in the regulation of gene transcription initiation. They bind to promoter elements and modulate the relative efficiency of transcription initiation by activation or repression. The search for transcription factor binding domains revealed that G to A transition at position -180 created a putative binding site for octamer transcription factor-1 (Oct-1). Oct-1 is a transcription factor belonging to the POU family that constitutively expressed in many cell types and specifically interacts with the octamer motif ATGCAAAT (24). Although multiple factors are involved in the control of human IL8 gene transcription (8), the Oct-1 transcriptional factor is particularly important because it can repress the expression of IL8 (2, 10, 26, 29) by displacing the C/EBP transcription enhancer from the IL8 gene promoter (28). Binding of this transcription factor may mediate the reduced sensitivity of promoter sequence of IL8 gene to exogenous and endogenous stimulation (17). In this study, cows with AA genotype at IL8-180 SNP showed significant lower SCS than GG and GA genotypes. The result here suggests that Oct-1 might act as a negative regulatory element for IL8 transcription.
    The IL8-180 significantly associated with average daily milk yield, while the associations with fat percentage and protein percentage were not significant. The significant associations were reported with the IL8 mutations and milk yield, 305 day milk protein yield, 305 day corrected milk yield, 305 day milk fat yield, SCS, and milk protein percentage, while their association with milk fat percentage was not significant (3). Leyva-Baca et al. (2007) also identified significant associations between the polymorphism of bovine IL8 (A/G at position 2647) with fat yield and udder depth. A possible association between IL8 and fat yield may be due to the IL8 angiogenic properties, including adipocyte movement and adipose tissue metabolism (23).
     The association of IL8 promoter region could be explained by the mapping quantitative trait loci (QTL) for milk production traits on bovine chromosome 6. Several studies have investigated segregating QTL with significant effects on milk production traits in the bovine chromosome 6. For example, three QTL affecting milk, fat, and protein production, as well as fat and protein concentration, were found on BTA6 in the Israeli Holstein population (22).  According to the confidence interval for QTL location on BTA6, several genes that have some physiological relevance to the milk production traits have been considered primary candidates for the QTL. ABCG2 (5, 13, 20), PPARGC1A (12, 27) and OPN (12) are located in BTA6 that they are potential candidate genes for milk production traits and SCC.
     In conclusion, the IL8-180 SNP (G/A transition) that lies in promoter region can be important in the regulation of IL8 transcription. Also, our results provide evidence that the IL8-180 SNP might have potential effects on milk yield and SCS. Therefore, the functional role of this SNP in bovine needs to be analyzed and confirmed by means of gene expression assays. In addition, further studies are needed to confirm the associated effects of the IL8-180 and other polymorphisms in bovine IL8 gene.