Polymorphisms in the 5′ Upstream Region of the CXCR1 Chemokine Receptor Gene, and Their Association with Somatic Cell Score in Holstein Cattle in Canada

Abstract

Identification of regulatory elements in 5' regions of chemokine genes is fundamental for understanding chemokine gene expression in response to infection diseases. The CXCR1 receptor is expressed on the surface of neutrophils and interacts primarily with CXCL8 (IL-8), the most potent chemoattractant for neutrophils. The aim of this study was to characterize the 5' upstream region (2.1 kb) of the bovine CXCR1 chemokine receptor gene for polymorphism content and to identify in silico potential transcription-factor binding sites. The 5' flanking region was found by mining the NCBI GenBank (www.ncbi.nlm.nih.gov/). A DNA sequence from the whole genome shotgun sequence project with reference number AC150887.4 contained the CXCR1 5' flanking sequence. Computer analysis revealed potential binding sites for the transcription factors nuclear factor kappaB (NF-kappaB), binding factor GATA-1, barbiturate inducible element (Barbie), nuclear factor of activated T-cells, and activator protein 1. Polymorphism discovery in this region was conducted by constructing an inclusive DNA pool including 2 phenotypic extreme groups, 20 bulls with high estimated breeding values (EBV) for somatic cell score (SCS), and 20 bulls with low EBV for SCS. Independent amplicons along the 5' flanking region of bovine CXCR1 were generated for polymorphism discovery by sequencing. Three novel single nucleotide polymorphisms (SNP), CXCR1c.-344T>C, CXCR1c.-1768T>A, and CXCR1c.-1830A>G, and a previously identified SNP in the coding region, CXCR1c.777G>C, were identified. The 4 SNP were genotyped in Canadian Holstein bulls (n = 338) using tetra-primer amplification refractory mutation system (ARMS)-PCR. Average allele substitution effects were estimated to investigate associations between the 4 SNP and EBV for SCS in first, second, and third and later lactations. Multiple trait analysis revealed that the SNP CXCR1c.-1768T>A was associated with EBV for SCS in the first and second lactations and over all 3 lactations. Haplotype analysis substantiated this association with EBV for SCS in the first lactation. Given the location of SNP CXCR1c.-1768T>A and the surrounding potential binding recognition sequences for NF-kappaB, GATA-1, and Barbie transcription-factors, this SNP may be implicated in gene regulation and warrants further research.

Single nucleotide polymorphisms in the CXCR1 gene and its association with clinical mastitis incidence in Polish Holstein-Friesian cows

“…Beecher et al (2010) revealed the association of the G allele with reduced SCS in cows of five breeds, but not in Holstein-Friesian bulls. The influence of mutation +735C>G on mastitis evaluated based on SCS was not confirmed neither by Leyva-Baca et al (2008) nor by Goertz et al (2009). Likewise, Verbeke et al (2012) did not find any association between the identified CXCR1 gene polymorphism and SCC.…”

“…Some authors demonstrated an association between CXCR1 gene polymorphism and udder health, evaluated using various parameters (e.g., SCS, somatic cell scores) (Youngerman et al, 2004a;Beecher et al, 2010;Galvão et al, 2011), but others (e.g., Leyva-Baca et al, 2008, Goertz et al, 2009) rejected this hypothesis. Recent studies have indicated a high degree of polymorphism in the CXCR1 gene.…”

Associations between CXCR1 polymorphisms and pathogen-specific incidence rate of clinical mastitis, test-day somatic cell count, and test-day milk yield

“…Several research groups studied associations between SNP in CXCR1 and udder health (Youngerman et al, 2004;Leyva-Baca et al, 2008b;Goertz et al, 2009;Beecher et al, 2010;Galvão et al, 2011;Verbeke et al, 2012). However, we were the first to study pathogenspecific data in a relative large population from multiple commercial dairy herds.…”

PCR-SSCP and Sequencing of CXCR1 (IL8RA) Gene in Indian Water Buffalo

“…But no report is available regarding SNP in buffalo yet. SNP (one) in the coding region (Youngerman et al 2004) and one SNP in the 5' upstream region (Leyva-Baca et al, 2008) of CXCR1 were found to be associated with mastitis susceptibility in case of Holstein Cattle. Zhou et al (2013) detected 4 single nucleotide polymorphisms (SNPs) of the CXCR1 gene in Chinese native cattle and found the significant association of c.337A>G and c.365C>T with the somatic cell score, which suggests the possible role of these SNPs in the host response against mastitis.…”