Eight polymorphisms were found within the promotor LTF region (Table 2). Four previously reported variations and four novel polymorphisms were identified. An insertion in -/G in the − 478 position has been previously reported by Li et al. [2004] and Daly et al. [2006]. Presumably, this insertion is the same reported by O’halloran [2009] in the − 479 position. A transition found in the − 271 position was reported by Daly et al. [2006], in Normande, Holstein Friesian, and New Zealand Holstein breeds, and might be the same reported in -270 by O’halloran et al. [2009]. Interestingly, a transition G/A polymorphism found in the − 190 position has been reported by Daly et al. [2006] and O’halloran et al. [2009]. The former study suggests the transcription importance of this variation by assessing its adjacent position to a potential SP1 transcription factor binding site, introducing an adenine nucleotide into this GC-rich region. Another previously reported polymorphism found, was a G/A transition in the − 156 position [Li et al., 2004, Daly et al., 2006, O’halloran et al., 2009].
Table 2
Confirmed and discovered polymorphisms in Lactoferrin promotor region in Holstein cows.
Position in AY319306.2*
|
Position from exon I
|
Nucleotide change
|
Variation
|
Allele frequency
|
3878
|
-4781,2
|
-/G
|
Insertion
|
G = 0.05
|
3935
|
-421
|
A/T
|
Transversion
|
T = 0.15
|
4083
|
-274
|
G/A
|
Transition
|
A = 0.35
|
4086
|
-2712
|
C/T
|
Transition
|
T= 0.09
|
4094
|
-265
|
C/A
|
Transversion
|
A = 0.25
|
4100
|
-256
|
A/C
|
Transversion
|
C = 0.27
|
4166
|
-1902,3
|
G/A
|
Transition
|
A = 0.27
|
4200
|
-1561,2,3
|
G/A
|
Transition
|
A = 0.54
|
*AY319306.2 Bos taurus lactoferrin (Lf) gene, 5' flanking region exons 1, 2 and partial cds (Zheng et al., 2005). 1Li et al. (2004), 2 Daly et al. (2006). 3O’halloran (2009)
|
Two novel variations showed a significant difference between the cow’s resistant and susceptible extreme groups. The variation in position − 421 A/T was significant (P = 0.0188) between groups, suggesting that the A allele carriers are susceptible to clinical mastitis. The variation in position − 256 A/C also showed a relevant difference (P = 0.0700) between groups. Li et al., (2004) reported three nucleotide variations in the 5’ region of the LTF gene of Holstein cows but not a significant relationship with somatic cell count. In another study, O’Halloran et al., [2009] found in a multi-breed dairy cattle sample twenty-nine polymorphisms within the regulatory region of the bovine LTF, from which 19 were novel variants; some associated with putative transcription factor binding sites, suggesting a potential effect on LTF expression levels. In a posterior analysis, Huang et al [2010], found three SNPs in the 5’ flanking region of the LTF gene in a Chinese Holstein population: these variations showed no individual association with somatic cell score but had a significant association in a combined haplotype. The present work outcomes significant association of at least one polymorphic variation in promoter region to clinical mastitis susceptibility by probability differences amongst extreme groups.
The interaction network assessment showed meaningful relationships among LTF gene and Cationic trypsin precursor mRNA, protein s100-a9, Lysozyme Z (LYZ), monocyte differentiation antigen cd14 precursor (CD14) and neutrophil gelatinase-associated lipocalin (LCN2). On the other hand, reactome analysis revealed a relationship with some antimicrobial peptides, Clusterin preproprotein (CLU), LYZ, and elastase, neutrophil expressed mRNA, but also specific granule lumen proteins, including the previously described proteins and LCN2 (Fig. 1). Direct association between these protein genes and mastitis incidence, somatic cell count, or related traits is not widely documented for all genes or proteins. Chen et al., (2010), found in a Chinese Holstein population polymorphism in the coding region of the LYZ gene associated with SCS, proposing its use as a candidate marker for mastitis. Ibeagha-Awemu et al. [1998] reported a significant association of genotype in 1908 non-synonym locus to a higher percentage of neutrophils expressing CD14 molecules on their surfaces and suggesting an important role of CD14 in mediating bacterial infections. On this basis, Kumar et al. (2014) demonstrated a significant association between polymorphisms in CL14 and mastitis frequency in Sahiwal cows. Similarly, Gupta et al. [2018], assessed the coding region of the CD14 gene and found a polymorphic pattern associated with SCS and differential expression between extreme genotypes indicating this gene is a candidate for mastitis resistance/susceptibility.
Enrichment analysis revealed nine related biological processes related to LTF, with a false discovery rate (FDR) < 0.001 and three ≤ 0.04. Most GO-terms were related to some degree of defense response (e.g bacterium, external stimulus, stress, and fungus). One of the most important GO terms (GO 0002523 FDR = 0.0226), was related to leukocyte migration involved in inflammatory response according to the AmiGO2 database. LTF is a protein present in milk, and interaction with other molecules and proteins is highly likely these evidence association outcomes indicate a gene concurrence on the potential typical role of these genes on the assistance of mastitis incidence in dairy cows, supporting further investigation for effective implementation of marker-assisted strategies.