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Neutrophil cytosolic factor 4 (NCF4) is component of the nicotinamide dinucleotide phosphate oxidase complex, a key factor in biochemical pathways and innate immune responses. In this study, splice variants and functional single-nucleotide polymorphism (SNP) of  NCF4  were identified to determine the variability and association of the gene with susceptibility to bovine mastitis characterized by inflammation. A novel splice variant, designated as  NCF4 - TV  and characterized by the retention of a 48 bp sequence in intron 9, was detected in the mammary gland tissues of infected cows. The expression of the  NCF4-reference  main transcript in the mastitic mammary tissues was higher than that in normal tissues. A novel SNP, g.18174 A>G, was also found in the retained 48 bp region of intron 9. To determine whether  NCF4 - TV  could be due to the g.18174 A>G mutation, we constructed two mini-gene expression vectors with the wild-type or mutant  NCF4  g.18174 A>G fragment. The vectors were then transiently transfected into 293T cells, and alternative splicing of  NCF4  was analyzed by reverse transcription-PCR and sequencing. Mini-gene splicing assay demonstrated that the aberrantly spliced  NCF4 - TV  with 48 bp retained fragment in intron 9 could be due to g.18174 A>G, which was associated with milk somatic count score and increased risk of mastitis infection in cows.  NCF4  expression was also regulated by alternative splicing. This study proposes that  NCF4  splice variants generated by functional SNP are important risk factors for mastitis susceptibility in dairy cows.

Role of an SNP in Alternative Splicing of Bovine NCF4 and Mastitis Susceptibility