Exploratory identification of coding and splicing-related SNV variants in A1A1 and A2A2 β-casein Holstein dairy cows

The A2 β-casein variant has gained considerable interest in the dairy industry due to proposed health related benefits, leading to an increasing frequency of the A2A2 genotype in dairy herds. Although the A1/A2 substitution in the β-casein gene (CSN2) does not directly affect gene regulation, previous transcriptomic studies have reported differences in mRNA isoform expression between A1A1 and A2A2 cows. Therefore, the objective of this study was to identify single nucleotide variants (SNVs) in A1A1 and A2A2 β-casein groups of cows using milk fat globule (MFG) RNA-seq data and to evaluate their predicted functional consequences. RNA sequencing was performed on MFG samples obtained from 14 lactating Holstein cows (A1A1, n = 7; A2A2, n = 7). Variants were classified according to their predicted effects as amino acid changing (AAC) variants, splice site effect (SSE) variants, or variants presenting both consequences. Additionally, variant data were integrated with previously reported mRNA isoform expression results, and only variants located in genes showing expression levels ≥ 0.2 FPKM were retained for further analysis. Candidate RNA-seq-derived variants differing between A1A1 and A2A2 β-casein genotype groups were identified in genes involved in mammary gland function and lactation, including mitochondrial function, lipid metabolism, vesicle trafficking and secretion, and RNA processing. Among the prioritised genes, A1A1 cows showed a greater representation of SNVs located in genes involved in mitochondrial oxidative phosphorylation (NDUFV2, NDUFAB1, COX7A2 and ATP5PF), while additional SNVs were identified in lipid metabolism-related genes (ACSL1, ATP10A and MFGE8). In contrast, A2A2 cows showed a greater representation of SNVs located in genes involved in lipid metabolism (LPIN1, FASN, SPTLC2 and ATP11B) and vesicle trafficking and secretion (SEC31A, LRRK2, DBNL, EIPR1 and ABCG2). Overall, these findings provide additional insight into the molecular differences detected between A1A1 and A2A2 groups of cows. Although the predicted functional consequences of the identified variants are currently based on in silico analyses, the novel SNVs reported here constitute a valuable resource for future studies investigating the biological consequences associated with selection for the A2A2 β-casein genotype in Holstein dairy cattle.