Identification of novel genes associated with atherosclerosis in Bama miniature pig

Abstract

Background: Atherosclerosis is a chronic cardiovascular disease of great concern. However, it is difficult to establish a direct connection between conventional small animal models and clinical practice. The pig's genome, physiology, and anatomy reflect human biology better than other laboratory animals, which is crucial for studying the pathogenesis of atherosclerosis.

Methods: We used whole-genome sequencing data from nine Bama minipigs to perform a genome-wide linkage analysis, and further used bioinformatic tools to filter and identify underlying candidate genes. Candidate gene function prediction was performed using the online prediction tool STRING 12.0. Immunohistochemistry and immunofluorescence were used to detect the expression of proteins encoded by candidate genes.

Results: We mapped differential single nucleotide polymorphisms (SNPs) to genes and obtained a total of 102 differential genes, then we used GO and KEGG pathway enrichment analysis to identify four candidate genes, including SLA-1, SLA-2, SLA-3, and TAP2. nsSNPs cause changes in the primary and tertiary structures of SLA-I and TAP2 proteins, the primary structures of these two proteins have undergone amino acid changes, and the tertiary structures also show slight changes. In addition, immunohistochemistry and immunofluorescence results showed that the expression changes of TAP2 protein in coronary arteries showed a trend of increasing from the middle layer to the inner layer.

Conclusions: We have identified SLA-I and TAP2 as potential susceptibility genes of atherosclerosis, highlighting the importance of antigen processing and immune response in atherogenesis.

Keywords: atherosclerosis; candidate genes; genome‐wide linkage analysis; major histocompatibility complex; whole genome sequencing.

FIGURE 1

Genetic susceptibility to diet‐induced atherosclerosis and genome‐wide linkage analysis. (A), Environmental factor (high‐cholesterol and high‐fat diet) affects the physiological state of pig blood vessels. (B), Atherosclerotic plaque lesions are more severe in the coronary arteries and abdominal aorta of SA pigs. (C), Sequencing of 9 samples was performed using NGS next generation sequencing, filters based on linkage analysis; 153 susceptible SNP sites were finally obtained.

FIGURE 2

Histogram of GO enrichment analysis and bubble chart of the KEGG pathway enrichment analysis. (A), Categories are divided according to color from top to bottom: Navy blue represents biological process; Red represents cellular component and Orange represents molecular function. (B), The x‐axis represents the proportion of genes enriched in the pathway out of the total enriched genes, while the y‐axis represents the name of the enriched KEGG pathway. The size of the dots corresponds to the number of genes enriched in the pathway, and the color indicates the p value.

FIGURE 3

Candidate gene function prediction. (A), Changes in amino acids in the primary structure of SLA‐1 protein. (B), Changes in amino acids in the primary structure of SLA‐2 protein. (C), Changes in amino acids in the primary structure of SLA‐3 protein. (D), Changes in amino acids in the primary structure of TAP2 protein. (E), Comparison of the tertiary structure of SLA‐1, SLA‐2, SLA‐3, and TAP2 protein in NSA and SA groups.

FIGURE 4

Expression of TAP2 protein in the coronary artery and PPI analysis. (A), TAP2 immunofluorescence in coronary artery of NSA and SA groups. Blue fluorescence represents the nucleus, and red fluorescence represents TAP2. (Scale bars: 50 μm). (B), The fluorescence area values of the two groups were statistically different (*p < 0.05). (C), TAP2 immunohistochemistry in coronary artery of NSA and SA groups. (Scale bars: 500 μm, 100 μm). (D), Protein–protein interaction network of candidate genes. The red line indicates evidence of gene fusion; the black line indicates co‐expression evidence; the blue line indicates co‐occurrence evidence; the light blue line indicates curated database evidence; the purple line indicates experimental evidence; the yellow line indicates text mining evidence; the green line indicates gene neighborhood evidence.