Transcriptome analysis identifies genes involved with the development of umbilical hernias in pigs

Abstract

Umbilical hernia (UH) is one of the most frequent defects affecting pig production, however, it also affects humans and other mammals. UH is characterized as an abnormal protrusion of the abdominal contents to the umbilical region, causing pain, discomfort and reduced performance in pigs. Some genomic regions associated to UH have already been identified, however, no study involving RNA sequencing was performed when umbilical tissue is considered. Therefore, here, we have sequenced the umbilical ring transcriptome of five normal and five UH-affected pigs to uncover genes and pathways involved with UH development. A total of 13,216 transcripts were expressed in the umbilical ring tissue. From those, 230 genes were differentially expressed (DE) between normal and UH-affected pigs (FDR <0.05), being 145 downregulated and 85 upregulated in the affected compared to the normal pigs. A total of 68 significant biological processes were identified and the most relevant were extracellular matrix, immune system, anatomical development, cell adhesion, membrane components, receptor activation, calcium binding and immune synapse. The results pointed out ACAN, MMPs, COLs, EPYC, VIT, CCBE1 and LGALS3 as strong candidates to trigger umbilical hernias in pigs since they act in the extracellular matrix remodeling and in the production, integrity and resistance of the collagen. We have generated the first transcriptome of the pig umbilical ring tissue, which allowed the identification of genes that had not yet been related to umbilical hernias in pigs. Nevertheless, further studies are needed to identify the causal mutations, SNPs and CNVs in these genes to improve our understanding of the mechanisms of gene regulation.

Fig 1. Hematoxylin & eosin stain histological section of the umbilical ring tissue sample from a normal (A) and an umbilical hernia-affected (B) piglet.

Fig 2. Heatmap with 230 differentially expressed genes between animals affected with umbilical hernia (21A, 27A, 27A, 29A, 31A and 36A) and normal piglets (20C, 26C, 28C, 30C and 37C).

The expression for each gene is shown in the rows and samples are visualized in the columns, showing a hierarchical clustering of genes and samples. Genes are upregulated (in green) and downregulated (in red) in the affected samples.

Fig 3. Comparison of Log2FC expressed values between the RNA-Seq and qPCR methodologies for the 12 target genes chosen for validation.

Fig 4. Pearson`s correlation (r) between Log2FC values of RNA-Seq and qPCR analyses for the 12 target genes selected for validation.

Fig 5. Significant biological processes of Differentially Expressed (DE) genes related to umbilical hernia in pigs.

The X-axis shows the total number of genes that were DE in each biological process, based on the genetic ontology using the David 6.8.

Fig 6. Significant cell components and molecular functions of Differentially Expressed (DE) genes related to umbilical hernia in pigs.

The X-axis shows the total number of genes that were DE in each of them, based on the genetic ontology using the David 6.8 database.

Fig 7. Gene network constructed with differentially expressed genes and main biological processes related to umbilical hernia using Cytoscape.

Differentially expressed genes are visualized in the circles and biological processes in the rectangles. Node sizes indicate the number of predicted gene interactions. The edge colors indicate the betweenness of the edges (low values are in small size and in bright colors).