The role of PRKG2 in Angus dwarfism

Dwarfism is an undesirable phenotype in beef cattle that has reduced profitability for both commercial and pure bred producers since the 1950.s. In 2002, a flare up of dwarfism on several ranches across the US led to new genomic research designed to find the genetic mutation. Linkage-mapping of Bos taurus autosome 6 (BTA6) indicated a LOD score of 7.34 between microsatellite markers AFR227 and BM4311. To expedite the fine-mapping process, the BLAST Extension and Alignment Program BEAP was developed to assist with sequence assembly of the Angus dwarfism locus prior to completion of the bovine genome project. Four positional candidate genes, bone morphogenetic protein (BMP) 2 kinase, bone morphogenetic protein (BMP) 3, fibroblast growth factor 5, and cGMP-dependant, type II, protein kinase (PRKG2 ), were re-sequenced using BEAP output as template. Within PRKG2, a C/T nonsense mutation was discovered (R678Z) that truncates PRKG2 by 85 amino acids (AA), including 25 AA of the kinase domain. A mouse knockout model and naturally occurring PRKG2 mutants demonstrate that lack of PRKG2 function causes dwarfism. Recent work in the rat indicates that PRKG2 signals SOX9 to regulate the transition from proliferative to hypertrophic growth in the growth plate. Gene expression data in the rat demonstrated that PRKG2 mutants lose the ability to regulate SOX9 mediated collagen (Col) 2 and Col10 transcription. To determine if the Angus R678Z PRKG2 mutation altered collagen expression, wild-type and R678Z bovine vectors were assayed in a cell culture transfection experiment. Real-time PCR results confirmed that COL2 was inappropriately over-expressed in cells transfected with the R678Z mutation as compared to cells transfected with wild-type PRKG2 (p\u3c0.01). Over-expression of COL2 in dwarf cattle was confirmed by real-time PCR of dwarf and normal cattle growth plate tissue samples (p\u3c0.0001). Expression of COL10 was found to be upregulated in dwarf animals as well (p=0.05). These experiments indicate that the R678Z mutation is a functional mutation, resulting in a loss of PRKG2 regulation of COL2 and COL10 mRNA expression. To determine if the R678Z marker was an accurate predictor of Angus dwarfism, we conducted a breeding experiment. Six calves were produced by embryo transfer and their phenotype was predicted with 100% accuracy prior to expression of the phenotype. Four animals were predicted to be dwarves, homozygous for the R678Z PRKG2 marker, and two were predicted to be normal, heterozygous for the R678Z PRKG2 marker. Differences in skeletal growth were characterized by measurements of gross differences in height, long bone and vertebral column length. Animals homozygous for the R678Z PRKG2 marker had shorter metatarsals (reduced 4.5cm; p\u3c0.01), and fused ulna/radius (reduced 7.5cm; p\u3c0.01) bones. No significant differences were observed between these animals for head length, calvarium (skull) width, or bone circumference (p\u3e0.25). Stature and body length were also compared between animals homozygous for the R678Z PRKG2 marker and the R678Z PRKG2 heterozygotes at approximately 210 days of age. Animals homozygous for the R678Z PRKG2 marker were 15.8cm shorter (p\u3c0.0001) and had spinal lengths reduced by 20.7 cm (p\u3c0.0001) (data not shown). Transcriptional profiling of the null PRKG2 dwarf versus wild-type animals was performed using a 70 mer cDNA microarray to assess global changes in gene expression. Both COL2 and COL10 were found to be differentially expressed in the same direction and similar magnitude as observed by real-time PCR (p\u3c0.05). In addition, non-sense mediated decay of dwarf PRKG2 and up regulation of Wnt5a, and collagen9, markers of proliferation, was observed (p\u3c0.05). Multiple genes were found to be differentially expressed in the MAPK and Wnt5a signaling cascades. In addition, many Golgi transport and neuronally expressed genes were observed as differentially expressed. These genes provide interesting candidates for understanding the intermediate processes in the PRKG2 intracellular signaling pathway that regulate proliferation and developmental timing of the growth plate. Implications of these discoveries will allow Angus breeders to eliminate dwarfism and present a model to study PRKG2 function in the growth plate

Different oil sources impacting brain lipid and transcriptome profiles of pigs

Lipids are fundamental to the structure and function of the brain, and their fatty acids (FA) composition is rich in polyunsaturated fatty acids (PUFA), which have protective effects and modulate gene transcription. For nutrigenomics studies, pigs (Sus scrofa) have been widely used as a biomedical model. Thus, the aimed to investigate whether different dietary oil sources modify the pig brain's lipid and transcriptomic profile. A 98-day study was performed using fifty-four male pigs. Treatments consisted of corn-soybean meal diets containing 3 % of soybean oil (SOY), canola oil (CO), or fish oil (FO). Total mRNA was extracted for sequencing. As a result, feeding diets with different oil sources affected the percentage of some FA. Palmitic acid showed a greater concentration in diets containing SOY with 27.037 %. Oleic acid and eicosenoic acid, showed a greater concentration in diets containing SOY, with 30.968 % and 2.096 %, respectively; and, total PUFA showed a better concentration in diets containing SOY and FO, with 11.685 % and 12.150 %, respectively. After quality control, considering the total reads obtained for the three groups, 94.87% were mapped against the reference genome SScrofa11.1. A comparison of gene expression between the groups of pigs was carried out by using the DESeq2 statistical package (R/Bioconductor). From SOY vs CO comparison, five differentially expressed genes (DEG, FDR < 0.05) were identified, from SOY vs FO forty-four DEG were verified, and from CO vs FO thirty-nine DEG were found. The functional enrichment analysis resulted in pathway maps (P < 0.05) related to apoptosis and cell proliferation, obesity and type 2 diabetes, neurophysiological process, and inflammation. The networks were associated with signal transduction, calcium transport, and oxidative stress. Overall, the results showed that diets with different oil sources could affect some brain tissue parameters and may help guide future research on the availability of dietary FA in the brain.This article is published as da Silva, Bruna Pereira Martins, Simara Larissa Fanalli, Julia Dezen Gomes, Vivian Vezzoni de Almeida, Heidge Fukumasu, Gabriel Costa Monteiro Moreira, Bárbara Silva-Vignato et al. "Different oil sources impacting brain lipid and transcriptome profiles of pigs." Livestock Science 284 (2024): 105490. doi:10.1016/j.livsci.2024.105490. © 2024 The Author(s). This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/)

Brain fatty acid and transcriptome profiles of pig fed diets with different levels of soybean oil

Background The high similarity in anatomical and neurophysiological processes between pigs and humans make pigs an excellent model for metabolic diseases and neurological disorders. Lipids are essential for brain structure and function. Polyunsaturated fatty acids (PUFA) have anti-inflammatory and positive effects against cognitive dysfunction in neurodegenerative diseases. Thus, our main goal was to evaluate the effect of different levels of dietary soybean oil on the lipid profile and brain tissue transcriptome in pigs. For this, thirty-six male pigs were used in a 98-day study. Treatments consisted of corn-soybean meal diets containing either 1.5% soybean oil (SOY1.5) or 3.0% soybean oil (SOY3.0). After slaughter, the brain samples were collected for total lipid content and fatty acid profile determination. Total mRNA extraction was performed for brain transcriptome sequencing. Results There were no differences for total lipid content and fatty acid profile between the two treatment groups. For differential expression analysis, a total of 34 differentially expressed genes (DEG, log2 fold change ≥ 1; ≤−1; FDR-corrected p-value < 0.05) between the SOY1.5 and SOY3.0 diets were identified by the DESeq2 statistical package. These DEG 25 were annotated, of which 11 were up-regulated and 14 were down-regulated for the SOY1.5 group compared to SOY3.0. The functional enrichment analysis performed by MetaCore with the 34 DEG (FDR < 0.05), identified four pathway maps (p-value < 0.05), which are related to the genes ALOX15B, CALB1 and CAST. The network calcium transport was also identified (p-value = 2.303e-2), with the CAST and CALB1 genes. Conclusion The results found in this study contribute to understanding the pathways and networks associated with processes involved intracellular calcium, lipid metabolism, and oxidative processes in brain tissue. Moreover, these results may aid in better understanding the modulating effects of soybean oil and its fatty acids (FA) composition on processes and diseases affecting brain tissue.This is a pre-print of the article da Silva, Bruna Pereira Martins, Simara Larissa Fanalli, Julia Dezen Gomes, Vivian Vezzoni de Almeida, Heidge Fukumasu, Felipe André Oliveira Freitas, Gabriel Costa Monteiro Moreira et al. "Brain fatty acid and transcriptome profiles of pig fed diets with different levels of soybean oil." Research Square (2022). DOI: 10.21203/rs.3.rs-1654172/v1 Copyright 2022 The Authors. Attribution 4.0 International (CC BY 4.0). Posted with permission