Calcium signaling genes in association with altitude-induced pulmonary hypertension in Angus cattle
Date
2019
Authors
Crawford, Natalie Faye, author
Thomas, Milton G., advisor
Coleman, Stephen J., advisor
Enns, R. Mark, committee member
Speidel, Scott E., committee member
Garry, Franklyn B., committee member
Journal Title
Journal ISSN
Volume Title
Abstract
This research used multi-omics technology (i.e., RNA-seq, qPCR for gene expression, SNP discovery and validation) to understand the influence of a particular subset genes on altitude-induced pulmonary hypertension susceptibility in Angus cattle. Three research aims were established to test the hypothesis that calcium-related genes may be associated with pulmonary hypertension in beef cattle. Data and samples utilized for the research came from the Colorado State University Beef Improvement Center Angus herd managed at 2,150 m of altitude. Transcriptome data from 6 tissues and 14 hypertensive and normotensive Angus steers were utilized for differential expression and pathway analyses. The objectives of the first aim were to: 1) to estimate and identify differentially expressed genes from RNA-Seq and pathway analyses, and 2) select putative candidate genes to analyze with qPCR (gene expression level). The largest number of DE genes was revealed in aorta (n = 631) and right ventricle (n = 2,183) samples. Top canonical pathways related to calcium signaling or utilization included: synaptic long-term depression, signaling by Rho family GTPases, and oxidative phosphorylation. Genes regulating calcium availability and utilization were expressed differently (log2 fold change > 0.589, < -0.589; P < 0.05) in Angus cattle with and without pulmonary hypertension. Isolated RNA from cardiac muscle (n = 9) and control muscle (n = 2) tissues from hypertensive and normotensive Angus steers were utilized to estimate gene expression using quantitative reverse transcription PCR in the candidate genes from Chapter 3. The objectives of this chapter were: 1) to establish the most appropriate reference genes in cardiac muscle tissues, and 2) to estimate and validated relative gene expression of calcium-related genes in cardiac muscle tissues using qPCR methods. Differences (P < 0.0055) among hypertensive and normotensive steers were estimated for right papillary muscle and right cardiac ventricle tissues (top, middle, and bottom) in candidate genes: ASIC2, EDN1, NOX4, PLA2G4A, RCAN1, and THBS4. Results of the current study validate the expression differences previously established of genes that regulate the availability and utilization of calcium with PH status in Angus steers at high altitude. Variant detection and association analyses were completed with 2 sets of available -omics data to identify opportunities for development of selection tools for reduced susceptibility to PH. The objectives of the third aim were to: 1) detect single nucleotide polymorphisms (SNP) in the transcriptome of 6 tissues, and 2) identify functional consequences of those variants associated with validated candidate genes from qPCR analyses. Pooled Angus sample analysis revealed 68 SNP in the 6 candidate genes: ASIC2, EDN1, NOX4, PLA2G4A, RCAN1, and THBS4. Thirty-eight SNP were revealed in the hypertensive group and 8 SNP in the normotensive steer group. Ten of the 68 identified SNP are utilized on large density commercially available bovine SNP chips (Illumina BovineHD BeadChip; GeneSeek Genomic Profiler HD; GeneSeek Genomic Profiler HDv2; Affymetrix Axiom Bovine). Analysis of transcriptome data identified SNP within genes regulating calcium availability and utilization, enhancing our understanding of sequence polymorphisms that may be involved in regulating pulmonary hypertension in Angus cattle raised at high altitude. These SNP are available for additional validation and potential use in genetic improvement programs.
Description
Rights Access
Subject
Angus
qRT-PCR
variants
pulmonary hypertension
altitude
RNA-Seq