Differential Expression Analysis of the Subchondral Bone and Articular Cartilage Transcriptomes in Neonatal and Adult Horses

Developmental orthopedic disease (DOD) represents a considerable health and welfare concern in the horse. The pathophysiology underlying this disease is unknown, but risk factors are thought to be both genetic and environmental. Tissue-specific gene expression will help elucidate important skeletal development pathways likely disrupted in DOD, but complete assembled transcriptomes are not publically available for relevant tissues at any age. The aims of this project are to 1) assemble complete transcriptomes of adult and neonatal articular cartilage and subchondral bone; 2) perform differential gene expression analysis between age groups within each tissue; and 3) perform network analysis of differentially expressed genes to identify key cellular pathways that may be disrupted in DOD.

Articular cartilage and subchondral bone was obtained from the fetlock, hock, and stifle of neonatal (n=4; ≤ 4 weeks old) and adult (n=4; 9-12 years old) horses. RNA was sequenced using an Illumina HiSeq, yielding ~22-38 million 100bp paired-end reads per sample. Reads underwent quality control prior to transcript quantification with Salmon. Transcript counts were converted to scaled gene counts using tximport. A MDS plot revealed clear clustering by age and tissue type. Differential expression analysis was performed using 4 software platforms (DEseq2, edgeR, limma, Sleuth). Differentially expressed gene sets were not identical between platforms; 1831 genes in bone and 2026 genes in cartilage were significantly differentially expressed between age groups across all analyses (q<0.05). Isoform-specific differential transcript expression between neonates and adults was also detected. This pilot work demonstrates proof-of-concept; additional sample collection and analysis is ongoing.