Expression of genes with biomarker potential identified in skin from DSLD-affected horses increases with age

Abstract

Degenerative Suspensory Ligament Desmitis (DSLD) negatively impacts connective tissues in horses, which often leads to progressive chronic pain and lameness. DSLD has been shown to be a systemic disorder that affects multiple body systems, including tendons, sclerae, and the aorta. Currently, the diagnosis is confirmed by post mortem histological examination of a tendon or suspensory ligament. Histology reveals inappropriate accumulations of proteoglycans in the tendons and other tissues in DSLD-affected horses. Unfortunately, there is no reliable method to diagnose DSLD in living horses. Recently, bone morphogenetic protein 2 (BMP2) was identified in active DSLD lesions. In addition, recent data from RNA sequencing (RNA-seq) showed overexpression of numerous genes, among them BMP2, FOS and genes for keratins in DSLD skin biopsies-derived RNA. We hypothesized that some of these genes can be used as biomarkers for diagnosis of DSLD in a panel. Overexpression of some of them was verified in quantitative real time PCR. Immunohistochemistry and RNAscope in-situ hybridization (ISH) assays were used to determine the level of overexpression of specific genes in skin biopsies from control and DSLD-affected horses. The RNAscope ISH assay has shown to be more reliable and more specific that immunohistochemistry. ISH confirmed a significant increase in KRT83 and BMP-2 in hair follicles in DSLD cases, as well as abnormally high expression of FOS in the epidermis, especially in aging horses. Because statistically relevant specificity and sensitivity was documented only for FOS and BMP2, but not KRT83 we recommend the use of FOS and BMP2 panel to diagnose DSLD. We conclude that a panel of two markers from the studied group (BMP2 and FOS) can serve as an additional diagnostic tool for DSLD in living horses, especially in older animals. Further studies are necessary to confirm if this biomarker panel could be used as a prospective tool to identify DSLD in horses as they age.

Fig 1. RT-qPCR results for selected individual biomarkers.

Blue—DSLD Cq for each biomarker. Orange—control Cq for each biomarker.

Fig 2. Summary of immunohistochemical staining for all markers.

A. Mean staining of each biomarker in follicles. (K39 control = mean 2.4, p = 0.06, 0.8 power n = 15, 0.9 power n = 20, p<0.05; K81 control = mean 2.2, p<0.05; K83 control = mean 2.6, p<0.05; BMP2 control = mean 0.6, p<0.05. K39 DSLD = 1.8, p = 0.06, 0.8 power n = 15, 0.9 power n = 20; K81 DSLD = mean 1.4, p<0.05; K83 DSLD = mean 2.6 p<0.05; BMP2 DSLD = mean 1.75, p<0.05). Star = highly significant difference between DSLD and corresponding control value. B. Mean staining of each biomarker in sebaceous glands. (K39 control = mean 2.3, p = 0.06, 0.8 power n = 15, 0.9 power n = 20, p<0.05; K81 control = mean 2.4, p<0.05; K83 control = mean 1.6, p<0.05; BMP2 control = mean 0.6, p<0.05. K39 DSLD = 1.9, p = 0.06, 0.8 power n = 15, 0.9 power n = 20; K81 DSLD = mean 1.6, p<0.05; K83 DSLD = mean 2.3 p<0.05; BMP2 DSLD = mean 2.0, p<0.05). C. Mean of FOS in horses with DSLD compared with controls. Control = 1.4 and DSLD = 2.5 (P<0.01). D. Immunohistochemistry staining parameters such as mean intensity, P-values, and percent differences between controls and DSLD cases or each biomarker. P values calculated using Wilcoxon rank sum test through JMP program.

Fig 3. Mean detection of each biomarker in horses based on age.

A. Mean biomarker detection through IHC based on horse age (controls only). Blue bar = ages 5–10 years. Orange bar = any horse over age 11. B. Mean biomarker detection through IHC based on age (DSLD only) in 5 year intervals. C. Mean biomarker age through RNAscope based on horse age (controls only). Blue bar = ages 5–10 years. Orange bar = any horse over age 11. D. Mean biomarker detection through RNAscope based on age (DSLD only) in 5 year intervals.

Fig 4. Percent positive follicles by biomarker for control and DSLD cases.

Fig 5. In situ hybridization with RNAscope.

A. BMP2 RNAscope in control skin, arrowheads point to negative hair follicles. B. BMP2 RNAscope in DSLD skin, arrowheads point to mild to moderately positive hair follicles. C. FOS RNAscope in control skin. D. FOS RNAscope in DSLD skin. E. KRT81 RNAscope in control skin. F. KRT81 RNAscope in DSLD skin. All, 200 x magnification.