Background

Phosphorylated neurofilament heavy, a marker of neuroaxonal damage, is increased in horses with equine neuroaxonal dystrophy. However, the temporal dynamics of this biomarker during the post-natal risk period are not understood.

Objective

To measure serum and cerebrospinal fluid phosphorylated neurofilament heavy concentrations in juvenile foals across the post-natal window of susceptibility for equine neuroaxonal dystrophy.

Study design

Case-control in vivo experimental study.

Methods

Concentrations of phosphorylated neurofilament heavy were measured using frozen serum and cerebrospinal fluid collected from 13 foals raised in a vitamin E deficient environment from 1 to 6 months of age. Four of these foals were produced by equine neuroaxonal dystrophy-affected dams, developed clinical signs consistent with equine neuroaxonal dystrophy and had a diagnosis confirmed by histopathology. The remaining nine foals, produced by healthy mares, were vitamin E depleted and remained clinically healthy. An additional cohort of foals, produced by healthy mares, were supplemented with vitamin E (α-tocopherol; α-TOH) from birth and sampled similarly.

Results

Serum α-TOH concentrations were significantly higher in vitamin E supplemented healthy foals. Serum phosphorylated neurofilament heavy concentrations did not differ significantly between groups at any time point. Cerebrospinal fluid phosphorylated neurofilament heavy concentrations increased with age in healthy vitamin E depleted foals (p < 0.001); an effect that was not observed in healthy vitamin E supplemented foals.

Main limitations

A genetically susceptible cohort supplemented with vitamin E was not available for comparison.

Conclusion

We demonstrate that vitamin E depletion may elevate cerebrospinal fluid phosphorylated neurofilament heavy in otherwise healthy juvenile foals by 6 months of age. We highlight an important cofactor to consider when interpreting cerebrospinal fluid phosphorylated neurofilament heavy concentrations in juvenile horses.

Background

Cervical vertebral compressive myelopathy (CVCM) and equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) are leading causes of spinal ataxia in horses. The conditions can be difficult to differentiate, and there is currently no diagnostic modality that offers a definitive antemortem diagnosis.

Objective

Evaluate novel proteomic techniques and machine learning algorithms to predict biomarkers that can aid in the antemortem diagnosis of noninfectious spinal ataxia in horses.

Animals

Banked serum and cerebrospinal fluid (CSF) samples from necropsy-confirmed adult eNAD/EDM (n = 47) and CVCM (n = 25) horses and neurologically normal adult horses (n = 45).

Methods

. A subset of serum and CSF samples from eNAD/EDM (n = 5) and normal (n = 5) horses was used to evaluate the proximity extension assay (PEA). All samples were assayed by PEA for 368 neurologically relevant proteins. Data were analyzed using machine learning algorithms to define potential diagnostic biomarkers.

Results

Of the 368 proteins, 84 were detected in CSF and 146 in serum. Eighteen of 84 proteins in CSF and 30/146 in serum were differentially abundant among the 3 groups, after correction for multiple testing. Modeling indicated that a 2-protein test using CSF had the highest accuracy for discriminating among all 3 groups. Cerebrospinal fluid R-spondin 1 (RSPO1) and neurofilament-light (NEFL), in parallel, predicted normal horses with an accuracy of 87.18%, CVCM with 84.62%, and eNAD/EDM with 73.5%.

Main limitations

Cross-species platform. Uneven sample size.

Conclusions and clinical importance

Proximity extension assay technology allows for rapid screening of equine biologic matrices for potential protein biomarkers. Machine learning analysis allows for unbiased selection of highly accurate biomarkers from high-dimensional data.

Background

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with vitamin E deficiency. In humans, polymorphisms in genes involved in vitamin E uptake and distribution determines individual vitamin E requirements.

Hypothesis/objectives

Genetic polymorphisms in genes involved in vitamin E metabolism would be associated with an increased risk of eNAD/EDM in Quarter Horses (QHs).

Animals

Whole-genome sequencing: eNAD/EDM affected (n = 9, postmortem [PM]-confirmed) and control (n = 32) QHs.

Validation

eNAD/EDM affected (n = 39, 23-PM confirmed) and control (n = 68, 7-PM confirmed) QHs. Allele frequency (AF): Publicly available data from 504 horses across 47 breeds.

Methods

Retrospective, case control study. Whole-genome sequencing was performed and genetic variants identified within 28 vitamin E candidate genes. These variants were subsequently genotyped in the validation cohort.

Results

Thirty-nine confirmed variants in 15 vitamin E candidate genes were significantly associated with eNAD/EDM (P < .01). In the validation cohort, 2 intronic CD36 variants (chr4:726485 and chr4:731082) were significantly associated with eNAD/EDM in clinical (P = 2.78 × 10^-4 and P = 4 × 10^-4 , respectively) and PM-confirmed cases (P = 6.32 × 10^-6 and 1.04 × 10^-5 , respectively). Despite the significant association, variant AFs were low in the postmortem-confirmed eNAD/EDM cases (0.22-0.26). In publicly available equine genomes, AFs ranged from 0.06 to 0.1.

Conclusions and clinical importance

Many PM-confirmed cases of eNAD/EDM were wild-type for the 2 intronic CD36 SNPs, suggesting either a false positive association or genetic heterogeneity of eNAD/EDM within the QH breed.

The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and regulatory elements, which are either not transcribed or transcribed at a very low level. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, presenting 39,625 novel transcripts, 84,613 candidate cis-regulatory elements (CRE) and their target genes, 332,115 open chromatin regions genome wide across a diverse set of tissues. We showed substantial concordance between chromatin accessibility, chromatin states in different genic features and gene expression. This comprehensive and expanded set of genomics resources will provide the equine research community ample opportunities for studies of complex traits in the horse.

Objectives

This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.

Procedures

A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.

Results

Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.

Conclusions

This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.

Following the successful creation of a biobank from two adult Thoroughbred mares, this study aimed to recapitulate sample collection in two adult Thoroughbred stallions as part of the Functional Annotation of the Animal Genome (FAANG) initiative. Both stallions underwent thorough physical, lameness, neurologic, and ophthalmic (including electroretinography) examinations prior to humane euthanasia. Epididymal sperm was recovered from both stallions immediately postmortem and cryopreserved. Aseptically collected full thickness skin biopsies were used to isolate, culture and cryopreserve dermal fibroblasts. Serum, plasma, cerebrospinal fluid, urine, and gastrointestinal content from various locations were collected and cryopreserved. Under guidance of a board-certified veterinary anatomic pathologist, 102 representative tissue samples were collected from both horses. Whole tissue samples were flash-frozen and prioritized tissues had nuclei isolated and cryopreserved. Spatially contemporaneous samples of each tissue were submitted for histologic examination. Antemortem and gross pathologic examination revealed mild abnormalities in both stallions. One stallion (ECA_UCD_AH3) had unilateral thoracic limb lameness and bilateral chorioretinal scars. The second stallion (ECA_UCD_AH4) had subtle symmetrical pelvic limb ataxia, symmetrical prostatomegally, and moderate gastrointestinal nematodiasis. DNA from each was whole-genome sequenced and genotyped using the GGP Equine 70K SNP array. The genomic resources and banked biological samples from these animals augments the existing resource available to the equine genomics community. Importantly we may now improve the resolution of tissue-specific gene regulation as affected by sex, as well as add sex-specific tissues and gametes.

Background

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM.

Hypothesis/objectives

Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses.

Animals

Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM-affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM-affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM-affected (n = 12) and age- and sex-matched control (n = 11-12) horses.

Methods

The rates of α-tocopherol/tocotrienol and γ-tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative reverse-transcriptase PCR (qRT-PCR) and droplet digital (dd)-PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog.

Results

Metabolic rate of α-tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ-tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number.

Conclusions and clinical importance

Increased α-tocopherol metabolism in eNAD/EDM-affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high-dose supplementation to prevent the clinical phenotype in genetically susceptible horses.