Genetic polymorphisms in vitamin E transport genes as determinants for risk of equine neuroaxonal dystrophy

1 INTRODUCTION

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disease associated with vitamin E deficiency during the first year of life.^{1, 2} Clinical signs of eNAD/EDM include symmetric ataxia (≥grade 2/5), wide-base stance at rest, proprioceptive deficits, and decreased serum vitamin E, specifically α-tocopherol, concentrations.^2-4 Even after 1 year of age, serum α-tocopherol concentrations typically remain lower in eNAD/EDM horse as compared to age-matched healthy controls.⁵ Equine NAD/EDM can be prevented in genetically susceptible foals by supplementing dams with high doses of water-soluble RRR-α-tocopherol during the last trimester of gestation, with continued supplementation in these foals through the first 2 years of life.³ Currently, the only way to conclusively diagnose eNAD/EDM is through postmortem histologic evaluation of the brainstem and spinal cord at necropsy.² While a recently developed biomarker test for phosphorylated neurofilament heavy chain (pNfH) has demonstrated some specificity for a diagnosis of eNAD/EDM in serum, the overall sensitivity is low.⁶

In humans, ataxia with vitamin E deficiency (AVED), an inherited disease caused by deleterious variants in tocopherol transfer protein (alpha; TTPA), shares clinicopathologic features with eNAD/EDM.^{7, 8} TTP(A) is the major protein involved in transferring α-tocopherol into liver secreted plasma lipoproteins.⁹ Equine NAD/EDM is not associated with genetic mutations in TTPA.¹⁰ However, polymorphisms in another gene involved in vitamin E uptake, distribution and metabolism could potentially modulate the risk of a horse developing eNAD/EDM. Vitamin E absorption¹¹ and transport to target tissues^{3, 12} are not altered with eNAD/EDM. However, vitamin E metabolism is increased in Quarter Horses (QHs) with eNAD/EDM.¹³ Additionally, there is greater hepatic expression of CYP4F2, the major metabolizer of vitamin E, in eNAD/EDM horses compared to unaffected horses.¹³ Thus, while we elected to profile all known vitamin E candidate genes, we hypothesized that genetic polymorphisms in genes involved in vitamin E metabolism, specifically CYP4F2, would be associated with increased risk of eNAD/EDM.

2 MATERIALS AND METHODS

3 RESULTS

3.2 Allele frequencies in public database

The top 3 SNPs (chr4:731082, chr4:726485, and chr26:39083023) from the clinical and postmortem-only analyses were genotyped using publicly available data from 504 horses across 47 breeds. Overall allele frequencies ranged from 0.06 to 0.1. Within-breed allele frequencies were calculated for breeds with ≥25 horses (Figure 1; Table S3). For chr4:731082, within-breed allele frequencies ranged from 0.03 (Franchese Montagne) to 0.30 (Warmbloods). For chr4: 726485, within-breed allele frequencies ranged from 0.02 (Shetland and Standardbred) to 0.23 (Warmbloods). Lastly, for chr26:39083023, within-breed allele frequencies ranged from 0.01 (Standardbred) to 0.3 (Franchese Montagne; Figure 1; Table S3).

4 DISCUSSION

In QHs, a postnatal vitamin E deficiency and genetic predisposition are both required to develop eNAD/EDM.^{2, 3} Abnormalities in vitamin E metabolism have been identified in QHs with eNAD/EDM, defined primarily by increased α-tocopherol metabolism in both serum and urine, after administration of an oral dose of RRR-α-tocopherol.¹³ In that same study, there was greater hepatic expression of CYP4F2, the major metabolizer of vitamin E, in eNAD/EDM horses compared to unaffected horses.¹³ We therefore profiled genetic variants in genes associated with vitamin E uptake, transport and metabolism, including CYP4F2, in this current study. Polymorphisms in these vitamin E genes are associated with varying vitamin E levels in humans.²⁴ While we did not identify any associated genetic variants in CYP4F2, 2 intronic CD36 SNPs were significantly associated with eNAD/EDM in QHs in both the clinical and postmortem-confirmed cohorts. A third SNP intronic in ABCG1 on chr26 was significantly associated with eNAD/EDM in the clinical cohort only.

Cluster determinant 36 (CD36) plays a key role in fatty acid uptake in many tissues. This transporter is a member of the scavenger receptor family and involved in uptake of oxidized low-density lipoproteins from the bloodstream. Alpha-tocopherol downregulates transcription of CD36, both in vitro³⁰ and in vivo.^{31, 32} In humans, a single intronic CD36 SNP (rs1527479) is associated with plasma α-tocopherol concentrations, and it is suggested that CD36 is involved in either intestinal absorption or tissue uptake of vitamin E.³³ Thus, multiple lines of evidence support that CD36 participates, either directly or indirectly, in vitamin E uptake. Since intestinal absorption of eNAD horses is comparable to unaffected horses¹¹ and tissue uptake into liver and CNS is also comparable after vitamin E supplementation,^{3, 12} variants in CD36 are unlikely be implicated in eNAD. Additionally, the 2 eNAD/EDM associated SNPs (chr4:731082 and chr4:726485) are intronic variants in CD36, with GERP scores less than 2. These GERP scores do not indicate high levels of evolutionary conservation. However, there are limitations to using this approach to identify deleterious mutations, particularly in noncoding sites, since regulatory elements are not typically highly sequence conserved.²⁸

While we identified a strong association between eNAD/EDM and these 2 intronic CD36 SNPs, many postmortem-confirmed cases did not have the alternate allele and allele frequencies were quite high for these variants in other breeds. In particular, Thoroughbreds are less likely to have eNAD/EDM³⁴ and allele frequencies for these CD36 variants on chr4 were high (0.15-0.18) in this breed. Detailed investigation of the genotypes obtained in the validation cohort revealed that no horses in that cohort were homozygous for the minor allele (GG) for either chr4:731082 or chr4:726485. In the public dataset of 504 horses, only n = 3 were homozygous for the minor allele in each SNP (chr4:731082; German Warmblood, Holsteiner and Saddle Trotter and chr4:726485; German Warmblood, Holsteiner and Thoroughbred). The lower minor allele frequency in QHs likely led to a false association, especially since the allele frequency for both SNPs ranged from 0.06 to 0.08 in the public database of QH samples (n = 61) and the fact that many other breeds had high allele frequencies for these SNPs. Additionally, our population of QHs may contain a higher subpopulation of QHs that have a higher frequency for these genetic variants. While we aimed to match our cases and controls by subpopulation, this stratification could have led to false positive results. This highlights the importance of assessing allele frequencies across breeds after identifying putative genetic associations in order to prevent false positive results.

A third SNP on chr26 was significantly associated with eNAD/EDM in the clinical cohort only. This SNP is intronic in ABCG1, an ATP binding cassette protein that transports various molecules across extra- and intra-cellular membranes. The transporter ABCG1 is involved in cellular vitamin E efflux and vitamin E metabolism is abnormal in Abcg1-deficient mice.³⁵ Due to the limited association for the ABCG1 SNP in only the clinical eNAD/EDM analysis, and the high allele frequencies within many breeds, this ABCG1 SNP is likely not a strong candidate variant for risk of developing eNAD/EDM.

A limitation of the current study is the biased preselection of candidate genes. While we attempted to include as many documented vitamin E candidate genes as possible, genes that warrant investigation may have been missed. Our selection of candidate genes was based on the most comprehensive review from the human literature.²⁴ Additionally, although we used both an algorithm and visual inspection of reads to identify larger structural variants, errors in the reference assembly could result in missing larger genomic rearrangements within these candidate genes that could be associated with disease. Long-range sequencing efforts are required to appropriately assemble genomes from affected and unaffected horses to identify these potential rearrangements. Despite the failure of this study to demonstrate a strong association for eNAD/EDM with any candidate variants, a genetic etiology for eNAD/EDM remains strongly supported by clinical cases in families^{1, 36, 37} and large-scale breeding farm investigations.^{2, 10, 14, 38, 39}

In conclusion, 2 intronic CD36 SNPs were significantly associated with eNAD/EDM in QHs. However, since many PM-confirmed cases of eNAD/EDM were wild-type for these variants, we either identified a false positive association or genetic heterogeneity exists for eNAD/EDM within the QH breed.

CONFLICT OF INTEREST DECLARATION

Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION

Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

Approved by the University of California-Davis IACUC (protocols #22427 and 22477).

HUMAN ETHICS APPROVAL DECLARATION

Authors declare human ethics approval was not needed for this study.