Equine neuroaxonal dystrophy (NAD) is an inherited neurodegenerative condition that has been described in young horses of various breeds.[1-4] The histologic lesions of NAD consist of neuronal degeneration within specific nuclei of the brainstem.[1, 3, 5] Equine degenerative myeloencephalopathy (EDM) is a pathologically more advanced form of NAD that also appears to be inherited. A histologic diagnosis of EDM is made when axonal necrosis and demyelination are found within the dorsal and ventral spinocerebellar tracts and ventromedial funiculi of the cervicothoracic spinal cord.[6-10] Both NAD and EDM have been associated with vitamin E deficiency.[3, 6] In American Quarter Horses (QHs) with histopathologic lesions consistent with EDM, we have demonstrated that there also are lesions consistent with NAD. Therefore, the term NAD encompasses the primary disease process, with EDM considered a more severe variant of NAD.
Comparatively, both the clinical and histologic findings in horses with NAD/EDM resemble ataxia with vitamin E deficiency (AVED) in humans. AVED is caused by various mutations in the alpha-tocopherol transfer protein gene (TTPA), which encodes for the protein responsible for vitamin E transport in the liver and incorporation of α-tocopherol, a main component of vitamin E, into very low-density liposomes for transport throughout the body.[11, 12] Human patients with AVED develop neurologic abnormalities similar to those seen in horses with NAD/EDM and pathologic findings in AVED are similar to those observed in NAD/EDM, including neuroaxonal dystrophy within the gracile and cuneate nuclei. Although we have previously demonstrated no difference in expression of TTPA between affected and unaffected horses, mutated genes can maintain normal expression levels while altering protein confirmation and their interactions, and therefore we aimed to definitively evaluate TTPA as a candidate gene for NAD/EDM.
A mode of inheritance for NAD/EDM is required to determine if variants uncovered in TTPA are putative mutations. Although previous studies have described pedigree analysis and a prospective breeding trial was performed in Morgan horses, the mode of inheritance for NAD/EDM has been difficult to determine. Either an autosomal dominant mode of inheritance with variable expression or a polygenic mode of inheritance was considered likely based on a breeding trial in Morgan horses. It is not known, however, if NAD/EDM is allelic (ie, if specific causal variants are shared) between Morgans and QHs. Using the large group of horses phenotyped in a previous study, we aimed to define the mode of inheritance of NAD/EDM in the QH.
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This study of NAD/EDM in the QH established a high heritability of 0.70 in this particular study population and investigated the mode of inheritance of NAD/EDM. An X-linked pattern of inheritance was excluded. In addition, we have excluded a fully penetrant autosomal dominant mode of inheritance based on 2 crosses by an unaffected stallion in this report, which is in agreement with the findings in Morgans with NAD by Beech et al. However, based on our previous research describing the clinical phenotype of NAD/EDM in the QH, including the effect that supplemental vitamin E, specifically α-tocopherol, during the 1st year of life may have on the overall phenotype of affected horses, it is likely that there is a strong environmental component in the development of NAD/EDM and the trait may be incompletely penetrant or polygenic. This theory is supported by the findings in this study. Therefore, a horse may have the putative genetic mutation, but not display a consistent moderate (grade 2) to severe (grade ≥3) ataxia.
Beech et al excluded an autosomal recessive mode of inheritance of NAD in the Morgan horse by producing unaffected foals when affected sires were bred to affected mares. In our population of horses, an autosomal recessive mode of inheritance cannot be excluded, as affected horses were not bred to affected horses. Based on the number of horses affected within these families, if the mode of inheritance was autosomal recessive, the carrier frequency for this mutation within this family would be quite high.
Heritability is defined as the proportion of the phenotype that is attributable to genetic variance. Recently, in genetic association studies, new methods that exploit the use of genetic marker data have been used to explain the fraction of phenotypic variance due to relatedness by generating a kinship matrix. This calculation is termed pseudoheritability because it resembles the heritability estimated from a pedigree, yet is not directly interchangeable with heritability of the trait because the estimated pairwise relatedness does not correspond exactly to the kinship coefficients. Pseudoheritability was estimated at 0.795 for NAD/EDM in this population of horses. To further evaluate this calculation, we performed an additional analysis, estimating heritability from phenotype and pedigree information alone, and obtained a comparable heritability estimate of 0.70. There was no significant effect of sex on this estimate. Such a value suggests a considerable amount of genetic variation, enough to promote a breeding program designed to substantially change disease risk. Moreover, a value of this magnitude would give support to a search for the genes responsible for this character, the so-called dissection of this complex trait. Ours was a relatively small sample and pedigree, and any values estimated from this sample may vary considerably from another selection of individuals. However, the behavior of this small sample, in the setting of this Bayesian estimation strategy, did yield stable and informative results. Heritability estimates may not be completely reliable in such a population as described here, where all horses were exposed to an environmental variable (ie, low dietary vitamin E) that may have impacted the incidence of NAD/EDM in a genetically susceptible population. Therefore, the high heritability as determined in this study should not be extrapolated to the entire QH population.
Complex segregation analysis determined that there was insufficient evidence for a single gene effect and therefore lends evidence to NAD/EDM being inherited as a complex trait. Polygenic traits may include the effects of more than 1 gene along with environmental influences to determine the phenotype. Susceptibility loci, or “risk alleles”, are base-pair variants that are found in a higher frequency in diseased individuals as compared with healthy individuals. Those individuals with the gene variant are at a higher risk to develop a particular disease, but the presence of the variant does not induce or cause the disease. Susceptibility loci have been associated with a wide variety of diseases in humans, including Alzhiemer disease, multiple sclerosis, and Parkinson's disease. We propose that the genetic variant associated with NAD/EDM may be a susceptibility locus and environmental effects, specifically the amount of α-tocopherol received by the foal during the 1st year of life, may play a role in determining the overall phenotype. The majority of randomly sampled horses in this study were α-tocopherol deficient, as determined by serum α-tocopherol concentrations, and there was no statistical difference between affected and unaffected horses, as previously reported.[9, 10]
Based on the association analysis performed by SNPs in the region and the direct sequencing of TTPA, it is unlikely that genetic mutations in TTPA are causative for NAD/EDM in the QH. A susceptibility locus for NAD/EDM in TTPA also is highly unlikely because none of the identified variants were significantly associated with the disease phenotype. Although 4 variants were unable to be genotyped on the multiplex assay, including 2 synonymous mutations in exon 1, these would be unlikely to be causative for NAD/EDM because there was no evidence of an association in the adjacent markers. Although potential regulatory mutations in TTPA cannot be completely ruled out based on this study, they are unlikely because of the fact that the cDNA from affected and unaffected horses has the same length (ie, contains the same number of exons). Size changes of 100 bp can be distinguished on a 2% agarose gel and none of the exons in TTPA were <100 BPM in length. In addition, we have previously demonstrated that there is no significant difference in the expression of TTPA between NAD/EDM-affected horses and controls.
Although TTPA was the strongest candidate gene for NAD/EDM and there are prominent similarities of NAD/EDM to AVED, there are distinct histologic differences between the diseases. Although lesions of AVED include spheroid formation within the gracile and cuneate nuclei of the brainstem, there also is mild Purkinje cell loss and axonal degeneration in the dorsal columns, features that are not found in horses with NAD/EDM.[5, 34] In addition, the axonal degeneration of the lateral and ventromedial funiculi seen in EDM is not observed in human patients with AVED. From a clinical perspective, the most striking distinction between the 2 diseases is that cases of AVED appear to stabilize or improve with supplemental α-tocopherol, whereas cases of NAD/EDM do not.[3, 36, 37]
A limitation of this study was the characterization of 29 affected NAD/EDM horses based on clinical examination and farm history alone. At this time, a definitive diagnosis of NAD/EDM can only be achieved by postmortem examination with careful histologic evaluation of the brainstem and spinal cord. All horses on this particular farm were vitamin E deficient at the time of diagnosis, which, in conjunction with the degree of relatedness among the horses, supports a likely clinical diagnosis of NAD/EDM as a cause of their ataxia. In addition, by classifying only the most severely affected horses as cases and horses with mild neurologic deficits as equivocal, we have minimized our chances of misphenotyping. The horses used for sequencing of TTPA were postmortem-confirmed cases and controls. Genetic heterogeneity in NAD/EDM within and across breeds is a distinct possibility, but we limited this study population to a small family of QH in an attempt to minimize this effect.
The association between α-tocopherol deficiency and the development of NAD/EDM remains unclear. There have been many reports describing α-tocopherol deficiency in NAD/EDM-affected horses,[6, 15] whereas other studies demonstrate that an α-tocopherol deficiency does not increase the risk of NAD/EDM development.[9, 38] There does appear to be strong evidence, however, that supplementation with α-tocopherol decreases the prevalence of NAD/EDM in genetically susceptible horses.[3, 6, 8] In addition to TTPA, there currently are over 30 proteins known to play a role in α-tocopherol absorption, transport, and metabolism and these genes can be considered additional candidates for NAD/EDM. Of interest, polymorphisms in many of these α-tocopherol-related genes have been associated with variable protective effects of supplemental vitamin E in humans.
In conclusion, NAD/EDM appears to be inherited as a complex trait and demonstrates an estimated heritability of 0.70 in a high-risk environment (ie, low dietary vitamin E). Variants in TTPA are not causative for NAD/EDM in this family of QHs. A genome-wide association study is necessary to provide insight into the genetic cause of NAD/EDM in QHs and additional breeds.