We have demonstrated in this study that a homozygous mutation in the SLC19A3.1 gene encoding a thiamine transport protein is strongly associated with AHE. Each of the 11 dogs with AHE had a homozygous mutation of SLC19A3.1, and 15/41 clinically normal AH dogs had a heterozygous mutation while the other 26 had wild type homozygosity. No such mutation was found in any of the 187 non-AH dog breeds. Although AHE has been claimed to be a primary mitochondrial encephalopathy based on clinical and pathological similarities to LS, no mutations were found in any of the candidate canine nuclear or mitochondrial genes linked to human LS. In dogs with AHE, biochemical analysis failed to demonstrate defects in the mitochondrial respiratory chain enzyme activities in liver, skeletal muscle or cultured fibroblasts, considered characteristic of LS. Together, these findings strongly suggest that the pathogenesis of AHE involves a primary defect in a thiamine transporter protein in the CNS and is not due to a primary mitochondrial encephalopathy. However, the AHE phenotype is consistent with a secondary mitochondrial disease.
Solute carrier family 19 (SLC19
) is a group of genes that transport water-soluble vitamins into cells. 
There are 3 members in the family, SLC19A1, SLC19A2
. The SLC19A1
gene encodes folate transporter 1 protein, regulating intracellular levels of folate, as well as mediating methotrexate transport. The SLC19A2
gene encodes the human thiamine transporter 1 (hTHTR1) protein whereas human thiamine transporter 2 (hTHTR2) protein is encoded by the SLC19A3
are structurally and functionally similar and thiamin (vitamin B1) is transported across the cell membrane by both thiamine transporters. All SLC19
family genes are expressed ubiquitously, although at variable levels in different tissues 
. The dog is unusual in that it has two SLC19A3
paralogs, compared with only one each in mice and people 
. In the dog, expression of SLC19A3
is also ubiquitously distributed, but the relative tissue expression varies depending on the specific paralog. In this study, we demonstrated that the CNS has high tissue expression of the SLC19A3.1
compared with SLC19A3.2
expressed at low levels in the CNS. This finding suggests that SLC19A3.2
in the CNS cannot compensate for a functional loss of thiamine transport caused by the defective SLC19A3.1
. In people, SLC19A3
is expressed at high levels in kidney, liver and placenta, with low levels in other tissues, including the brain 
. The highest level of SLC19A3
RNA expression in the human brain is in the thalamus 
. If the same holds true in the dog, the need for relatively high SLC19A3
RNA expression in the thalamus could explain the location of the most severe lesions in dogs with AHE 
Thiamine is an essential nutrient and thiamine deficiency (TD) may cause bilaterally symmetrical brain lesions in domestic animals and people 
. Thiamine, a B-complex, water-soluble essential vitamin, has a fundamental role in carbohydrate metabolism in all cells and a critical role in mitochondrial metabolism 
. Because of the critical dependence of the CNS on mitochondria for energy metabolism, TD can lead rapidly to severe neurological deficits. In dogs, the classical histological lesion of TD (both experimental and spontaneous) is bilaterally symmetrical polioencephalomalacia, confined to the brain stem nuclei, periventricular grey matter, claustrum, lateral geniculate nuclei, caudal colliculi, occipital and parietal cortex, and cerebellar nodulus. Lesions in the caudal colliculus are the hallmark of dietary thiamine deficiency encephalopathy in dogs. Dogs with experimental TD also have cardiac lesions 
. The distribution of these lesions in the dog differs from those in people, in whom the mammillary bodies are mainly affected, and involvement of the cerebral cortex is more pronounced.
While dogs with TD and AHE have the same histological lesions of bilaterally symmetrical encephalomalacia, anatomic patterns of lesion distribution are distinctively different. Dogs with AHE do not have lesions in the caudal colliculus or lateral geniculate nucleus, and dogs with TD do not have thalamic lesions 
. In people with spontaneous TD (Wernicke's encephalopathy) who are still responsive to thiamine administration, the lesions are mainly confined to the mammillary bodies. While it is unlikely that the pathogenesis of AHE is due to an absolute global TD, these findings may be consistent with a localized TD, resulting from the functional mutations apparently restricted to the tissue distribution of the SLC19A3.1
There is a phenotypic spectrum of diseases in people associated with abnormalities in different regions of the SLC19A3
gene, including biotin-responsive basal ganglia disease (BBGD) 
, Wernicke's-like encephalopathy (WLE)
, and an encephalopathy described in 4 related Japanese boys 
. People with BBGD have vague clinical signs including confusion and vomiting, progressive loss of motor function, dysarthria, dysphagia, cogwheel rigidity, and seizures, which are ultimately fatal if untreated. The major features on brain MRI are areas of bilaterally symmetrical polioencephalomalacia of the caudate and putamen nuclei 
. Two homozygous missense mutations (c.68G>T; p.G23V; and c.1264A>G, p.T422A) in SLC19A3
have been identified. In patients with either mutation, no clinical response is noted following thiamine administration. However, if treated with biotin early in the course of disease, the clinical signs may be reversed 
. In two adult siblings with BBGD, two heterozygous mutations in SLC19A3
(c.74dupT/p.Ser26LeufsX19 and c.980-14 A>G) led to a premature termination codon (PTC), and a loss of function mutation of SLC19A3
. One patient responded to biotin, but the other did not improve with biotin administration until thiamine was concurrently supplemented.
However, since hTHTR2 does not transport biotin, both the pathophysiology of BBGD, and the response to therapy with biotin imply that a more complex mechanism than simple thiamine deficiency causes these lesions 
. For example, perhaps the truncated form of the transporter is still able to transport biotin albeit not to the same level as the wild type form, or perhaps since biotin is an important cofactor of many carboxylases in mitochondria, biotin supplementation may exert a compensatory effect.
WLE was reported in 2 Japanese brothers in their second decade of life. Clinical signs included diplopia, external ophthalmoplegia, ptosis, seizures, nystagmus and ataxia. On brain MRI there were hyperintensities on the FLAIR (fluid attenuated inversion recovery) sequences bilaterally in the thalamus and periaqueductal region, which are entirely consistent with actual Wernicke's encephalopathy (thiamine deficiency encephalopathy). A compound heterozygous mutation of p.K44E and p.E320Q was found in SLC19A3
, resulting in approximately 60% thiamine intracellular uptake in Chinese hamster cells transfected with constructs containing either p.K44E or p.E320Q cDNA compared to the wild-type. Neither patient had serum thiamine deficiency, but both patients responded clinically to thiamine 
In four related Japanese boys with infantile seizures, psychomotor retardation, and characteristic lesions on brain MRI (focal T2W hyperintensity in bilateral symmetrical thalamic and basal nuclei, with cerebellar and cerebral cortical atrophy), a homozygous mutation was found (c.958G>C, p.E320Q) in SLC19A3.
There was no change in either neurological symptoms or brain MRI lesions in response to biotin administration in the one boy who was treated 
In BBGD, WLE, and in the Japanese boy encephalopathy, there is a distinct genotype-phenotype correlation. Patients affected by each disease have a remarkably similar phenotype. While the BBGD phenotype is thought to be secondary to a loss-of-function mutation, the Japanese boy encephalopathy may be due to a toxic gain-of-function secondary to the SLC19A3
. Further investigations are needed to characterize the function effects of these SLC19A3
mutations and the associated genotypic-phenotypic correlation.
Dogs with AHE did not have any biochemical evidence of primary mitochondrial disease in liver, skeletal muscle or fibroblasts, but did have subtle evidence of mitochondrial pathology. By light microscopy, there was prominent mitochondrial hyperplasia in muscle and liver and ultrastructural examination of muscle revealed “megamitochondria” and abnormal glycogen deposits. However such abnormal mitochondrial morphology is not necessarily evidence of a primary mitochondrial disease. The observed mitochondrial changes could be epiphenomenal, reflective of a response to a defect in energy metabolic pathways as expected with a defective SLC19A3 gene, especially in an active racing breed.
In conclusion, we have demonstrated that a homozygous mutation in SLC19A3.1, a gene encoding a thiamine transport protein in the brain, is strongly associated with AHE. The mutation is homozygous in dogs with AHE while unaffected AH are either homozygous wild type or heterozygous for the mutation. While our findings are consistent with the mutation identified being widespread in AH from North America, we do not know to what extent this or similar mutations might be causal to AHE worldwide. We are hopeful that the findings from this study will help in future studies to test for this mutation in AH from Europe and other regions of the world. While the pathologic findings in AHE are not consistent with classical experimental or spontaneous global TD, they may be consistent with localized TD or may suggest more complex pathways involved with thiamine metabolism in the CNS. This large animal model may allow for prospective investigations into the mechanisms of SLC19A3 related syndromes and the potential role of thiamine and/or biotin as a therapeutic strategy.