Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy

et al. (2013) Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy. PLoS ONE 8(3): e57195. doi:10.1371/journal.pone.0057195 Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy Karen M. Vernau 0 Jonathan A. Runstadler 0 Emily A. Brown 0 Jessie M. Cameron 0 Heather J. Huson 0 Robert J. Higgins 0 Cameron Ackerley 0 Beverly K. Sturges 0 Peter J. Dickinson 0 Birgit Puschner 0 Cecilia Giulivi 0 G. Diane Shelton 0 Brian H. Robinson 0 Salvatore DiMauro 0 Andrew W. Bollen 0 Danika L. Bannasch 0 Gerard Roel Rutteman, Utrecht University, The Netherlands 0 1 University of California Davis, Davis, California, United States of America, 2 University of Alaska Fairbanks, Fairbanks, Alaska, United States of America, 3 Research Institute, The Hospital for Sick Children , Toronto, Ontario , Canada , 4 Department of Pathology, University of California San Diego, La Jolla, California, United States of America, 5 Department of Neurology, Columbia University Medical Center , New York , New York, United States of America, 6 Department of Pathology, School of Medicine, University of California San Francisco , San Francisco, California , United States of America Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 - 43.36-43.38 Mb and SLC19A3.1 - 43.411-43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C.A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE. - Funding: This study was supported by a grant from The Center for Companion Animal Health, from the School of Veterinary Medicine, UC Davis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Alaskan Husky Encephalopathy (AHE) is a fatal brain disease in young Alaskan Husky (AH) dogs, often affecting multiple dogs from the same litter [1,2]. AHE was initially described in 13 dogs from the northern United States, including Alaska, Massachusetts, New York, Wyoming, Maine and Minnesota [1]. Based on the clinical and neuropathological similarities to Leigh Syndrome (LS) in people, AHE was proposed to be a mitochondrial encephalopathy. Human LS includes a group of diseases with heterogeneous clinical symptoms, usually characterized by elevations in blood lactate and respiratory chain enzyme dysfunction(s), and due to various mutations in either nuclear or mitochondrial DNA. An almost identical clinicopathological disease to AHE is described in 11 European Yorkshire terriers without a defined underlying cause [3]. Dogs with AHE may have acute onset of clinical signs, or chronic progressive waxing and waning clinical history. Typically, they have multifocal central nervous system deficits including seizures, altered mentation, dysphagia, absent menace response, central blindness, hypermetria, proprioceptive positioning deficits, facial hypoalgesia, ataxia and tetraparesis. Diagnostic testing reported in dogs with AHE was limited to normal serum and cerebrospinal fluid pyruvate and lactate concentrations; evaluation of mitochondrial respiratory chain enzymes was not done. Two dogs also had intracranial imaging. Computed tomography images in one dog had bilateral hypoattenuating lesions in the thalamus, and MR images of the other dog had bilateral hyperintense lesions in the brainstem on T2 weighted images. All dogs died or were euthanized, most within 27 months, however one dog lived for 1 year after the onset of clinical signs when it died of natural causes [1,2]. In people and domestic animals, a multitude of uncommon diseases are associated with bilateral and symmetrically distributed brain lesions, apparent on magnetic resonance imaging and/or neuropathology. Examples include toxicities (carbon monoxide poisoning), metabolic (Leigh Syndrome [4], hypoxic-ischemic encephalopathy, hypoglycemia, biotin responsive basal ganglia disease [5], Wernickes-like encephalopathy [6]), inflammatory (Neuro Behcet disease), nutritional (thiamine deficiency encephalopathy [7]), vascular (deep cerebral vein thrombosis), infectious (bovine spongiform encephalopathy, Creutzfeld-Jakob disease), and inborn errors of metabolism (L-2 hydoxyglutamic aciduria [8], citrullinemia). A systematic diagnostic approach to this group of disorders is essential to ensure that a specific diagnosis can be rendered in a timely manner. In people, mutations in the SLC19A3 gene (encoding a thiamine transporter protein) are associated with encephalopathies that have some clinical and neuropathologic similarities to AHE [9]. In this study, 11 dogs with neuropathologically and/or neuroradiologically confirmed AHE were evaluated for 1) mitochondrial respiratory chain enzyme functions; 2) light and electron microscopical pathological studies on skeletal muscle and liver, and 3) a genome wide association study utilizing the Illumina canine HD array. Together, our findings suggest that the pathogenesis of AHE results from a genetic defect in a thiamine transporter (SLC19A3), and is not a primary mitochondrial encephalopathy. Clinical Findings and Outcome in dogs with AHE All 11 dogs with AHE included in this study showed typical clinical signs (Table 1), as well as MR imaging (Figure 1) and/or neuropathological features of AHE in the brain (Figure 2). Of these 11 dogs, 6 were euthanized either immediately after their clinical evaluation (dogs # 1, 3, 6, 8, 9, 11), or six months ( (...truncated)