COMMD1 Exemplifies the Power of Inbred Dogs to Dissect Genetic Causes of Rare Copper-Related Disorders

Wilson’s Disease is a rare autosomal recessive disorder in humans, often presenting with hepatic copper overload. Finding the genetic cause of a rare disease, especially if it is related to food constituents like the trace element copper, is a Herculean task. This review describes examples of how the unique population structure of in-bred dog strains led to the discovery of a novel gene and two modifier genes involved in inherited copper toxicosis. COMMD1, after the discovery in 2002, was shown to be a highly promiscuous protein involved in copper transport, protein trafficking/degradation, regulation of virus replication, and inflammation. Mutations in the ATP7A and ATP7B proteins in Labrador retrievers and Dobermann dogs resulted in a wide variation in hepatic copper levels in these breeds. To our knowledge, numerous dog breeds with inherited copper toxicosis of unknown genetic origin exist. Therefore, the possibility that men’s best friend will provide new leads in rare copper storage diseases seems realistic.

Investigation of Genetic Modifiers of Copper Toxicosis in Labrador Retrievers

Copper toxicosis is a complex genetic disorder in Labrador retrievers characterized by hepatic copper accumulation eventually leading to liver cirrhosis. The variation of hepatic copper levels in Labrador retrievers has been partly explained by mutations in ATP7A c.980C>T and ATP7B c.4358G>A. To further elucidate the genetic background of this disease, we used targeted Next Generation Sequencing (NGS) in a cohort of 95 Labrador retrievers to analyze 72 potential modifier genes for variations associated with hepatic copper levels. Variants associated with copper levels were subsequently evaluated in a replication cohort of 144 Labrador retrievers. A total of 44 variants in 25 different genes were identified, of which four showed significant association with copper levels. Of the four variants found associated with hepatic copper levels in the NGS cohort, one was validated in the replication cohort. The non-reference allele of the variant NC_006602.3.g.52434480C>T in RETN resulting in amino-acid change p.Leu7Phe was associated with decreased hepatic copper levels. In humans, resistin is associated with severity of non-alcoholic fatty liver disease, fibrosis, cirrhosis and mitochondrial dysfunction in hepatocytes. Further studies are needed to investigate the biological function of RETN p.Leu7Phe in the development of copper toxicosis in Labrador retrievers.

Chronic copper toxicosis in a crossbred heifer calf

An 8-mo-old, crossbred, heifer calf was presented to the Heeke Animal Disease Diagnostic Laboratory with a history of ataxia and altered mentation. Grossly, the liver was diffusely yellow-orange, turgid, and exuded watery, thin blood on cut section. The cortex and medulla in both kidneys were diffusely and markedly dark brown to black. The urinary bladder was filled with dark red urine. Histologically, centrilobular hepatocellular degeneration was observed, but these sections lacked necrosis. In the kidney, numerous cortical tubules contained intraluminal bright eosinophilic fluid and red-orange granular casts that stained positive for hemoglobin with the Dunn–Thompson method. The gross and histologic lesions supported a high level of suspicion for copper toxicosis. Feed and water samples from the farm were submitted for mineral analysis. The copper concentration in the feed was 118 mg/kg, and the molybdenum concentration was 0.9 mg/kg. Chronic copper toxicosis is rarely reported in cattle. The gross lesions in our case are a departure from, although similar to, previously reported cases, including lack of histologic hepatocellular necrosis. Collectively, gross and histologic lesions were compatible with copper toxicosis in this calf, and copper concentrations in the feed samples suggest a feed-mixing error.

Towards Bioengineered Liver Stem Cell Transplantation Studies in a Preclinical Dog Model for Inherited Copper Toxicosis

Wilson Disease is a rare autosomal recessive liver disorder in humans. Although its clinical presentation and age of onset are highly variable, hallmarks include signs of liver disease, neurological features and so-called Kayser-Fleischer rings in the eyes of the patient. Hepatic copper accumulation leads to liver disease and eventually to liver cirrhosis. Treatment options include life-long copper chelation therapy and/or decrease in copper intake. Eventually liver transplantations are indicated. Although clinical outcome of liver transplantations is favorable, the lack of suitable donor livers hampers large numbers of transplantations. As an alternative, cell therapies with hepatocytes or liver stem cells are currently under investigation. Stem cell biology in relation to pets is in its infancy. Due to the specific population structure of dogs, canine copper toxicosis is frequently encountered in various dog breeds. Since the histology and clinical presentation resemble Wilson Disease, we combined genetics, gene-editing, and matrices-based stem cell cultures to develop a translational preclinical transplantation model for inherited copper toxicosis in dogs. Here we describe the roadmap followed, starting from the discovery of a causative copper toxicosis mutation in a specific dog breed and culminating in transplantation of genetically-engineered autologous liver stem cells.