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John C. Fyfe, D.V.M, Ph.D.

Research

Genetic diseases are nature's "knockout" experiments, occurring spontaneously in all species. The purpose of the Laboratory of Comparative Medical Genetics is to define inherited disorders that occur in companion animals. Investigation of these disorders provides novel insight into normal physiologic functions and the benefits of improved control of the disease in animal populations. Well-characterized disorders also serve as models of human genetic disease. Those animals that are determined to exhibit a true orthologue of human disease are a valuable resource for developing understanding of underlying mechanisms of disease and innovative treatment modalities, including gene therapy. In other instances the investigation reveals a previously unrecognized disease gene and new aspects of cell biology. Characterization of these disorders requires an integrated, multidisciplinary approach that may begin in the clinic but subsequently involves laboratory techniques of biochemistry, cell biology, and molecular genetics. The rapidly improving canine and feline genome maps and recent sequencing of these genomes are providing exciting new prospects for these investigations.

Several disorders are currently under investigation. Spinal muscular atrophy (SMA) is the most common recessive disorder lethal to infants and the second most common pediatric neuromuscular disorder. SMA is a lower motor neuron disorder which results in denervation atrophy of skeletal muscle. In humans, most cases are due to mutations of the survival of motor neuron gene ( SMN1 ), but the disorder has wide phenotypic variability and is genetically heterogeneous. We recently investigated a form of juvenile-onset SMA that occurs as an autosomal recessive trait in Maine coon cats. Published results (He Q et al., 2005) indicate that the SMN locus is not altered in the affected cats. Rather, a whole genome scan for linkage revealed a large deletion that abrogates expression of two genes (Fyfe et al ., 2006), one of which is preferentially expressed in postnatal lower motor neurons but which is currently a gene of unknown structure or function. Further investigation of the molecular basis of SMA in this cat model is ongoing. Additionally, discovery of the disease-causing mutation has allowed us to create a carrier test for SMA in Maine coon cats that is now available through our laboratory.

Keeping with the theme of neuromuscular disease, we are also investigating an autosomal recessive neurodevelopmental abnormality in a family of dogs that affects function of motor neurons throughout the central nervous system. Affected fetuses become immobile late in gestation and are born with global contracture of skeletal muscle. In addition there is hypoplasia of the pontocerebellar regions of the brain; thus the disorder resembles pontocerebellar hypoplasia type I of humans. The molecular basis of this disorder remains to be discovered in any species, and a whole genome scan for linkage to the canine disorder is under way.

Glycogen is the storage form of readily available glucose in animals. The glycogen storage disorders (GSD) are metabolic disorders that typically result in tissue accumulation of excessive or abnormally structured glycogen. These often cause liver and muscle disease and may or may not cause neuronal degeneration, hypoglycemia, and/or heart failure. GSD type IV is an autosomal recessive disorder of Norwegian forest cats previously defined in the United States (Fyfe JC et al ., 1992) that causes degeneration of neurons and cardiac and skeletal muscle with death ensuing before a year of age. Hypoglycemia in the immediate postnatal period often causes weakness and death unless treated. The disorder is due to a mutation of the glycogen branching enzyme gene (Fyfe JC et al ., 2007) and storage of abnormally structured glycogen (long outer chains).

In contrast GSD type IIIa (GSDIIIa) is an autosomal recessive disorder that causes liver and skeletal muscle disease due to deficiency of the glycogen debranching enzyme (GDE) and tissue storage of abnormally structured glycogen (short outer chains, a.k.a. limit dextrin). This disorder was recently discovered in an extended family of curly coated retrievers (CCR), with carriers found in USA, Canada, Finland, Australia, and New Zealand (Gregory BL et al . 2007), and is due to a single based deletion in the GDE gene. Affected dogs experience episodic hypoglycemia, and liver and muscle disease evidenced by abnormal leakage of liver and muscle enzymes into plasma. The laboratory also provides carrier testing for GSD IIIa in curly coated retrievers .

Hypothyroidism in the neonatal period results in dwarfism and severe mental retardation. Therefore, it is imperative that congenital hypothyroidism (CH) be diagnosed and treatment instituted early in life. Inherited CH may be due to defects of the hypothalamus, pituitary, or thyroid glands. We are presently investigating two forms of CH in dogs, both of which are recessive traits resulting in growth and behavioral abnormalities. One is a signal transduction defect of thyrotrophs, the pituitary cells responsible for production of thyroid stimulating hormone. Investigation has ruled out involvement of all loci known to cause a similar disorder in humans or mice, and a genome scan for linkage is under way. The second is severe CH with goiter (CHG) apparent by a week of life due to thyroid follicular cell failure to utilize iodide in production of thyroid hormones. Biochemical analysis suggested involvement of the thyroid peroxidase gene, a well-described origin of CH in humans, and we found a nonsense mutation in this gene (Fyfe JC et al ., 2003) that is the basis of a CHG carrier test available to Toy Fox Terrier breeders . Subsequently, the same mutation was discovered to cause CHG in rat terriers (Pettigrew R et al ., 2007), so the same carrier test is applicable to both breeds.

Selective intestinal cobalamin (vitamin B12) malabsorption with proteinuria in dogs is an autosomal recessive defect in the expression of the intrinsic factor-cobalamin receptor in intestine and the same complex that functions as a multiligand receptor in kidney tubules. The functional receptor complex is made of 2 proteins, cubilin and amnionless, and the complex is referred to as cubam Fyfe JC et al ., 2004). To date the canine disorder is the only known spontaneously occurring animal model of a vitamin malabsorption as well as of the human disorder called Imerslund-Gräsbeck syndrome. Analysis initiated by whole genome scan for linkage led to the discovery that affected dogs have mutations in amnionless ( AMN ), a gene expressed in polarized epithelia but of previously unknown function. AMN mutations causing the disorder have been determined in both giant schnauzer and Australian shepherd kindreds (He Q, et al ., 2005). We are currently investigating the disorder in other dog breeds, including Hungarian komondor, beagle and border collie.

In each of the disorders described above, definition of the molecular basis of the disorder is a primary objective, not only for the expected insights into pathophysiology and the development of valuable animal models, but also so that molecular tests may be developed to determine which animals in a breeding population are carriers of these recessive disorders. In our opinion, prevention through carrier detection is the only real solution to the problem of genetic disease in companion animal populations.