Hereditary & Genetic Diseases Modern Diagnostics for Hereditary Disorders in Dogs and Cats
Urs Giger, PD, Dr. med. vet., MS, FVH, DACVIM, DECVIM
University of Pennsylvania
Philadelphia, PA, USA

Because of the increased awareness of breeders, pet owners, and veterinarians of genetic defects and the improved diagnostic abilities in clinical practice, the number of reported hereditary diseases in small animals is rapidly growing. At present, approximately 430 hereditary diseases in dogs and 180 disorders in cats have been adequately documented, and every year over a dozen new defects are being reported. Although any genetic defect may occur in any animal, many have been documented only in one family or breed. For the small animal practitioner, it can be a daunting, nearly impossible task to remember all these diseases.

Genetic diseases are caused by chromosomal alterations or gene mutations. Disease-causing mutations are heritable changes in the sequence of genomic DNA that alter the expression, structure, and function of the coded protein. The genotype refers to the animal's genetic makeup, reflected by its DNA sequence, whereas the phenotype relates to the clinical manifestation of specific gene(s) and environment, or both. The molecular genetic defect is now known for more than 40 hereditary disorders in small animals (Table). These molecular genetic changes include point mutations, deletions, and insertions in the DNA sequence that result in a missense or nonsense sequence with an altered codon sequence. For approximately half of the disorders suspected to be of a genetic nature, however, the mode of inheritance remains unknown. The dog has 76 autosomes (38 pairs) and 2 sex chromosomes (78XX or 78XY), whereas the cat has 38XX or 38XY. The human genome project has also allowed major progress in canine and feline gene mapping. Through physical and genetic mapping strategies, genes can now be assigned to and localized along a chromosome, and new genes can be identified. During 2003 the entire sequence of the canine genome (Boxer) will be sequenced which will greatly facilitate the characterization of molecular bases of hereditary diseases in dogs. The pattern of inheritance depends mainly on two factors: 1) whether the mutation is located on an autosome (autosomal) or on the X chromosome (X-linked), and 2) whether the phenotype, the observable expression of a genotype as a disease trait, is dominant, i.e., expressed when only one chromosome of a pair carries the mutation, or recessive, i.e., expressed when both chromosomes of a pair carry the mutation. Thus, it is the phenotype rather than the mutant gene or protein that is dominant or recessive. Whereas in humans most diseases are dominantly inherited, recessive traits are favored by the common inbreeding practices in small animals.

Most genetic defects cause clinical signs early in life. The term congenital only implies that the disease is present at birth, however, and does not necessarily mean it is hereditary. A common presentation is failure to thrive. They are poor doers, often fade (hence the term fading puppy or kitten syndrome), and finally die. Failure-to-thrive should not be confused with growth retardation. In addition to these relatively unspecific clinical signs, some defects may cause specific clinical manifestations. Easy to recognize are malformations that involve any part of the skeleton and lead to disproportionate dwarfism, gait abnormalities, and/or facial dysmorphia. A large number of hereditary eye diseases have been described in dogs, some of which are not recognized until adulthood. Neuromuscular signs may vary from exercise intolerance to ataxia and seizures. Defects of many other internal organs are associated with unspecific clinical signs.

Diagnostic tests generally are required to further support a genetic disorder in a diseased animal. Radiology and other imaging techniques may reveal skeletal malformations or cardiac anomalies, and ophthalmologic examination may further define an inherited eye disease, although some are not recognized before several years of age. Routine tests such as complete blood cell count, chemistry screen, and urinalysis may suggest some specific hematologic or metabolic disorders or rule out many acquired disorders. Furthermore, clinical function studies may more clearly define a gastrointestinal, liver, kidney, or endocrine problem. Histopathology and/or electron microscopy of a tissue biopsy from an affected animal or from the necropsy of a littermate or relative may give the first clue as to a genetic defect.

A few laboratories provide special diagnostic tests that allow a specific diagnosis of an inborn error of metabolism. Inborn errors of metabolism include all biochemical disorders due to a genetically determined, specific defect in the structure and/or function of a protein molecule. Aside from the classical enzyme deficiencies, genetic defects in structural protein receptors, plasma and membrane transport proteins, and other proteins covered by this definition will result in biochemical disturbances. The laboratories' approach is to detect the failing system or to determine the specific protein or gene defect. Disorders of intermediary metabolism typically produce a metabolic block in a biochemical pathway leading to product deficiency, accumulation of substrates, and production of substances via alternative pathways. The most useful specimen to detect biochemical derangements is urine because abnormal metabolites in the blood will be filtered through the glomeruli, but fail to be reabsorbed, as no specific renal transport system exist for most abnormal metabolites.

Once the failing system has been identified, the defect can be determined at the protein level. These protein assays include the classic enzyme function tests as well as immunologic assays. Because most enzymes are present in abundant amounts, no major functional abnormalities are generally observed unless the enzyme activity is severely reduced, usually to less than 20 percent of normal value. Thus, homozygously affected animals have very low protein activity and/or quantities, often in the range of 0 to 5 percent. These tests may also be used to detect carriers (heterozygotes), who typically have intermediate quantities at the protein level (30-70 percent), but no clinical signs. Unfortunately, protein assays require submission of appropriate tissue or fluid under special conditions to specialized laboratories along with a control sample, and are labor intensive. The Section of Medical Genetics at the School of Veterinary Medicine of the University of Pennsylvania is one of the few places that performs such tests to diagnose known as well as to discover novel hereditary disorders www.vet.upenn.edu/penngen .

The molecular defect has been identified for over 3 dozen hereditary diseases in companion animals, and thus DNA screening tests have been developed. These tests are mutation specific and can therefore only be used in animals suspected to have the exact same gene defect. Small animals within the same or a closely related breed will likely have the same disease-causing mutation for a particular disease. However, dogs and cats as well as unrelated breeds of a species with the same disorder will likely have different mutations.

DNA tests have several advantages over other biochemical tests. The test results are independent of the age of the animals, thus, the tests can be performed at birth or at least long before an animal is placed in a new home as well as before clinical signs become apparent. DNA is very stable and only the smallest quantities are needed; hence, there are no special shipping requirements as long as one follows the specific instructions for biological products. DNA can be extracted from any nucleated cell, e.g., blood, buccal mucosa (cheek swabs), hair follicle, semen, and even formalinized tissue. For instance, blood can be sent in an EDTA tube or a drop of blood can be applied to a special filter paper. Buccal swabs can be obtained with a special cytobrushes, although this method should not be used in nursing animals, or if absolutely necessary, only after flushing the oral cavity. The DNA segment of interest is amplified with appropriate primers and polymerase chain reaction (PCR). The mutant and/or normal allele are identified by DNA size difference directly on a gel in case of deletions or insertions or after restriction enzyme digestion for point mutations. These tests are generally simple, robust, and accurate as long as appropriate techniques and controls are used. Furthermore, they can be used not only for the detection of affected animals but also for carriers and thus are extremely valuable to select breeding animals that will not cause disease or further spread the disease causing allele. For instance, phosphofructokinase deficiency was recognized to cause intermittent anemia and myopathy in English Springer spaniels and a DNA based test has become available in the early 1990s, there were still 4% and 1% carriers in the field trial and conformation lines, respectively, in the first randomized survey performed in 1998. If an animal with all the desirable qualities is found to be a carrier, it could be bred to a clear animal (homozygous normal), as this would not result in any affected and as long as all offspring would be tested and only clear animals were going to be used in the next generation.

For many inherited disorders, the defective gene remains unknown; however, for a few a polymorphic DNA marker that is linked to the mutant allele has been discovered. Such linkage tests were first developed for copper toxicosis in Bedlington terriers and are now available for some forms of retinopathy and renal carcinoma and nodular dermatitis in German Shepherds, and are accurate for a particular patient as long as there is a known affected animal in its family (informative family). At present, mutation-specific and linkage tests are available only for single gene defects in small animals; however, complex genetic traits may also soon be approached by these methods as they are for humans.

In order to reduce the frequency or eliminate altogether a genetic defect, the further spread of the mutant gene has to be prevented in a family or entire breed. It is obvious that affected animals of any genetic disease should not be used for breeding. This approach is simple and effectively eliminates disorders with a dominant trait. For recessively inherited disorders, however, the elimination of affected animals is not sufficient to markedly reduce the prevalence of a defect within a breed or kennel/cattery. Although it may be safest not to breed any related animals of affected animals, as requested by some kennel clubs, this practice may, because of inbreeding and narrow gene pools in some breeds, eliminate all breeders in an entire kennel or cattery, and may severely reduce the genetic diversity of a breed. Thus, it will be pivotal to detect carriers (heterozygotes) and truly "clear" animals (homozygous normal). Obligate carriers can be readily identified for autosomal (both parents of affected) and X chromosomal recessive (mother of affected) disorders. As mentioned above, for some diseases, reliable carrier detection tests are available and many breeders know about them and inform the veterinarian. For instance, carriers have halfnormal (~50%) enzyme activity by functional assays, or have a normal and mutant DNA sequence for the diseased gene on a DNA test. Breeders should, therefore, be encouraged to screen their animals before breeding for know genetic diseases whenever carrier tests are available. Their availability is also listed on several web sites including www.vet.upenn.edu/penngen. Unfortunately, many breeders mistrust these newer tests; either they were disappointed by the inaccuracy of early tests, such as the radiographic examination for hip dysplasia, or they fear that the results may become public and could hurt their business. Thus, breeders need to be educated by well-informed veterinarians. If a carrier needs to be used because of a narrow gene pool and many other desirable traits, it should be bred with a homozygously normal (clear) animal; all its offspring need to be tested, and only clear animals should be used in future breedings. If no carrier tests are available, a test mating between the dog in question and a known carrier or affected could be performed, and no affected and at least 5 and 11 healthy puppies/kittens, respectively, need to be born to "clear" an animal of a carrier state. For many breeders, this approach is ethically unacceptable because it may produce affecteds.

Examples of Hereditary Disorders Characterized At the Molecular Genetic Level in Dogs and Cats

Disorder

Breed

Hematologic disorders

Elliptocytosis (band 4.1)

Mixed breed

Pyruvate kinase (PK) deficiency

Basenji, West Highland white terrier, Dachshund, Abyssinian, Somali, DSH cat

Phosphofructokinase deficiency (PFK)

English Springer and cocker spaniel, mixed breed dog

Hemophilia A (Factor VIII)

Mixed breed

Hemophilia B (Factor IX)

Cairn terrier, Labrador retriever, mixed breed

von Willebrand disease (vWD)Type 1

Doberman, Manchester and Cairn terrier, Pembroke Welsh Corgi

von Willebrand disease (vWD) Type 2

German shorthair & wirehair pointer

von Willebrand disease (vWD)Type 3

Dutch Kookier, Scottish terrier, Sheltie

Severe x-linked combined immunodeficiency (SCID)

Basset, Cardigan Welsh Corgi

Leukocyte adhesion deficiency (CLAD)

Irish setter, Red & White setter

Complement component 3 deficiency

Brittany spaniel

Hereditary eye diseases

Progressive retinal atrophy

Irish setter (ß-phosphodiesterase)

Rod cone dysplasia

Cardigan Welsh Corgi, Chesapeake Bay & Labrador retriever, English cocker spaniel, Portuguese Waterdog

Stationary night blindness

Briard

Neuromuscular diseases

Shaking puppy syndrome

English Springer spaniel

Dystrophin muscular dystrophy

Golden retriever, Rottweiler, DSH cat

Mucopolysaccharidosis I

Plott hound

Mucopolysaccharidosis IIIA

Wirehaired Dachshund, New Zealand Huntaway dog

Mucopolysaccharidosis IIIB

Schipperke

Mucopolysaccharidosis VI

Siamese cat (two mutations), Miniature pinscher

Mucopolysaccharidosis VII

German shepherd, mixed breed dog, DSH cat

Alpha mannosidosis

Persian, DSH cat

Gangliosidosis GM1

Siamese, Korat cat

Gangliosidosis GM2

Korat cat

Globoid cell leukodystrophy (Krabbe)

West Highland white and Cairn terrier

Glycogenosis type IV

Norwegian Forest cat

Alpha fucosidosis

English Springer spaniel

Neuronal ceroid lipofuscinosis

English setter

Myotonia congenita

Miniature schnauzer

Narcolepsy

Doberman, Labrador retriever

Ivermectin toxicity (MDR-1 gene)

Collie, Sheltie, Australian kettle dog

Malignant hyperthermia

Hepatic diseases

Hyperchylomicronemia

DSH cat

Glycogenosis type Ia

Maltese

Copper toxicosis

Bedlington terrier

Renal diseases

Cystinuria type I

Newfoundland, Labrador retriever

Renal adenocarcinoma and nodular dermatitis

German Shepherd (linkage test)

x-linked nephropathy

Samoyed

Speaker Information
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Urs Giger, PD, Dr. med. vet., MS, FVH, DACVIM, DECVIM
University of Pennsylvania
Philadelphia, PA, USA


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