Proceedings of the 33rd World Small Animal Veterinary Congress
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1 Proceedings of the 33rd World Small Animal Veterinary Congress Dublin, Ireland Next WSAVA Congress : Reprinted in IVIS with the permission of the Congress Organizers
2 15 Hereditary Gen e t i c d i s e a s e t e s t i n g m a d e c l i n i c a l ly p r a c t i c a l Urs Giger Dr.med.vet., MS, FVH, Dipl.ACVIM, ECVIM-CA & ECVCP, School of Veterinary Medicine, University of Pennsylvania, 3900, Delancey Street, 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, >900 hereditary diseases in dogs and >200 disorders in cats have been adequately documented, and every year over a dozen new defects are being reported. For the small animal practitioner, it can be a daunting, nearly impossible task to remember all these diseases and approach them appropriately in clinical practice. Beyond physical examination and imaging tools, genetic, metabolic, and other specific laboratory techniques are used to diagnose hereditary disorders in companion animals. Practical aspects of screening for hereditary diseases from sample collection to genetic test principles and interpreting results will be reviewed and illustrated by case presentations. Associated topics on advances in canine and feline hereditary disorders as well as genetic control of hereditary diseases for the clinician will be discussed in adjacent sessions. 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 inherited. A common presentation is failure to thrive. They are poor doers, often fade (hence the term fading puppy or fading kitten syndrome ), and finally die (puppy and kitten mortality complex). Failure-tothrive should not be confused with growth retardation which may be proportionate (dwarf) or disproportionate (chondrodysplastic). In addition to these relatively vague and nonspecific clinical signs, some defects may cause specific clinical manifestations. Easy to recognize are malformations that involve any part of the skeleton and can lead to disproportionate dwarfism, limb and 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 nonspecific clinical signs and often require intense work ups unless one is aware of a set of signs being responsible for a syndrome or a particular predilection for a disease in a breed. Many acquired diseases may mimic the clinical signs of a genetic defect. Diagnostic tests Diagnostic tests are generally required to further support a genetic disorder in a diseased animal. Radiology and other imaging techniques may reveal skeletal malformations or cardiac anomalies, and an ophthalmologic examination may further define an inherited eye disease, although some are not recognized until several years of age. Routine tests such as complete blood cell count, chemistry screen, and urinalysis may suggest some specific hematological or metabolic disorders or rule out many acquired disorders. Furthermore, clinical function studies may more clearly define a gastrointestinal, liver, kidney, or endocrine problem. Finally, 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 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. 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 first 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 these abnormal metabolites. One such laboratory that offers metabolic genetic screening to discover novel hereditary diseases is in the Section of Medical Genetics at the School of Veterinary Medicine of the University of Pennsylvania (www. vet.upenn.edu/penngen for information on sample submissions). Once the failing system has been identified, the defect can be determined at the protein level. Homozygously affected animals have very low protein activity and/ or quantities (0-10%). These tests may also be used to detect carriers (heterozygotes), who typically have intermediate quantities at the protein level (30-70%), but no clinical signs (carriers of recessive disorders are asymptomatic). Unfortunately, protein assays require submission of appropriate tissue or fluid under specific conditions to specialized laboratories along with a control sample, and are labor intensive. The molecular defect has been identified for 60 and 20 hereditary diseases in dogs and cats, respectively, and thus DNA screening tests have been developed. These 312 WSAVA / FECAVA Programme 2008
3 Hereditary 15 Alport syndrome Centronuclear myopathy (CNM) Ceroid lipofucinosis Cobalamin malabsorption Complement 3 Deficiency Congenital hypothyroidism with goiter Copper toxicosis Cyclic neutropenia / gray collie syndrome Cystinuria Type I Dilated juvenile cardiomyopathy X-linked ectodermal dysplasia Epidermolysis bullosa Epilepsy (Lafora type) Factor VII deficiency Factor XI deficiency Fucosidosis Glanzmann thrombasthenia Globoid cell leukodystrophy GM1 gangliosidosis Glycogenosis Ia Glycogenosis IIIA Hemophilia A Hemophilia B Samoyed, mixed breed dog Labrador retriever English setter, dachshund Giant schnauzer, Australian shepherd Brittany spaniel Toy fox terrier, rat terrier Bedlington terrier Collie, rough and smooth Labrador, Newfoundland Portuguese water dog German shepherd Golden retriever, German shorthaired pointer Miniature wirehaired dachshund Beagle, Alaskan klee kai, Scottish deerhounds Kerry blue terrier English springer spaniel Great Pyrenees, otterhound West Highland white terrier, cairn terrier Portuguese water dog, shiba inu, Alaskan husky Maltese terrier Curly-coated retriever Mixed breeds Airedale terrier, bull terrier, Lhasa apso, Labrador retriever L-2 Hydroxyglutaric aciduria Staffordshire bull terrier, West Highland white terrier Leukocyte adhesion deficiency (CLAD) Malignant hyperthermia MDR1 drug sensitivity (ivermectin) Mitochondrial myopathia Mucopolysaccharidosis (MPS) I Mucopolysaccharidosis (MPS) IIIA Mucopolysaccharidosis (MPS) IIIB Mucopolysaccharidosis (MPS) VI Mucopolysaccharidosis (MPS) VII Muscular dystrophy (Duchenne, x-chrom.) Irish setter, Irish red and white setter Greyhound Collie related breeds Clumber spaniel Plott hound Dachshund, New Zealand huntaway Schipperke Miniature pinschers, miniature schnauzer German shepherd, mixed breed dog Golden retriever, Rottweiler, German shorthair pointer, others Table 1: Examples of hereditary diseases in dogs with known mutations (except eyes) Part 1 WSAVA / FECAVA Programme
4 15 Hereditary Myotonia congenita Narcolepsy Osteogenesis imperfecta, dominant Phosophofructokinase deficiency Primary hyperparathyroidism Pyruvate kinase deficiency (PK) Renal adenocarcinoma and nodular hyperplasia Renal dysplasia, juvenile X-Linked severe combined immunodeficiency (SCID) Autosomal recessive SCID Shaking puppy Von Willebrand s disease (vwd) Type I Von Willebrand s disease (vwd) Type II Von Willebrand s disease (vwd) Type III Miniature schnauzer, Australian cattle dog Dachshund, Doberman pinscher, Labrador retriever Beagle, Golden retriever American cocker spaniel, English springer spaniel, mixed breed dog Kerry blue terrier Basenji, beagle, dachshund, Eskimo toy, West Highland white terrier German shepherd Lhasa apso, Shih tzu, soft coated wheaten terrier Basset hound, Cardigan Welsh corgi Jack Russell terrier English springer spaniel Doberman pinscher, Drentsche patrijshond, Kerry blue terrier, Manchester terrier, papillon, Pembroke Welsh corgi, poodles, Shetland sheepdog, West Highland white German shorthaired and wirehaired pointer Scottish terrier, kooiker Table 1: Examples of hereditary diseases in dogs with known mutations (except eyes) Part 2 tests are mutation or rarely DNA marker 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, although exceptions are being recognized where the same mutation may be responsible for a particular disease in several breeds. DNA tests 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 mailing instructions for the particular laboratory and for biological products. DNA can be extracted from any nucleated cells, e.g., EDTA blood, buccal mucosa (using cheek swabs or brushes), 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 for DNA extraction; buccal swabs can be obtained with special cytobrushes - the cheek cells and not the saliva is needed and swabs need to be completely dried before being wrapped and shipped. Cheek swabs should be collected after a couple of hours fasting; there is a slight concern that in nursing animals, material from the mother s colostrum or milk could be collected which could give rise to false results and thus again a brief fasting period is required. The DNA segment, which is surrounding the mutation in the gene of interest, is amplified with appropriate DNA primers utilizing the polymerase chain reaction (PCR). The mutant and/ or normal alleles are identified by various techniques such as sequencing or DNA fragment size or base pair differences. 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 from birth on, and thus are extremely valuable to select breeding animals that will not cause disease or further spread the disease-causing allele. 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 offspring. However, all offspring should be tested and only clear animals should 314 WSAVA / FECAVA Programme 2008
5 Hereditary 15 Acute intermittent porphyria Alpha mannosidase Dominant cardiomyopathy Gangliosidosis GM1 Gangliosidosis GM2 Glycogenosis Type IV Hemophilia B Hypertriglyceremia/-chylomicronemia Mucolipidosis II Mucopolysaccharidosis I Mucopolysaccharidosis VI Mucopolysaccharidosis VII Muscular dystrophy, x-chromosomal Niemann-Pick disease C Polycystic kidney disease Primary hyperoxaluria Pyruvate kinase deficiency Retinitis pigmentosa Spinal muscular atrophy Siamese Persian, domestic shorthair Maine coon Siamese, Korat Korat Norwegian forest Siamese Persian, and others Abyssinian, Somali, domestic shorthair Abyssinian Maine coon Table 2: Examples of hereditary diseases in cats with known mutations be used in the next generation to ultimately eliminate the disease-causing mutation from the breed. For many inherited disorders, the defective gene remains unknown and research is ongoing. In a few cases a polymorphic DNA marker that is linked to the mutant allele has been discovered that can be used for screening purposes. However, depending on how far the linked marker is to the disease-causing mutation the more likely crossovers can occur which affect the test accuracy. 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. Markers have been proposed for a few complex traits such as hip dysplasia. Many predispositions such as inflammatory, immune-mediated, and malignant disorders have a genetic basis and it is hoped that molecular tools will be developed to determine the risk for such diseases in a particular animal. WSAVA / FECAVA Programme
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