Diagnosis And Control Of Hereditary Diseases
Urs Giger, PD Dr. med. vet. MS FVH, DACVIM, DECVIM
Clinical Genetics is involved in the diagnosis, management, and control of hereditary disorders and has emerged as an important discipline in small animal practice for several reasons. Effective preventative measures reduced the frequency of infections, nutritional disturbances, and intoxication. Furthermore, life-saving advances in medicine and surgery increase the chance of survival and thus tend to raise the recognition of genetic defects. Inbreeding practices to preserve the desirable traits in certain breeds favor the occurrence of recessively inherited diseases. The recent technologic advances have allowed the recognition and characterization of the clinicopathologic, biochemical, and molecular basis of many hereditary diseases. This presentation reviews the characteristic clinical features of hereditary diseases in small animals, the various modes of inheritance, diagnostic tests, and management and control of these diseases by utilizing different case examples. The importance of genetic counseling is addressed, i.e., providing information for pet owners and breeders of animal afflicted with a hereditary disorder concerning the consequences of such a disorder and the ways in which it can be prevented in future generations.
Because of the increased awareness of breeders, pet owners, and veterinarians, and the improved diagnostic abilities in clinical practice, the number of reported hereditary diseases in small animals is rapidly growing. Originally, diseases with apparent clinical manifestations affecting the appearance and gait of an animal were recognized. Thus, it is not surprising that skeletal malformations, skin and eye abnormalities, as well as neuromuscular defects were more frequently reported than disorders involving internal organs. Furthermore, we recognize now that animals with recurrent of chronic infections or immune-mediated diseases may have a genetic defect that deregulates their immune function. Finally, a variety of other genetic predispositions of certain animals, families, or breeds to develop disorders such as hip dysplasia, gastric torsion, drug idiosyncracies, and cancer have been clearly established.
Presently, >400 hereditary diseases in dogs and >170 disorders in cats have been documented, and every year over a dozen new defects are being reported. These numbers are much higher than in food animals, where economic pressures rapidly eliminate and abolish investigation of diseased animals. In contrast, several thousand hereditary disorders have been accumulated in McKusicks’s Catalog of Mendelian Inheritance in Man. Thus, practically all diseases described in small animals have also been seen in humans and generally represent close homologues.
Although any genetic defect may occur in any animal, many have only been documented in a family or breed. In fact, in some breeds, the frequency of a particular disorder may be very high. This may be due to a founder effect where one or more of the founders of a small ancestral group was a carrier or even affected, or as observed in several smaller breeds where a popular sire was later determined to be a carrier of a mutant gene. Unfortunately, genetic disease frequencies are generally not available or are biased because of data collection. Large scale screening programs and open registries have rarely been established. For instance, the prevalence of hip dysplasia may differ greatly depending on methods used to reach a diagnosis and whether a registry requires or only encourages recording of every examined animal.
Although many hereditary diseases occur rarely and often in only one breed, all together they represent an important clinical problem. For the small animal practitioner, it can be a daunting, nearly impossible task to remember all these diseases. Recently, however, various resources became available to obtain genetic information. A list of hereditary diseases and associated breeds is in the appendix of Ettinger’s Textbook of Veterinary Internal Medicine, and there are other published lists organized by breed or disease. A list of genetic diseases in all species with references assembled by Frank W. Nicholas, known as Mendelian Inheritance in Animals, can be obtained online. The most comprehensive and updated searchable information, however, is Donald F. Patterson’s Canine Genetic Disease Information System, available on disk and in book format from Mosby in 2003.
Genetic diseases are caused by chromosomal alterations of gene mutations. Disease-causing mutations are heritable changes in the sequence of genomic DNA that alter the structure and function of the coded protein. These changes include point mutations, deletions, and insertions in the DNA sequence that result in a missense or nonsense sequence. The molecular genetic defect is now known for more than 30 hereditary disorders in small animals (Table 1). Among the disorders caused entirely or partly by genetic factors, three main types are recognized: chromosomal, single gene, and complex or multifactorial disorders. 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 major human genome project has also allowed rapid progress in canine and feline gene mapping, and a first partial genome sequence from one dog has been accomplished. Through physical and genetic mapping strategies, genes can now be assigned to and localized along a chromosome, and new genes can be identified.
Chromosomal disorders are caused by an excess or deficiency of genes contained in a chromosome or chromosomal segment. Understandably, such defects may result in severe, often lethal clinical symptoms. Although alterations of autosomes have only rarely been reported in small animals – some had syndromes with multiple defects – they are common in infants and are often responsible for fetal losses. In contrast, abnormalities involving the X- and Y-chromosomes leading to sex development disorders are well recognized. The best example is the tricolored (calico, tortoiseshell) male cat with testicular hypoplasia and an XXY chromosome set. However, not every sex developmental disorder is due to a defect in the sex chromosomes, e.g., XX-sex reversal.
Single Gene Traits
The inheritance of a single gene defect is often called Mendelian and involves one mutant gene (allele) at a single locus. When an animal has a pair of identically mutant alleles, it is said to be homozygous (a homozygote), whereas when only one of the genes is mutated, it is said to be heterozygous (a heterozygote) at that gene locus. 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.
Autosomal Recessive Inheritance
Autosomal recessive inherited traits are most common in small animals. The parents of affected animals have to be carriers (heterozygotes), therefore, called obligate carriers. Typically one fourth of males and females in a litter are equally likely expected to be affected. Phenotypically normal offspring may be in a ratio of 2:1 either carriers (heterozygotes) or free of a mutant allele (“clear,” homozygous normal). Although the parents could also be affected, diseased animals generally are not used for breeding, unless they do not develop signs until later in life.
X-Linked Recessive Inheritance
In X-linked recessively inherited disorders males who are hemizygous for the X-chromosome typically are affected. When heterozygous females (carriers) are mated to a normal male, half of their male offspring will be affected and half of their female offspring will be phenotypically normal carriers, whereas the other males and females will be “clear”. The mutant X-chromosomal gene is never passed on from the sire to a male offspring, but is transmitted by an affected male to all its female offspring (obligate carriers). Affected females would only occur, if a carrier female were mated with an affected male. Heterozygous females are usually unaffected, although some manifestations may occur because of X-chromosomal inactivation. In addition, an X-linked dominant trait may need to be considered, but has only been reported in Samoyeds with a glomerulonephropathy. X-linked disorders should not be confused with sex-limited disorders, such as diseases related to the primary and secondary sex organs. Finally, Y-chromosomal diseases have not been reported in animals.
Autosomal Dominant Inheritance
In autosomal dominant traits the disease appears in every generation. An affected animal generally has one affected parent unless this animal has a new mutation in the gamete of a phenotypically normal parent or when the disease is variably expressed (non-penetrant in parent). Males and females are equally likely to transmit the disease to an offspring of either sex. Because affected animals are generally heterozygous, however, half of all offspring will be affected. Affected animals generally are not used in breeding programs. Furthermore, homozygous states of dominant traits are often lethal.
Mitochondrial inheritance is a very rare and atypical Mendelian inheritance of disorders involving the mitochondrial DNA. Because all mitochondrial DNA is transmitted from the ova, all offspring from an affected female, but none from an affected male, will be diseased. In humans several neuromuscular diseases are known to be associated with mutations in mitochondrial DNA, and in dogs some myopathies may be caused by a mitochondrial defect.
Complex or Multifactorial Inheritance
A number of developmental disorders resulting in congenital malformations are caused by complex or multifactorial inheritance, as well as other disorders in adult animals. Rather than having one single gene error, several minor defects (polygenic) in the genetic information together with certain environmental factors can produce or predispose to a serious illness. Hip- and other dysplasias as well as certain congenital heart defects (conotruncal defect) are examples, and the degree to which a trait (e.g., hip dysplasia) is genetically determined may greatly vary between breeds (heritability).
Thus, the hereditary nature of a particular disease may be suggested or established by a familial occurrence, breed predilection, breed studies, an established mode of inheritance and/or an identified gene defect.
Molecular genetic characterization of hereditary disorders in companion animals and DNA tests (examples)
Gene defects can involve any gene or organ; therefore, the clinical signs of hereditary diseases are extremely variable and may mimic other acquired disorders. Some typical features, however, may raise our suspicion of a genetic disorder.
In contrast to infectious diseases, intoxications, and nutritional imbalances, which generally affect an entire litter, hereditary diseases often involve only a few in a litter. Furthermore, the age of onset of clinical signs for a particular gene defect is rather specific and independent of environmental factors.
Most genetic defects cause clinical signs early in life. In fact, fetal resorptions, abortions, and stillborns may also be caused by genetic traits, but are rarely determined. Most puppy and kitten losses occur during the first week of life, shortly after the maternal homeostatic system can no longer compensate for an endogenous defect. Certain congenital malformations also may not be compatible with life, such as severe cleft palates and hernias. 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. These animals lag behind their healthy littermates in their development; they do not gain weight at a normal rate and are generally lethargic. 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, which refers to a proportionally stunted growth that may or may not be associated with other clinical signs. 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. Many disorders cause an isolated typical sign, whereas others produce a characteristic overall pattern of anomalies known as syndromes.
Clinical manifestations of hereditary diseases are extremely variable ranging from benign to debilitating and lethal. They are usually chronic and progressive, i.e., once an animal shows signs it probably will not recover, and often cause death at an early age. A few hereditary defects, however, result in intermittent or recurrent problems, such as hereditary bleeding disorders and primary immunodeficiencies.
Diagnostic tests generally are required to further support a genetic disorder in a diseased animal. Radiology and other imaging techniques may reveal skeletal and CNS 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 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. 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 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 tests include the classic enzyme function tests as well as immunological assays. Because most enzymes are present in abundant amounts, no major functional abnormalities are observed unless the enzyme activity is severely reduced, usually to less than 20% of normal value. Thus, homozygously affected animals have very low protein activity and/or quantities, often in the range of 0 to 5%. These tests may also be used to detect carriers (heterozygotes), who typically have intermediate quantities at the protein level (40-60%). 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. (NIH #02512, http://www.vet.upenn.edu/penngen)
The molecular defect has been identified for several hereditary diseases, 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 mutation for a particular disease, e.g., phosphofructokinase deficiency in English springer and American Cocker spaniels. However, dogs and cats as well as unrelated breeds of a species with the same disorder will likely have different mutations, as shown with X-linked muscular dystrophy and erythrocyte pyruvate kinase deficiency in various dog breeds and cats.
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, before clinical signs become apparent, or at least long before an animal is placed in a new home. DNA is very stable and only the smallest quantities are needed, hence, there are no special shipping requirements. DNA can be extracted from any nucleated cell, e.g., blood, buccal mucosa (cheek swab), 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 brush, 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 directly by DNA sequencing or size differences 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.
For a few inherited disorders, the defective gene remains unknown; however, 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 (but the mutation is now known) and are available for renal carcinoma and some forms of renal dysplasia and retinopathy, and may be accurate for a particular patient as long as there is a known affected animal in its family (informative family). Presently, mutation-specific and linkage tests are only available for single gene defects in small animals; however, complex genetic traits may also soon be approached by these methods as they are for humans.
Therapy and Control
Because the clinical consequences of the many hereditary disorders vary greatly, it is not surprising that the prognosis for survival and quality of life ranges from excellent to grave. The clinical course and outcome for a particular defect is rather similar among affected animals. Some defects are recognized as an incidental finding, e.g., microcytosis in Akitas, whereas others are progressive and lead to severe organ dysfunction and death, e.g., lysosomal storage diseases.
Presently, the therapeutic options in the treatment of hereditary diseases are limited and ethical principles need to be carefully considered. Although several structural malformations can be surgically corrected, such as cryptorchism, hernias, hepatic shunts, and a patent ductus arteriosus, these animals should not be shown or bred. In a few cases a deficient protein, cofactor, substrate, or metabolite can be supplemented to correct the defect. For instance, vitamin B12 deficiency in cachectic and lethargic Giant Schnauzers, Beagles, and Border collies with an ileal receptor defect can be helped by monthly cobalamin injections. Pancreatic enzyme supplementation and daily insulin injections are used to manage animals with exocrine or endocrine pancreatic insufficiency, respectively. Fresh frozen plasma is administered in the treatment of hereditary coagulopathies and vonWillebrand disease whenever animals excessively bleed. Other enzyme and protein replacements are also experimentally attempted.
Several hereditary disorders of hematopoetic cells have been experimentally corrected by bone marrow transplantation, e.g., pyruvate and phosphofructokinase deficiency, cyclic hematopoesis, and interleukin-2 (IL-2) receptor defects. Furthermore, bone marrow transplantation is being attempted to deliver functional cells or active proteins to other tissues including liver, bone, and brain, e.g., in lysosomal storage disease. Finally, gene therapy experiments, with the aim to integrate a functional gene into the patient’s own defective cells, appear encouraging based on experimental studies in dogs with hemophilia B and dogs with mucopolysaccharidosis VII. However, the technology needs to be improved to achieve persistent and regulated gene expression and to assure safety.
Much more important than the treatment of hereditary disorders is the control of these traits in breeding programs. Thus, 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, 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-linked 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 half-normal (~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 known genetic diseases, whenever carrier tests are available. Unfortunately, many breeders mistrust these newer tests; either they were disappointed by the inaccuracy of earlier tests (HD testing) or they fear that the results may become public and would hurt their business. Thus, breeders need to be educated by well-informed veterinarians. 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 or at least 5 and 11 healthy puppies/kittens, respectively, need to be produced to “clear” an animal. For many breeders, this approach is ethically unacceptable because it may produce affected animals. Furthermore, 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 and all its offspring need to be tested. Only clear animals should be used in future breedings.
conclusion, it is most exciting to learn about many recent advances
in clinical genetics in small animal practice, be it for the diagnostic
approach to a hereditary disease, the understanding of its pathophysiology,
or its control to improve the future health of animals. In
addition to their responsibility to suspect a genetic disease and to appropriately
diagnose it with modern techniques, clinicians must become involved in
the control of these disorders in the breeders’ kennels or catteries.
Practitioners thus can make an important contribution toward controlling
the further spread of mutant genes and reducing future suffering of animals.
Giger Urs (2000), “Clinical Genetics,” in Textbook of Veterinary Internal Medicine,
S.J. Ettinger and E.C. Feldman, ed. Philadelphia, PA, Saunders.
Dr. Urs Giger, Veterinary Hospital University of Pennsylvania,