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A New Age: Veterinary Molecular Genetics
Wendy Shepard Chisholm, V. M.D.
"It's time to ring in the century of
biotechnology ... Now we're just a few years away from one of the most
important breakthroughs of all time: deciphering the human genome,
the 100,000 genes encoded by 3 billion chemical pairs in our DNA."10
(Time Magazine "The Future of Medicine: How genetic engineering
will change us in the next century." Jan. 11, 1999). What follows
is a technical discussion of genetic information important to all breeders.
Most of this material I was privileged to hear at the North American
Veterinary Conference, January, 1999, in Orlando, Florida. I have tried
wherever possible to include explanations and definitions of medical
terms, so all readers can follow the discussion.With the recent availability
of molecular markers for the canine genome, and the ongoing work to
develop a comprehensive canine genetic map, we are at the beginnings
of a revolution in canine health care.1 With the control
of most infectious diseases, nutritional problems, and intoxications,
hereditary problems have emerged as a major problem in small animal
medicine. More that 500 hereditary disorders have been recognized in
dogs and cats, and represent a large, heterogeneous, and growing group.8 In
the dog alone, there are 65 genetic orthopedic diseases. With the common
practice of breeding closely related animals, there is a lack of selection
against genetic disorders. Many breeds of dogs are characterized by
genetic diseases, many of which are inherited as autosomal recessive
or apparently complex traits (autosomes are all chromosomes, excluding
the X and Y sex chromosomes). In the coming months, it is expected
that diagnostic tests will be developed for many common canine diseases,
as they are currently available for progressive retinal atrophy, progressive
rod cone degeneration, copper toxicosis, Von Willebrands disease and
more. Most recently, over a dozen hereditary disorders have been characterized
with the development of the simple and accurate DNA-based polymerase
chain reaction technique (PCR). Breeders will have the option and responsibility,
to test key dogs in their breeding programs to determine carrier status
for a variety of inherited disorders. A dog will be able to be clearly
identified as being carrier or "clean" of a particular genetic
disorder.
The Canine Genome
and Map
For most canine diseases, the underlying
genetic cause is yet to be determined. Historically, the canine genome
has been difficult to study. The dog has 38 pairs of chromosomes. Standards
for chromosome identification by G-banding have been established for
only the largest 22 canine autosomes by the committee for the Standardized
Karyotype of the Dog.1,3 The remainder of the chromosomes
are expected to be identified in the very near future. With genetic
mapping of disease genes, an effort is made to find polymorphic markers
which are linked to disease loci. Markers with the best predictive
value are those which are located very close to the disease gene in
question.
Genetic Tests
Genetic tests are not all sophisticated
polymerase chain reaction (PCR) based7 assays. While some genetic tests
identify the animals genotype (affected, carrier, or normal), others
indicate the phenotype of the disorder. Note: (The genotype is the
genetic constitution of the dog. It is unique to each dog. A comparison
of genotype can determine if a dog is the offspring of a sire or dam
tested, thus verifying parentage. The phenotype is the clinical appearance
of the dog.)
Many traits may be complex in origin, with
several genes predicted to contribute to the final phenotype. These
traits often appear in the population with significant variability
in the phenotype. Screening for cataracts, ausculting for heart murmurs,
hip and elbow radiographs, and observation on behavioral traits are
all tests of the phenotype.7
PCV based tests are true tests of the genotype.
The test is specific for a defective gene, and can differentiate between
genetically normal, carrier, and affected individuals. These can be
performed at any age, regardless of the onset of the disorder. Off-spring
can be tested before placement into breeding versus non-breeding situations.
Molecular screening tests, utilizing the polymerase chain reaction
techniques (PCR tests), are now available to simply and accurately
diagnose affected and carrier animals. Examples of PCR based tests
are phosphofructokinase deficiency in Spaniels, pyruvate kinase deficiency
in Basenji is, several storage diseases (fucosidosis, mucopolysacchardoses,
glycogenesis, gangliosidoses), cystinuria, and type 1 and 3 Von Willebrands
disease, as well as several x-linked inherited disorders (hemophilia
B, severe combined immunodeficiency).8 These tests are specific
for a particular mutation and are therefore usually breed specific.
One recent successful genome screen was
the mapping of the progressive rod-cone degeneration (PRA) locus to
canine chromosome 9. PRA (progressive retinal atrophy) is the most
widespread hereditary retinal disease leading to blindness in dogs.
Phenotypically it is the canine counterpart of retinitis pigmentosa
(RP) in humans.1,4 With the rapid progress in the field
of canine genetics, the identification of genes underlying many of
the inherited traits, makes the dog a unique asset for the study of
mammalian genetics in general.5 The majority of the problematic
genetic disorders in domestic animals have a recessive component, with
inapparent carriers being used for breeding. Most dominant genetic
disorders are easier to control, as the defective traits are apparent.
PCR based tests can also be used for linkage
studies to polymorphic genetic markers if a defective gene has not
been identified. If the genotype is linked to a genetic marker, the
defective gene is located close to the marker on the same chromosome.
While not an exact test of the genotype, marker based tests can successfully
be used to identify genotypes. Examples of marker-based tests are copper
toxicosis in Bedlington Terriers, and progressive retinal atrophy (PRA),
in many breeds of dogs.
Other phenotypic tests, such as enzyme
storage diseases and blood factor assays, can identify heterozygous
carriers of defective genes. A problem with these tests is that sometimes
carriers and normal individuals cannot be separated. Other phenotypic
tests, such as electroretinograrn for PRA, or pelvic radiographs for
canine hip dysplasia, only identify the affected phenotype and not
carriers. Phenotypic tests may also have certain age requirements for
their validity.
Polygenic Disorders
Polygenic disorders, such as epilepsy, hip
dysplasia, elbow dysplasia, osteochondrosis, and congenital heart defects
historically have been difficult for breeders to control. For example,
in canine hip dysplasia, there is no one "normal hip" gene.
A number of genes must combine to produce an affected, dysplastic individual.
If phenotypically normal parents produce affected offspring, both should
be considered to carry a genetic load for the disorder. In polygenic
disorders, the phenotype of the individual does not provide all of
the necessary information for genetic control. Many polygenic disorders
have a major recessive or dominant "trigger gene" that must
be present to produce an affected individual. The trigger gene in one
breed or family may be different that the gene in another. The identification
of these genes will provide better control in the future.7
DNA Fingerprinting
With the application of molecular genetics
to veterinary medicine, and the availability of DNA certification programs
to preserve the integrity of breed registries, the ability now exists
to reliably identify individuals and to deduce their relatedness to
others (pedigree) by DNA analysis.9
DNA fingerprinting is being used widely
to identify individuals, breeds, and strains, as well as to determine
the parentage of not only domestic and wild animals, but microorganisms,
insects and plants. "All forms of DNA fingerprinting are based
upon detection of a specific segment of the DNA (alleles) or the relative
location of repeated nucleotide sequences which are scattered randomly
throughout the genome of animals."9 The DNA alleles
are inherited, approximately 50% from each parent. They provide a reliable
means of identifying individuals as well as determining the pedigree
of individuals, even in highly inbred populations.
With the advances in mapping the canine
genome, we will have the ability to identify the genes responsible
for over 350 identified, inherited diseases in dogs, as well as the
genes that affect infectious diseases, cancer and reproduction.
MHC Complex
The major histocompatibility complex (MHC)
is a multi-allelic group of genes present in all animals.9 It
is a polymorphic system, with thousands of potential allelic combinations.
The genes of the MHC are involved in controlling disease resistance,
immune function, and reproduction. MHC haplotypes are associated with
a number of significant diseases (arthritis, thyroid diseases, autoimmune
disease, ocular disease, intestinal diseases, mastitis, some forms
of cancer, and infertility). It is thought that similar disease associations
can be made with specific canine MHC haplotype. The development of
canine MHC genetic markers will soon allow these associations to be
identified, and thus controlled or eliminated by selective breeding.
The long term viability of any population
of animals (breed or species) depends on maintaining a high degree
of genetic diversity (polymorphism) in the MHC. There are powerful
selective pressures to keep the MHC as diverse as possible. The MHC
is responsible for "hybrid vigor', disease resistance, as well
as performance traits and heritable defects.9 As polymorphism
decreases (usually related to inbreeding), the survivability of the
individual, and the long term health of the population is incrementally
reduced. This loss of MHC genetic diversity is responsible for a portion
of the reduced "hybrid vigor" in inbred or highly selected
animals, including some dog breeds.
One of the major reasons breed registries
and methods to reliably document pedigree were developed, was recognition
of the detrimental effect of inbreeding on disease resistance and reproduction.
However, such systems do not prevent the breeding of individuals with
similar MHC haplotypes. This is a problem when the genetic basis of
the breed in narrow (due to inbreeding and line-breeding). With the
advances in genetic research, information will soon be available to
breeders, so they can make informed decisions on breeding.
One of the most powerful applications of
DNA fingerprinting is the identification of individuals and tracing
their pedigree through several generations. Breeders, recognizing the
importance of pedigree in selecting breeding animals, will have a reliable
means to trace pedigree, to measure and record performance criteria,
as well as to work to improve the overall health and maintain the "hybrid
vigor" of the breed.
Open versus Closed
Registries
An open genetic disease registry is a data
book of genetic history for any breed and for specific genetic diseases.
In an open registry, owners, breeders, veterinarians and scientists
can trace the genetic history of any particular dog, once that dog
and close relatives have been registered. In order to control genetic
diseases, we must know how prevalent the diseases are within the breed
and in any particular blood-line.6
Since June of 1990, 3 genetic registries
are available to dog breeders. The Institute of Genetic Disease Control
in Animals (GDC), in Davis, California, is an open registry. Here,
information about each dog is automatically linked by a computer, with
other relatives in the registry. This information is available to people
so they can choose which bloodlines indicate a reduced risk of producing
genetic disease. This information is available because the owner has
signed a release so their dog may be placed in the open registry. This
type of information is not available in a closed or confidential registry.
Only when conscientious breeders submit all the information to the
registry, on both normal and abnormal individuals, is this information
available.
The GDC open registry is similar to breed
registries in Europe, such as Sweden. With polygenic traits such as
hip dysplasia, elbow dysplasia, epilepsy, and congenital heart defects,
an excellent phenotype does not guarantee excellent genotypes or progeny.
Breeding for an excellent "genotype" can only be determined
after a review of as many relatives as possible. The GDC gives a report,
after enough individuals in a line of dogs have been reported. For
a fee of $10.00, one can obtain a report for prospective males to select
for breeding This type of information is not available in a closed
or confidential registry (Since 1990, the GDC has maintained a registry
for orthopedic diseases, in 1992: soft tissue diseases were added,
1993: CMO, Perthes, and medial patella luxation were added, 1994: eyes
and tumors, 1995: globoid dystrophy, 1997: tricuspid valve dysplasia,
and deafness were added. These are done at the requests of breed clubs,
with the GDC working with the veterinarians, dog owners and breed clubs).
Both the Orthopedic Foundation for Animals
(OFA) and the Canine Eye Registration Foundation (CERF) are closed
or confidential registries. They provide only phenotypic information.
Information is provided if the individual is free of signs of the disease,
but the status of the parents, siblings, half- siblings or progeny
is unknown. And it is well-known that the mating of phenotypic, unaffected
dogs, may result in offspring that are affected, unaffected, or a combination
of both.
Positive Identification
of Dogs for Registration
The AKC, OFA and GDC all recognize the importance
of positively identifying an individual dog for, registration. Current
means for identification are tattooing, micro-chipping, and DNA finger-printing.
Retrospective pedigree evaluation of some genetic registries (e.g.
dairy cattle) have shown that in many cases the reported pedigree is
incorrect.9 DNA certification ensures the integrity of a
registry in a way never before possible.DNA ''fingerprinting'' has
been required by the Australian and New Zealand Greyhound Association
for registration since 1994 for alI sires, and 1996 for all dams. Over
2000 greyhounds have been "fingerprinted''- The system has effectively
resolved disputes related to identification and pedigree. The Irish
Coursing Club also has a DNA "fingerprinting" program for
greyhound registrations.9 Both systems assure the validity
of the sampling by having a veterinarian submit the samples. Having
a licensed professional collect the sample, from a legal perspective,
addresses the "chain of custody" issue when submitting the
sample.The AKC and the United Kingdom Kennel Club also officially accept
the use of DNA ''fingerprinting'' in the resolution of identity and
pedigree disputes These organizations do not require DNA analysis as
a condition for registration. The AKC has a voluntary DNA certification
program. There are two inherent problems with the AKC program:1. The
AKC does not address the legal problem of "chain of custody" of
the sample. There is poor sample security, with anyone able to submit
a sample of saliva by the cheek-swab method.2. The second Problem is
the possibility of cross-contamination. With the AKC "cheek-swab" method,
if a dog licks another dog, the sample may be cross-contaminated with
DNA from the saliva of the second dog.Research scientists currently
working on genetic markers for inherited eye diseases, only accept
blood samples submitted by a veterinarian, to avoid the possibility
of either of these problems (chain of custody and crosscontamination)
from occurring.
Recommendation to
Breeders and Parent Breed Clubs
Because attempts to control inherited diseases
have largely failed, in part due to inaccurate reporting of a pedigree
to genetic registries, breed organizations are beginning to adopt the
use of DNA "fingerprinting" to protect pedigree integrity. "DNA
fingerprinting provides the best method to measure relative genetic
relatedness."9 It allows the breeder to compare genetic
composition of breeding animals, allowing them to maintain as much
hybrid vigor as possible by avoiding inbreeding. DNA fingerprinting
provides the ability to map specific performance traits and genetic
diseases to the responsible genes. Being able to identify carriers
of specific genetic diseases, these systems have the ability to influence
breeding programs in order to select for disease resistance.
The most
important factor in the control of genetic disease is to know the status
of the entire litter from which the problem came rather than the status
of the individual parents. Genetic counselors advise owners to breed
from phenotypically normal individuals where the majority of full-siblings
are also phenotypically normal. While some genetic tests (PCR) accurately
identify an animal's genotype (affected, carrier), others indicate
the phenotype of the disorder. On the other hand, a "relative risk" pedigree
analysis identifies the minimum age of the mutated defective gene in
the population, providing a closest common ancestor analysis. The minimum
age of the defective gene in the population helps to identify the genetic
spread of the defective gene in the gene pool. The closest common ancestor
analysis in pedigrees does not identify carriers of a defective gene,
and its use for this purpose (witch-hunting or finger-pointing) is
counterproductive. This point can not be over emphasized. However,
the closest common ancestor analysis can be used in a positive manner
with genetic counseling. For example, carriers of a genetic trait (as
determined by a blood test), used in breeding, should be accompanied
by the recommendations to replace carrier breeding stock with normal
testing off-spring. This selects against the defective gene , but allows
a breeding program to progress without limiting genetic diversity.
Recommendations to eliminate all carriers and affected individuals
from breeding can significantly limit genetic diversity.
Genetic diversity
concerns are also compounded with the widespread use of frozen and
fresh shipped semen, where individual males can have a profound input
on a breed's gene pool. This has become especially evident with the "favorite
sire syndrome", with detrimental recessive genes becoming widespread
due to prolific breeding of popular sires in many breeds. Any major
shift in breeding choices to a limited number of males will restrict
genetic diversity and increase the possibility of fixing undetected,
defective, recessive genes in the population.
Breed-wide genetic disease
control programs should monitor the frequency of the defective genes
in the population, and work to diminish them without affecting the
overall genetic diversity of the gene pool. High frequency defective
genes require breed-wide counseling, so that selective pressure does
not significantly shift the gene pool. Rare defective genes, regardless
of their genetic spread, should be closely controlled. The test and
slaughter system should no longer be used. We cannot afford to eliminate
every affected dog, and carrier dog from breeding, but instead have
to learn to live with genetics. In some cases there may be a truly extraordinary
dog, who exemplifies the breed standard, and is found to be a carrier
of a highly undesirable trait. Owners and breeders will have to make
the difficult decision how to modify their breeding program, and what
sort of risks they are willing to take. From a veterinarian's perspective,
clients will expect accurate risk assessment for their dogs, and will
want guidelines on how best to proceed with their breeding programs.Some
breed clubs have their own genetic registries. OFA, CERF and GDC are
examples of multi-breed registries. The Canine Genetics Laboratory,
Baker Institute, Cornell University, developed and established the
DNA tests for hereditary eye disorders. The Josephine Deubler Genetic
Disease Testing Laboratory, at the University of Pennsylvania, offers
biochemical, hematologic and molecular genetic tests for many hereditary
disorders of companion animals. One example of successful control of
a genetic problem by a breed club is the control of PRA in Irish Setters
by a blood test. While PRA was once a major problem in the breed, the
availability of the blood test has reduced the carrier rate to 7%.
Conclusion
Genetic disease control must be balanced
with the need to breed individuals whose form, structure and function,
and performance, improves the breed. Breeders should select for healthier
breeding stock, while slowly working away from genetic defects. And
the most successful endeavors to map disease genes will be those which
are based on high quality diagnostic data from veterinarians. "Having
absolutely accurate information about dogs in a family affected, and
how the disease state is expressed, will be the key to unraveling the
genetics of any canine disease trait".1
References
- Ostrander, Elaine Ph.D. "Forward:
Clinical Approaches To Genetic Diseases, Diagnosis & Control" Proceedings:
The North American Veterinary Conference January 9-13, 1999,
Orlando, Florida.
Mellersh, C.S., and Ostrander, E.A. (1197) "The Canine Genome-" In: "Advances
in Veterinary Medicine." J. Womack, (ed.), Academic Press 40:191-215
Switonski M., N. Reimann, A.A. Bossa, S. Long, S. Bartnitzke, A. Pienkowska,
N.M. Moreno-Milan, and P. Fischer. 1996. Report on the progress of standardization
of the G-banded canine (Canins familiaris) karyotype. Chromosome Reg.
4:306-309.
Acland, G.M., Ray, K., Mellersh, C.S., Weikuan G., Langston, A.A., Rine J.,
Ostrander, E.A. and Aguirre, G.D. (1988). Comparative mapping and linkage
analysis of canine progressive rod-cone degeneration (prcd) establishes locus
homology with retinitis pigmentosa (RP17) in humans. Proc.
Natl. Acad. Sci. USA 95:3048-3053.
Ostrander, E.A_ and Giniger, E. (1997). Semper Fidelis: What man's
friend can teach us about human biology and disease. American Journal
of Human Genetics 61:475-480
Poulos, Paul, D.V.M., PhD., "The Genetic Diseases of Bones and Joints
- Diagnosis and Control". The Institute for Genetic Disease Control
in Animals, Davis, California. Proceedings: The North American Conference January
9-13, 1999, Orlando, Florida.
Bell, Jerold, D.V.M., The Dept. of Clinical Science. Tufts University. School
of Vet. Medicine, North Grafton, Mass. "Genetic Counseling and the Use
of Genetic Tests in Genetic Disease Control". Proceedings: The North
American Veterinary Conference January 9-13, 1999, Orlando, Florida.
Giger, Urs, PD. Dr. med vet., University of Pennsylvania School of Veterinary
Medicine. "Recent Advances in the Diagnosis of Hereditary Diseases." Proceedings:
The North American Veterinary Conference January 9-13, 1999, Orlando,
Florida.
Fenwick, Brad, D.V.M., Ph.D. College of Veterinary Medicine, Kansas State
University, Manhattan, Kansas. "Genetics, Pedigree Analysis, and DNA-Based
Strategies for Breed Improvement." Proceedings: Canine Sports Medicine
Symposium, at the North American Veterinary Conference, 1999, Orlando,
Florida.
- Time Magazine, "The Future
of Medicine: How Genetic Engineering Will Change Us in the Next Century".
January 11, 1999.
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