Department of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis
Objectives of the Presentation
To provide breeders and veterinarians the tools necessary to determine which genetic tests are most relevant to them.
To introduce basic modes of inheritance and interpretation of test results.
To introduce non-mendelian testing options and interpretation of these tests (odds ratios, incomplete penetrance, and relative risk).
To introduce allele frequency and its relevance to genetic testing.
Overview of the Issue
There are now over 300 DNA based genetic tests available to breeders and veterinarians. Not all of these tests are relevant to every breed.
Complex mode of inheritance DNA tests are now available and their interpretation is more challenging.
Some diseases are extremely rare and may not warrant testing in some breeds.
Background Facts About DNA.
The nucleus of each somatic cell in a dog's body has all 78 chromosomes (38 pairs of autosomes and one pair of sex chromosomes). Each chromosome is one very long strand of DNA. The DNA of each cell nucleus has all the information necessary to control the metabolic processes of that individual's body.
One chromosome of each pair was originally obtained from the sire and the other from the dam at the time of fertilization. Each dog carries two copies of all of its genes and each copy is located at the same position on the same chromosome. The location of a gene on each chromosome is termed the gene locus. The copies of the gene are called the alleles. Although the alleles are for the same gene, meaning they encode the same protein, one allele may be normal and the other may have a mutation. One can differentiate the normal from the mutant allele by examining the nucleotide sequence of the DNA for each allele.
Inherited Diseases in Dogs
There have been over 400 inherited diseases described in dogs with additional diseases recognized each year. Inherited diseases are common among domestic dogs due to the population structure of dog breeds. In contrast to people, there is a high level of inbreeding in purebred dogs leading to higher levels of autosomal recessive disorders. The establishment of a breed begins when dogs with similar physical and behavioral characteristics are bred to each other to create more dogs with those characteristics. Once the breed has been established there are a limited number of individuals available for breeding purposes, therefore related dogs are bred together which leads to decreased heterogeneity (genetic differences). Using this breeding practice, traits with value to the breeder can be fixed in the progeny leading to a consistent type of dog. Unfortunately this practice, called "line breeding", also uncovers recessive alleles explaining why most genetic diseases are recessively inherited in purebred dogs. The overuse of popular sires further limits the gene pool within a breed, which can be deleterious for the breed if the sire carries the allele for a recessive disease.
Mode of Inheritance
The mode of inheritance of a particular disease is important to understand in order to interpret test results. The majority of DNA-based tests are for simple autosomal recessive disorders. If a disease is inherited as a simple autosomal recessive then the animal most have two copies of the mutant allele to express the disease. If the animal only has one copy of the disease allele and the other copy is normal the animal will appear normal. Animals that have one normal allele and one mutant allele are called carriers. Identification of these animals is important to a breeding program since they appear completely normal but can produce affected offspring.
The alleles responsible for polygenic disease are much more difficult to determine, therefore to date there are few DNA tests available for these types of diseases even though they are common in many breeds. Examples of diseases that are polygenic are hip dysplasia, elbow dysplasia and epilepsy. Polygenic disorders are caused by mutations in more than one gene and the affects are additive between the different genes. This makes it much more difficult to predict which animals might produce affected offspring. Rather genetic risk is given as odds ratios. If the dog has a particular allele then its risk of developing a particular disease is increased by a certain amount. It does not mean that the dog will get the disease just that it has increased risk of getting the disease.
Penetrance is a term that indicates how often the dog will have the disease if it has the mutation. You might think this should always be all the time but in fact that is not the case. Penetrance values can vary from 0 to 100%. If a disease is 100% penetrant then every time the animal has the disease genotype it will have the disease. If the penetrance is 50% then only half the animals with the disease genotype will have the disease. Diseases with low penetrance occur in dogs and make DNA testing decisions difficult.
A measure commonly used to describe the parameters of a genetic disease is the allele frequency. Allele frequency is used broadly for both dominant and recessive diseases and estimates the percentage of disease-causing alleles (variants) of a gene among all the alleles within the population. High allele frequency indicates that the disease is common in the population. Allele frequency is reported as a number between 0 and 1. Within a breed, an estimated allele frequency of 0.5 means that half of the copies of the gene are disease copies and half of the copies are normal.
Mode of Inheritance Interpretation
The interpretation of test results should be based on the mode of inheritance of the disease and the assumption that the disease is fully penetrant. The following tables are written in a general fashion so that they could be used for either a desirable trait or an undesirable trait. In the case of a disease, breeding goals should be centered on reducing the disease allele frequency as well as elimination of the occurrence of the disease itself entirely. Towards this end, a reduction in allele frequency can be obtained by not breeding animals with dominant diseases as well as not breeding carriers of recessive diseases. In a breed with low diversity, the goal might be to reduce the allele frequency of the disease allele more slowly to avoid a reduction in diversity within the breed. In that case, a strategy of breeding an individual, testing its genotype and replacing that individual with an unaffected offspring can be used where carriers are bred but non-carrier offspring are retained for future breeding. In the case of a dominant trait, heterozygotes are bred and unaffected progeny are retained in the breeding program.
Guidelines for Reliability and Accuracy of DNA-Based Tests
Error rates are associated with all clinical diagnostic testing including DNA-based genetic testing. Errors can occur in sample handling and labeling prior to or after submission to the testing laboratory. Quality control is up to the discretion of each testing laboratory and no standardized guidelines exist for DNA tests for inherited diseases in animals. Many testing companies are not even associated with the laboratories that performed the research that lead to the development of the test. In addition, many tests have been developed through research that has not been published. In the case of DNA testing for inherited diseases there should be an error rate available for each individual test performed, however the experiments necessary to determine the error rate may be difficult to perform.
In general DNA tests based on the mutation that causes the disease should have a low error rate. However, false negative errors can result when a mutation which has occurred in a different gene causes the same disease the test is being used to identify (phenocopy). A good example of this type of error is in the case of the disease progressive retinal atrophy, which has over 20 different genes that can be mutated to cause the same disease. It is possible for a dog to test normal for the particular allele that the test was developed to evaluate but still have the disease caused by a mutation in a different gene. In addition, it is possible that a false negative could occur if a different site in the same gene was mutated. The chance that these types of errors would occur is based on the mutation rate of DNA and the target size, which is the size of the genes that could cause a similar disease when mutated. These false negatives are very unlikely to occur especially for recessive diseases. False positive errors can occur if there is a contamination problem since PCR is a very sensitive assay. Contamination of a sample used for PCR of DNA can come from just a few cells from a different individual. For example, cheek swabs taken from a puppy can be contaminated with the mother cells if the puppy is still nursing.
Marker tests can have the same errors as the mutation tests but there are additional sources of errors with these types of tests. The linked marker used to infer the status of the disease gene is located either downstream or upstream from the gene. The distance between the gene and the marker can serve as a source of error due to recombination of the chromosomes during meiosis. Researchers should have some idea of the distance a marker is from a disease-causing gene and should provide an error rate for the distance. The distance between the marker and the gene can cause both false positive and false negative results. In addition, linked marker tests can contain another source of error. Geneticists term the other type of error linkage phase error. The linkage phase is wrong if the copy of the marker identified with the disease copy of the gene is different in different families. This can occur since all dogs of a single breed are not necessarily related to one another. Phase errors can lead to both false negative and false positive results. Phase must be established in a family using affected individuals and three generations of animals. It is best to use marker tests on families of dogs with affected animals rather than on an individual animal alone because of the potential for phase errors to occur.
Since genetic tests are available in dogs, there can now be less guesswork in predicting breeding outcomes. DNA tests are available for many different coat colors as well as diseases. Simple charts specific for coat colors are available in order to allow determination of the results of crosses with respect to the many color genes. Coat colors for the most part to do not affect an animal's quality of life. Merle color may be an exception to this statement since animals with two copies of the Merle mutation may be blind and deaf.
DNA-based tests for inherited diseases in dogs are available from many different laboratories. Currently, there are no governing bodies approving tests or maintaining quality control for those testing laboratories. As mutations that cause diseases or coat colors are identified, many are published in peer-reviewed journals prior to tests being offered. However, some tests are offered without supporting scientific publications that indicate what is being tested. As many advances are made in the field of dog genetics, tests that are patent protected may be offered and never published in the scientific literature.
Once the mutation that causes a disease has been identified and a test developed and offered, there is no reason that animals with that disease should ever be knowingly produced. This is very simple and straightforward for lethal diseases; however, some more complex diseases with complex phenotypes can muddy breeding decisions. Some smaller dog breeds may have high carrier rates and quick elimination of carriers could have an effect on genetic diversity within the breed. In those breeds, it is best to counsel breeders to replace carriers in their breeding programs with non-carrier offspring rather than not breeding carriers. Ultimately, the best thing for dogs is to eliminate serious medical conditions from the breed; however, the complicated nature of breeding decisions may make this impossible.
Not all genetic tests should be run for every breed of dog or for every individual. The severity of the disease, treatment options, the frequency in the breed and the type of test (linked marker, direct simple or complex) all need to be considered prior to making testing decisions and interpretation.