(The article below, written by Danika Bannasch, DVM, PhD is part of Eukanuba’s Breed Smart Program and is reprinted below with their permission.)
Molecular Genetic Testing in Dogs
Danika Bannasch, DVM, PhD
Inherited Diseases in Dogs
There have been over 300 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 must 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 no DNA tests available for these types of diseases even though they are common in many breeds. Polygenic disorders are caused by mutations in more then 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. Examples of diseases that are polygenic are hip dysplasia, elbow dysplasia, and epilepsy. The introduction of a molecular genetic test to determine a dog’s genetic status with respect to a particular inherited disease will facilitate the elimination of these diseases.
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.
It is important that veterinarians and breeders understand the basis for the DNA tests that are becoming available in dogs. There are two different types of tests available; the direct and the indirect DNA test. Each of these different types of tests has its own sources of error and therefore has different implications for breeding decisions. Both of these types of testing could be used for clinical diagnosis of diseases when normal diagnostic tools might be invasive and carry with them life-threatening complications. Presently DNA based genetic tests are available for over 50 inherited diseases in dogs and the number of tests increases all the time.
Direct DNA Tests
Direct DNA test are designed to assay the change in DNA sequence in a particular gene that leads to a disease. These tests use molecular biology techniques to assay the exact change in DNA that has been demonstrated to cause an inherited disease. Different types of changes in the DNA can cause disease. The changes that are easiest to identify involve mutations in the coding DNA that result in a change in the amino acid chain either by causing early chain termination or by causing a change in the amino acid sequence. The tests use polymerase chain reaction (PCR) to amplify small amounts of dog DNA isolated either from EDTA preserved whole blood or from a cheek swab. PCR is carried out using primers (short single stranded pieces of DNA) as the basis for the amplification of the dog’s DNA. In some cases the mutation is a deletion of nucleotides. This type of mutation can be detected as a size difference in the PCR product. The advantage of this type of mutation is that there will be a PCR product in both the mutated allele and the normal allele. Other mutations change just a single base pair of DNA from one base to another. These require more complicated assays and sometimes require two different PCR reactions — one for the mutant allele and one for the normal allele. Ideally, direct DNA tests have a positive control for PCR amplification.
Indirect DNA Tests
The basis for indirect tests are DNA markers called microsatellite markers. These markers are small pieces of DNA sequence that contain repeats of two to four nucleotides. The number of repeats can vary in length between individual animals. The variation in individuals is used by geneticists to follow these regions of the chromosome through pedigrees in order to identify the region of a particular chromosome that is associated with the disease of interest. Analyzing the segregation of markers with disease is called linkage analysis. Because the markers show differences between individuals they are commonly used for individual identification and parentage testing. A set of microsatellite markers is used to identify individual animals for registration purposes, forensics, and permanent identification. The orthopedic foundation for animals (OFA) accepts “DNA testing” as a form of permanent identification for radiographs. Currently, the American Kennel Club (AKC) requires mandatory DNA testing for frequently used sires as well as animals bred by the use of frozen semen. There have been numerous criminal cases where the individual identification of dog hairs, blood, and even urine has been used to solve cases. Although the AKC does not require parentage verification for registration purposes, some registries for other purebred animals require it for registration to ensure the validity of the registry.
Microsatellite markers are excellent tools for individual identification, parentage analysis, and for linkage analysis to a disease gene. Once a linked microsatellite for a particular disease is identified in one species, the equivalent regions in the genomes of other species are located and candidate genes investigated. If there are no candidate genes based on comparison to other species, then the area near the disease gene is narrowed by checking additional nearby microsatellites. The techniques used to identify a gene without candidates can take many years and can be very expensive. In the meantime, researchers can offer tests to breeders based on the linked microsatellites that they have identified. These tests are called indirect tests since the disease-causing gene is unknown and the status of the disease-causing gene; mutant or normal, is inferred from the microsatellite markers. Since these tests are indirect there is a higher error rate associated with them compared to a direct DNA test.
Guidelines for Reliability and Accuracy
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, direct DNA tests should have a low error rate. However, false negative errors can result for a direct test from a mutation, which has occurred in a different gene, which 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.
Indirect tests can have the same errors as the direct 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 indirect tests on families of dogs with affected animals rather than on an individual animal alone because of the potential for phase errors to occur.
During the last five years, major advances have been made in our understanding of the molecular basis for inherited diseases in dogs. These advances, in the form of DNA-based tests for breeding animals, are just the beginning of the genomics revolution in veterinary medicine. Most of the tests available today have been developed using a comparative medicine approach to canine genetics. This method utilizes previously identified human diseases similar to the canine ones and tests the gene or genes responsible for the human disease to see if they are also mutated in the canine disease. This type of approach is very helpful for veterinary medicine but does not have beneficial implications for human medicine.
Recently researchers have taken a different approach to inherited diseases in dog. Human populations tend to be more outbred then purebred dogs making a genetic analysis easier in dogs than in people. By taking advantage of the fact that a dog breed is a large family of related individuals, researchers have been able to identify genes that cause diseases in dogs which have not previously been implicated in the equivalent human diseases. This approach has already resulted in the development of genetic test for narcolepsy and copper toxicosis.5,6 Based on the utility of the dog as a model for inherited diseases in people, the National Institute of Health has placed the dog at the top of the list for a full genome sequence. The canine genome project will change the way we practice veterinary medicine as well as the way we breed animals. As more tests are developed, the link between genetics and veterinary medicine will grow even stronger. Veterinarians will have the tools at their disposal that will allow them to prevent inherited diseases through the use of these genetic tests.
(Articles on the USKBTC website do not necessarily represent the opinions of any person other than the writers. The information provided in this article is intended for general information and guidance and, in the case of articles relating to veterinary care, is not meant to be a substitute for professional veterinary advice. Statements or expressions of opinion or comments appearing herein are those of the authors and are not necessarily those of the United States Kerry Blue Terrier Club.)