By Casey R. Carl, DVM and Blake Blake Ballif, PhD
A topic in veterinary genetic testing currently getting significant attention is the difference between direct and indirect genetic testing for mutations associated with various diseases and traits. Although both types of testing can play a useful role in determining a dog’s genetic health status, the use of indirect genetic testing comes with some additional caveats that need to be considered when selecting the best testing strategy for a particular dog.
Direct Genetic Testing
As the name indicates, direct genetic testing is a general term for any genetic testing technique which looks for the presence of the specific genetic variant (mutation) known to play a causal role in a particular disease or trait. Therefore, regardless of which genotyping technique used, test results obtained from direct testing identify the presence or absence of the specific mutation that has been associated with the disease or trait. Barring differences in quality of laboratory practices and test development, direct testing is therefore the ideal method to detect a specific mutation. Furthermore, identifying the precise causative mutation in a DNA sample from a dog allows one to draw appropriate conclusions about the implications of this mutation in this dog and it’s offspring based on the previously published scientific studies. However, this type of testing is not always available for every disease or trait.
Unknown Causal Mutation
In some cases, indirect testing is performed because the specific causal mutation is not known. Genetic association studies play a crucial role in discovery of disease or trait-causing mutations. To find a causal mutation, the genomes of many individuals are examined by a variety of techniques to determine which genetic variants are present in every animal with the disease or trait being evaluated. To complete the correlation, these same genetic variants must also be absent from animals which have not developed that disease or trait.
Sometimes researchers can make educated guesses as to where a disease or trait-causing mutation might be located by searching for mutations in “candidate genes” that are known to play a role in the body system or biological pathway which is dysfunctional. Unfortunately, the canine genome is large and there are many thousands of genes in the genome about which very little is known. Fortunately, recent advances in genomic technologies allow researchers to perform genome-wide association studies (GWAS) that look at DNA variations in hundreds of thousands of locations across the genome. In addition, some researchers may even sequence the entire genomes of the animals being studied in an attempt to identify DNA variants that are present in dogs presenting with the disease or trait compared to those who are not. These types of studies can identify many variants that are statistically associated with the disease or trait but may still not be the actual causative mutation. Often DNA variants found to be associated with a disease or trait in a GWAS or whole-genome sequencing study are not located in genes or regions of the genome that would suggest the mutation is truly causing the disease or trait. These DNA variants are often located very close to the actual causative mutation and are referred to as “linked markers”. Although these linked markers can be tested for and, depending on the proximity of the linked marker to the actual causative mutation, can be somewhat informative, there will eventually be cases where the “linked marker” in a particular dog is not associated with the disease or trait in question. Thus, these linked markers are “indirect” targets for testing because linked markers are not the causative mutation and do not associate with the disease or trait 100% of the time. The use of linkage information for breeding purposes can lead to inadvertently maintaining a disease mutation in a breeding program and even producing pups with the disease. This is why, when the actual mutation is known, testing should only be conducted with the direct assessment of that known mutation and not with linked markers.
Limitation of Technology
In some cases, indirect testing is performed because methodologies employed by a laboratory may be limited in their ability to detect certain types of mutations or the specific tests developed by the laboratory were not designed to detect the precise causative mutation. For example, microarrays are a high throughput testing technology with profound power for examining large numbers of genes simultaneously from a single DNA sample. Despite their exceptional utility in genetic testing, microarrays also have some limitations. Without going into a complicated explanation of microarray technology, one specific limitation of microarrays is that direct genetic testing for a specific genetic mutation may not always be possible if the microarray was designed without that mutation in mind. Therefore, if a disease-associated genetic mutation is discovered after the microarray is designed, direct testing for that mutation is not possible if a test for that specific mutation is not present on the microarray. Thus, a linked marker that is located near the causative mutation and present on the microarray can serve as an indirect test for the disease or trait. Using a linked marker may be the only possible way for that laboratory to assess a dog’s sample unless the microarray is redesigned to detect the precise causative mutation. In ideal situations, the linked marker selected would be 100% correlated with the disease and therefore, 100% correlated with the inheritance of the causal mutation. However, it is not always possible to find a marker with such a reliable and strong linkage. Laboratories using linked markers should provide the reliability of the information being provided so that breeders understand if the information can be used in their breeding program.
Linked Markers and Limitations of Indirect Testing
For a linked marker and causal mutation to be found in strong linkage disequilibrium, the two variants must be in close genomic proximity. Linkage disequilibrium refers to the nonrandom association of a marker with the actual disease or trait variant. The closer the marker and actual mutation are found on the chromosome, the tighter the linkage disequilibrium. The reasons behind this are based in reproduction. During the formation of gametes in meiosis, homologous chromosomes (the chromosome pair) undergo a process known as crossing over, marked by exchange of genetic material between the pair of chromosomes. Crossing over is an important process for increasing genetic diversity within a population through creation of unique genetic sequences. Although increases in genetic diversity are typically considered advantageous for a species, in some cases, crossing over can wreak havoc on the use of an indirect genetic test. The larger the distance between a linked marker and causal mutation, the higher the likelihood that a crossover event could occur between the variants. Thus, breaking the linkage between the previously linked marker and the causal mutation and providing the incorrect interpretation of the genotype on the dog.
Testing with Paw Print Genetics and Canine HealthCheck
Due to the potential concerns involving indirect genetic testing, all tests offered at Paw Print Genetics are direct genetic tests for the causal mutation unless only a linked marker for the disease is known. This is true whether ordering breed-specific testing through the Paw Print Genetics website or genetic screening through our popular Canine HealthCheck. If you have additional questions about genetic testing, please feel free to contact us via email at AskUs@pawprintgenetics.com or give us a call at 509-483-5950 during business hours (8 am to 5 pm Pacific time, Monday to Friday) to speak with one of our knowledgeable and friendly staff.