Paula S. Henthorn, PhD
Objectives of the Presentation
The objectives of the presentation are to provide answers to the following questions, using real examples to illustrate concepts:
Why study genetic diseases in dogs and cats?
How are genetic studies conducted?
How does the study of genetic disease relate to genetic testing?
Overview of the Issue
There are many reasons to study genetic diseases in dogs and cats. As the understanding of and treatment of infectious and other environmentally caused disease has progressed, diseases with a genetic component are more commonly recognized. The natural history of canine and feline breed development, and the breed barriers (especially in dogs, where to be registered as a particular breed, both parents have to be registered members of the breed), and the basic concept of like begets like, which increases the tendency for inbreeding to be practiced by breeders, explains why recessively inherited diseases tend to be more prevalent than in outbred populations, such as human populations. In domesticated animals, genetic diseases are often recognized when multiple family members suffer from the same disease or when it is recognized that a particular breed has an increased frequency of a particular disease. You will spend the next two days hearing about the study of these diseases, what has been accomplished, what will be possible in the near future, and what impact these studies will have on the health of our beloved pets. What I will try to do in the next 40 minutes is to review how genetic diseases have been studied in mammals, and are likely to be studied in the near future, with reference to basic genetic principles, and using various real world examples. What I hope you will recognize is that, while the technologies that we use are advancing by leaps and bounds, the basic approach really doesn't change, and is absolutely dependent on practicing veterinarians to recognize and diagnose animals with disease, for research veterinarians to study the biology and pathology of these diseases, and for breeders to maintain good records and cooperate with veterinarians and geneticists to study and eliminate these diseases. This participation by breeders and veterinarians will only become more critical as, with advancing technologies, we will be able to tackle the more common and more complex genetic diseases.
It is worth going back to the basics to think about how the DNA is organized in the cell, and what DNA does. DNA is a long polymer of four similar but distinct subunits called nucleotide bases, and abbreviated A, C, G, and T. DNA is double stranded, and the bases on each strand pair up, forming a structure much like a twisted ladder, therefore the size of a DNA fragment is often given in units of base pairs or bp. The DNA is found in the nucleus of the cell, and is organized into multiple different strands, called chromosomes. The number of chromosomes is different for each species, and most cells have two copies of each chromosome, with the exception of the sex chromosomes (males have one copy each of the X and Y chromosomes, females have two copies of the X chromosome). Eggs and sperm have only one copy of each chromosome. The process by which eggs and sperm are formed, each with only one copy of every chromosome, is called meiosis, and has special importance for geneticists. During this process, the two chromosomes of each pair line up exactly, basepair by basepair, with one another and DNA can be exchanged, in a process called recombination. Because the probability of recombination between bases increases at increasing distance, genes (and the traits they determine) that are located near each other on the same chromosome are more often inherited together from parent to offspring, than expected by chance. If one has access to many families in which various genetic traits can be detected and followed from generation to generation, the frequencies at which traits are inherited together can be observed accurately, and can be used to determine the relative locations of the genes encoding the traits. Geneticists call this genetic mapping. This can be accomplished without any knowledge of DNA sequence, an example of which was reported by Drs. O'Brien and Patterson and colleagues (O'Brien et al., 1986) for the beta hemoglobin and albino gene loci in cats.
Happily, we DO have knowledge of DNA sequence of dogs and cats. The entire DNA content of one copy of each of the chromosomes (including the sex chromosomes) is referred to as the genome. In mammalian species, the total amount of DNA in the genome is between 2.4 and 3.0 x 109 bp. Only about 1% to 2% of this DNA is composed of protein-encoding genes. Much of the remaining 98% is free to accumulate some amount of variation between the individuals of a species. Again, geneticists take advantage of this situation. We started by identifying fragments of DNA that contained simple sequence repeats, such as CACACACACACACACACACA, written as (CA)n, with n referring to the number of times CA is repeated. Simple sequences often vary among individuals, with different alleles having different integer values for n, and with many different alleles possible in a population of individuals. In mammalian genomes, thousands of instances of instances of (CA)n repeats are scattered around the genome, and in each instance, are surrounded by DNA of unique sequence that can be the basis for specifically detecting different alleles. These uniquely identifiable variable repeat sequences therefore "mark" the genome, and can be followed from generation to generation in dog and cat pedigrees, just as the inheritance of visible traits or diseases can be followed through pedigrees. A whole genome scan refers to the VERY large undertaking where hundreds of simple sequence "markers" distributed across all of the chromosomes are assayed in pedigrees of dogs or cats containing individuals affected with a particular genetic disease of interest (up to hundreds of individuals), and analyzed statistically to detect instances where marker alleles "follow" the disease through the pedigrees more consistently than expected by chance. A marker that behaves this way is located near the gene that has a disease-causing mutation. In the past, the marker or markers could then be used to isolate the nearby DNA and genes that it contains in order to find the appropriate gene and mutation. Today, the DNA sequence of several mammalian species has been determined, dog and cat among others such as human, mouse, rat, cow, and chimpanzee, and most of the genes contained in these genomes have been identified. Therefore, now we can know precisely where the polymorphic (the geneticists term for markers with many different alleles or variants) markers are located, and which genes are near them. Therefore, the process of going from an associated or "linked" marker to finding the disease-associated gene and mutation is much less laborious than it was before the whole genome sequences were determined.
The dog genome sequencing project was conducted in a way that will allow dog geneticists to take advantage of more efficient technologies. DNA from dogs of several breeds was sequenced, and revealed millions of single bases that differed between individual dogs, and are referred to as single nucleotide polymorphism (SNPs, pronounced "snips"). These SNPs can be used in the same way as the simple sequence repeat markers, that is as markers to examine for association with disease. The technology now exists to assay hundreds of thousands of SNPs in a single experiment, and you will hear more details in later talks on how this approach is conducted, and how much more powerful it will be.
With this brief review of the basis of genetic approaches, let us turn our attention more specifically to genetic diseases, with specific emphasis on genetic testing. Because most dogs and cats don't get to pick their mating partners, there exists the unique opportunity to eliminate particular genetic diseases If the cause of the genetic disease can be identified. This cause will be a DNA change that deleteriously affects expression of the gene or the function of the protein gene product, or some non-deleterious DNA-change that is located near and associated with the disease-causing mutation. Once a disease-causing mutation, or sequence variation associated with genetic disease, is identified, it can be used as the basis of a genetic test of the animals that may be used for future breeding. Among the tested animals, breeding pairs can be chosen that will not produce offspring that will be affected with the particular genetic diseases. This is particularly important for recessively inherited diseases, where animals carrying one mutant allele are not otherwise distinguishable from normal animals. Therefore, at least in the present day, an important outcome of canine and feline genetic studies is the development of genetic tests.
All information that can be collected about a disease is useful for genetic studies, not just pedigree information. The information available can be loosely placed into one of two general categories, which can be thought of as "positional" information, represented on the left side of the figure below, or "biological" information, represented on the right side. Collecting DNA from affected animals and their unaffected relatives, combined with genome mapping and sequencing information allows whole genome studies that locate the approximate chromosomal position of a disease-causing mutation. Depending on the study, the region identified may contain few or many (up to hundreds) of genes. Any clinical and laboratory information also contributes to genetic studies, as indicated on the right, and results in hypotheses on what protein or biological process is likely to cause the disease. Both positional and biological information point to one or more genes that are good candidates for further study, including sequencing to identify DNA sequence differences that may cause the disease. The study of any genetic disease will be based on some combination of information of both types. Also, information concerning phenotypically similar diseases in humans can be critical to approaching genetic disease in dogs and cats.
Our first experience with cystinuria in the Section of Medical Genetics at the University of Pennsylvania occurred when a young male Newfoundland dog came to our clinic with urinary tract obstruction due to cystine stones. Cystinuria is a defect of amino acid transport in the renal tubules, and affects cystine, which has low solubility at low pH, and the dibasic amino acids ornithine, lysine, and arginine. In humans, the disease is inherited as an autosomal recessive trait. However, at that time, it was assumed but not proven to be X-linked recessive in dogs. Dr. Casal performed breeding studies that demonstrated cystinuria in Newfoundlands was autosomal recessive (Casal et al., 1995). At about that time, mutations in the SLC3A1 gene were identified in human cystinuria patients, so we cloned and sequenced this gene in affected Newfoundland dogs, identifying a disease-causing mutation that is the basis for a genetic test. Cystinuria is found in many other breeds, but does not have the same genetic defect as in the Newfoundlands. Additional studies are ongoing to determine the genetic basis of this disease in other breeds.
X-linked Severe Combined Immunodeficiency (XSCID)
In XSCID, both cellular and humoral immunity are essentially absent, and affected individuals succumb to overwhelming infection early in life, if not treated. In humans, this disease is referred to as the "boy in the bubble" disease, and was recognized in Basset hounds in the late 1970's in the Section of Medical Genetics (Jezyk et al., 1978). After the X-linked recessive mode of inheritance was confirmed in dogs, we undertook experiments using polymorphic markers on the X chromosome to show that the disease in dogs mapped to the analogous region of the X chromosome as it did in humans (Deschenes et al., 1994). As with cystinuria, a candidate gene was discovered in humans, and a mutation in this gene also proved to be responsible for XSCID in the Basset hounds (Henthorn et al., 1994). Coincidentally, we identified XSCID in Cardigan Welsh corgi dogs (Pullen et al., 1997) identified a different mutation in the same gene as in the Basset hounds (Somberg et al., 1995). Because the mutation was a recent event in that breed, and a group of breeders worked very hard to identify dogs at risk of being carriers, we were able to develop a genetic test and eliminate the mutant allele from the Cardigan Welsh Corgi breeding population.
Selective Malabsorption of Cobalamin
Selective cobalamin (vitamin B12) malabsorption, known as Imerslund-Grasbeck syndrome, is a recessive disorder in man and also well characterized in giant Schnauzer dogs (Fyfe et al., 1991). Before 2000, mutations in only one gene, cubilin gene (CUBN), had been identified in human patients, and the cause this disease in dogs was unknown. Genetic studies in the dog, including CUBN sequencing and linkage analysis in the region of the CUBN gene, ruled out this gene as a cause of the canine form of the disease. A whole genome scan in a large canine pedigree demonstrated linkage of markers on dog chromosome 8 to the disease gene. This region contained the amnionless gene, AMN, which had concurrently been shown to harbor disease-causing mutations in some human patients (He et al, 2003 and references therein). Thus, these comparative-mapping data provide evidence that canine Imerslund-Grasbeck syndrome was a homologue of one form of the human disease and provided a useful system for understanding the molecular mechanisms underlying the disease in humans.
Technologic advances in the study of genetic disease in humans have been translating to veterinary medicine. Genetic studies in dogs and cats are becoming increasingly more promising, and will rely even more heavily on positional information for the identification of candidate genes. While the most immediate uses of the information gained from these studies will be in genetic testing to allow for selective breeding, future advances in genetics will also play a role in the understanding of disease mechanisms that will lead to new therapies.
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