Candidate gene screening and genetic mapping of loci for feline autosomal dominant polycystic kidney disease.
Leslie A. Lyons, PhD1, Marilyn M. Raymond, PhD1, David S. Biller, DVM2, Stephen DiBartola, DVM3 and Stephen J. O=Brien, PhD1
1Laboratory of Genomic Diversity, National Cancer Institute, FCRDC, Building 560, Room 11-10, Frederick, MD 21702-1201
2Kansas State University, Department of Clinical Sciences, College of Veterinary Medicine, Manhattan, Kansas 66506-5606
3Ohio State University, College of Veterinary Medicine, Department of Veterinary Hospital, 601 Vernon L. Tharp Street, Columbus, OH 43210-1089
$15,000.00 per year, 2 years.
Scientific Abstract
An autosomal dominant form of polycystic kidney disease (PKD) has been characterized in the Persian cat breed. This non-syndromic form of polycystic kidney disease has an early onset with multiple bilateral cysts. Severity of the condition is highly variable although the disease is fully penetrant. Disease heterogeneity has not been evaluated although only Persian cats and cats of Persian origin, such as Exotics, appear to have the heritable form. Sporadic kidney cysts do occur in other cat populations but are not generally bilateral and multiple in presentation, nor at an early age. Ultrasonic evaluation of the kidneys can reveal cysts as early as 6 to 8 months of age and diagnosis is certain by 12 months. Clinical severity has a broad range and a majority of cats can go undiagnosed throughout their life. Hepatic cysts are occasionally present. Treatment is restricted to management of poor kidney function and any resulting secondary conditions. Several inherited forms of polycystic kidney disease are known in other species, including humans, mice, and rats. Several genes have been isolated in these species for single gene defects, both autosomal dominant and recessive in inheritance. A large outbred PKD pedigree exists that originated from Persians. We propose to augment the present pedigree to allow a more powerful linkage analysis to verify locus selection, to identify a causative gene and develop a rapid, robust molecular screening technique to identify carrier animals for selective breeding. We will also be interacting with PKD researchers in human medicine in order to further delineate the differences between the feline and human conditions in an effort develop drug therapies for the cat.
Layman Abstract
Some Persian cats have been shown to have a disease of the kidneys (PKD) that is inherited is a dominant fashion, only one copy of the mutation is required to cause disease. The disease causes cysts to occur in the kidneys that impairs the normal function. The cysts start to occur very early and they can generally be detected by 6 to 8 months of age using ultrasound detection. Cats can be considered free of the disease if cysts have not formed by 12 to 18 months. Although kidney cysts can be found in many types of cats, the cysts found in the Persians occur very early, will be found in both kidneys, and generally more than one cyst will be present. Since only Persians and cats that originated from Persians, Exotics, have been shown to have this inherited form, probably only one mutation is causing the disease. Some cats can have very mild cysts while others have very severe problems, so many cats can go undiagnosed for the disease. Treatment is limited to assisting the cats kidney functions. Liver cysts can also occur, thus some cats show liver problems too. Secondary health problems can arise due to poor kidney or liver function. Inherited kidney diseases are well documented in humans, mice and rats. Several types of kidney diseases are know and a causative gene has been found for two forms. Several kidney diseases have been shown to not be caused by the two know genes, thus others must be in the genome. We propose to test if the two known genes that cause kidney disease in humans are the cause of feline PKD. We will also develop a pedigree study that will prove or disprove an association of feline PKD with the known genes, this will also help to identify other genes that may be involved.
Background
Polycystic kidney disease (PKD) is a well documented abnormality in domestic cats, particularly of Persian breed origin. Cystic kidneys can sporadically occur in any population of cats, but early onset and bilateral presentation is hallmark to the hereditary form. In Persians, the condition has been shown to be inherited as a single autosomal dominant gene. It is esttimated over 37% of the Persians have PKD, a breed that accounts for nearly 80% of the cat fancy. Although the disease in Persians is inherited as an autosomal dominant, cystic kidneys are not usually life threatening until later years and a majority of cats have gone undiagnosed for the condition. No cure is available and clinical management is designed to prevent or minimize kidney failure and secondary conditions arising from poor kidney function. Other clinical findings have included hepatic cysts. Extensive clinical diagnostics and managements have been under investigation on a large cat colony of Persian origin at Ohio and Kansas State Universities for nearly a decade. These studies have lead to reliable and robust diagnostic methods and standards. The presence of cystic kidneys can be determined by 6 to 8 months of age by ultrasonic techniques and affection diagnosis is generally certain by one to two years. These ultrasonic evaluations have a 91% sensitivity and 100% specificity at 9 months. The investigators at Kansas and Ohio State have been involved with researchers at the Laboratory of Genomic Diversity (LGD) of the National Cancer Institute for several years regarding the identification of candidate genes for feline polycystic kidney disease. Blood samples, pedigree information and disease status has been relayed to the LGD to develop linkage studies and mutation screening of candidate genes. There are a plethora of conditions in humans that have an association of polycystic kidneys. Several conditions that have major kidney involvement and are predominantly diagnosed as polycystic kidney diseases have been shown to be controlled by single gene defects, some dominantly inherited, some recessively inherited. Table 1 lists details of these conditions.
Clinical Design
The continuation of the Persian PKD study can be approached by several different laboratory methods but they are all dependent on reliable clinical determination of disease status. The Persian breed accounts for nearly 80% of the cats in the cat fancy, thus sample availability is not of concern, but affection diagnosis must be reliable. We would like to focus our project by working with several large Persian catteries so that reliable and extensive pedigree and closely related individuals can be evaluated using very standard and reproducible diagnostic criteria. This can be best accomplished by indentifying cooperative breeders with large Persian catteries and setting up an ultrasound clinical with particular equipment and personnel. We will interact with the Persian breed council to help establish the catteries for inclusion in the study. Inclusion will require large catteries that have already been shown to have PKD in their Persians. Three generations of cats will be required, in the form of siblings, parents and grandparents. Half-sibling families will be acceptable. Priority will be given to the most extensive pedigrees and the availability of the parents and grandparents. Not all cats need to reside in the same cattery, but must be readily available for blood sample collection and ultrasonic evaluations. Pedigrees that are also segregating for other conditions, such as retinal atrophies, heart disease, cryptorchidism, or coat colors will be extremely valuable since these samples could then be used by other projects.
Laboratory and Analysis Design
The laboratory procedures will follow two approaches to find the feline gene for PKD. Firstly, since specific human and mouse genes have already been isolated for human and mouse PKD, these genes will be isolated from the cat and screened for specific mutations in affected cats. The feline specific genes will be isolated from the Purina cat-BAC genomic library and from PCR amplification of feline genomic DNA using primers that were designed from analyses of the human and mouse sequence of the gene of interest. This type of procedure is currently ongoing for the cat gene for PKD1. Concurrently, we will map the candidate genes in the feline interspecific hybrid pedigree. Once the genes are mapped, we can identify specific microsatellite markers that are located very close to the genes of interest. These markers will then be screened in the collected PKD families for an association with the disease. This approach will at least provide information as to which gene may or may not be involved with feline PKD. With this information, more exhaustive techniques can be applied to the candidate gene identified by the linkage analysis. The linkage analysis should suggest one of the known human or mouse genes to be the causative gene for cats, but it may also suggested a totally different gene. The success of the linkage study will be dependent on sample size, proximity of genetic markers, and marker polymorphism. Close markers can be isolated using the Purina cat-BAC library, thus although 250 markers are available for cats, better and specific markers can be readily isolated. Marker polymorphism is always a concern when working with closed populations. We must maximize polymorphism, thus selected catteries will be diverse, covering many different lines and could extend to related breeds, such as Exotics. Exotics may be of particular value due to their relatively more recent outcrossing.
Animal (Sample) Requirements
The current pedigree under analysis was produced from outcrossing a Persian cat that had PKD. The pedigree has approximately 150 cats that were used to determine mode of inheritance and clinical presentation. DNA samples are available from only a small portion of this pedigree and a 38 member pedigree is currently being used for the linkage analysis. This pedigree has the power to determine linkage with markers very close to the gene of interest with a 87% probability for markers with a distance of less than 5cM, but the power significantly decreases as markers become more distant. This pedigree can exclude a candidate gene with an 75% probability for markers with a distance of up to 10 cM. Since are current genetic map does not have adequate coverage to ensure markers are only 5 cM in distance, we require more pedigrees to increase the power of the study. With the addition of 4 three-generation pedigrees with 6 offspring each, the power of the study increases significantly. Linkage could be detected with markers as far as 15 cM and excluded at up to 25 cM distances with even higher probabilities. This is well within the range of our current map.
Result Expectations
The current colony provides a strong foundation for the study as does the knowledge of specific genes that cause PKD in other species. Since Persians are an extremely large breed, identification and acquisition of samples should be not be difficult but will require large efforts in coordination, particularly since clinical evaluations are also required. The linkage analyses should definitively determine the genetic region of interest provided enough families are collected, and the genetic markers are polymorphic. Polymorphism can be evaluated concurrently and can be somewhat over come by including more families. The linkage analyses should provide a marker that can be immediately used for genetic testing for early identification of affected cats. A linked marker can have an accuracy of 80 - 99%. If one of the candidate genes is suggested as being the gene of interest, mutation screening will be required to identify the exact genetic problem. This would provide a test of 99% accuracy but finding mutations is more difficult to predict. If the gene is identified, the expectations are extremely high, but if the gene is not known, then extensive work will have to be performed to first find the gene. Thus, the PKD project has many advantages over other genetic studies currently ongoing in the cat and has a very high probability of success. Genetic testing could then be accomplished by cheek swabs after kittens are weaned or from blood samples at an earlier age. Earlier detection will assist breeders with the management of their cattery and the development of their showing program. Project Length: 2 years Since samples are currently available, all procedures can proceed concurrently. Newly collected samples will augment the currently available colony. Candidate genes are currently being screened. Should the gene be identified prior to 2 years, funding will be directed at carrier test development if the gene is known or gene isolation if the genetic region is known but the gene is not yet isolated.
Project Budget:
The project funds will primarily be used to establish ultrasound clinics for the pedigrees of interest. A mailing and or website could be established to solicit assistance from Persian and Exotic breeders. The specifics of the pedigree structure and participation requirements would be presented. At least four 12 member pedigrees will be required but sufficient cats will have to be screened to identify appropriate pedigrees and to confirm diagnoses. This may necessitate hundreds of screenings. Our lab had already expended over $3,000.00 to the collection of PKD samples. The cost of a clinic will be highly dependent on the cooperating veterinary facility but it can be estimated to be $50.00 - 100.00 per cat, including travel for the investigators to the clinic. Funding can be awarded to any of the cooperating research facilities or to a third party (Winn Foundation, CFA, or the Persian Breed Council) that would expedite payment to veterinary clinics.
150 cats x $100.00 = $15,000
PKD Project Progress Report:
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