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Identification of Genetic Markers for an Inherited Craniofacial Deformity Syndrome in Burmese Cats.

Leslie A. Lyons, Ph.D., Marilyn Menotti-Raymond, Ph.D., and Stephen J. O'Brien, Ph.D.

Laboratory of Viral Carcinogenesis, Frederick Cancer Research and Development Center, National Cancer Institute, Building 560, Room 21-105, Frederick, MD 21702-1201

Requested Funding: $15,000.00

Proposal is in response to the NABB request for proposal: A Study of Craniofacial Malformations Occurring in Burmese Cats.

Introduction

The Burmese became an established breed of pedigreed domestic cat in the late 1930's. The foundation of this breed in the United States originated with an importation of a single female, "Wong Mau", from the capital of Rangoon. Wong Mau was phenotypically distinct from the Siamese cats of her homeland in that she had a distinctively more cobby body frame with a walnut-brown coat color, exhibiting darker brown points. A breeding program was established initially between Wong Mau and Siamese cats, then with successive backcrosses and brother-sister matings (1). Over several generations, it was established that Siamese, intermediate, and Burmese type cats could be produced. The Siamese cats express coloration most markedly at their points; the intermediates, like Wong Mau, (now know as Tonkinese) express coloration throughout their coat with darkening at the points; and the Burmese express a deeper, richer, coat color with less point demarcation. These cats are now known to possess different alleles at the albino locus, C, for coat color (2,3). The Siamese carrying cscs, the Burmese carrying cbcb, and Tonkinese expresses the incomplete dominance of the alleles, carrying cscb. Each genotype expresses less than full production of pigment in the coat color. Along with the cobbier body frame, the cats with the cbcb genotype established the Burmese breed, and were accepted for stud book registration by the Cat Fancy Association (CFA) in 1936. The present CFA standard for the Burmese breed reflects a cat of medium size with substantial bone structure, good muscular development and a surprising weight for its size. The head should be pleasingly rounded without flat planes. The face is full with considerable breadth between the eyes and blends gently onto a broad, well developed short muzzle. A visible nose break is present, the eyes are large, set far apart, with rounded aperture (4).

During the 1970's, a alternative style Burmese cat was established. Phenotypically still within the CFA standard, this strain of Burmese expresses a more rounded head with a higher frontal prominence, a shorter, broader muzzle, seeming larger and more prominent eyes, and generally a more demarcated nose break. This shorter, broader muzzle form has been referred to as the "Eastern", "new look", "Contemporary", or "more extreme". The longer, narrower muzzle form is referred to as "Traditional" or "less extreme". The "more extreme" strain of the Burmese quickly became popular in the show ring and intensive breeding programs ensued. Shortly after the widespread establishment of the "more extreme" strain, an increasing proportion of litters involving the "more extreme" cats were producing kittens with a severe congenital craniofacial deformity. An investigation of the defect by Zook et. al (5), provided a detailed clinical description of the defect as well as a suggestion that the deformity may elicit an autosomal recessive mode of inheritance. By 1983, over 90 purebred Burmese from a disperse group of catteries had been afflicted with the deformity. No obvious infections, toxic agents or environmental conditions could be correlated with the deformity. The common genealogy of the cats producing the deformity in their litters revealed cats of common ancestry that had been extremely proliferative, including a line of show-winning cats that had been extensively bred. This suggests that the Burmese have experienced inbreeding depression within a sub-population of the breed and the "more extreme" cats were producing deleterious homozygous recessive offspring as a consequence of inbreeding.

A research cooperative was established to investigate causation and the mode of inheritance of the craniofacial deformity (6). The cooperative was known as The Burmese Cooperative Research Project, and a cattery, Searchcore, was established to oversee the test breedings for the project. Under the assumption of a recessive mode of inheritance, Searchcore established matings between known carriers (all "more extreme") and non-carriers and/or cats of unknown status (both of the "less extreme" phenotype). The kittens of the resulting litters were then bred to known carrier cats, which all were of the "more extreme" phenotype. The 33 second generation matings produced 151 kittens of which 20 expressed the craniofacial deformity. The interpretation of the test matings and data collected from 46 questionnaires to breeders is convoluted and unclear. Frances O. Smith, D.V.M., of the University of Minnesota, was enlisted by the Searchcore to interpret the pedigree analysis. Dr. Smith suggested an incomplete dominant mode of inheritance and that the deformity was a result of the "more extreme" phenotype (6). Deformed kittens being homozygous and the heterozygous form to be expressed as the "more extreme" phenotypic cats. Searchcore also established a collaboration with Cornell University to investigate the developmental mechanism of the deformity (7). Over 40 of the deformed kittens were examined. The defect was originally described as either maxillonasal hypoplasia (5) or incomplete diprosopus (8). The Cornell study initially suggested a mechanism of transformation of the medial nasal part of the frontonasal process, naming the defect: telencephalic meningohydroencephalocele. This defect was also referred to as: Incomplete conjoined twinning, by the Cornell group. This group restated Dr. Smith's interpretation that the "more extreme" phenotype is a less severe expression of the deformity, and some homozygotes cross a threshold which results in the lethal malformation. This may be tested by examining facial and cranial measurements in an attempt to qualitate the structure variations. The inheritance pattern was informally addressed by Sponenberg and Graf-Webster in 1986 (9). Of 22 litters born to matings between Burmese parents which previously had produced the craniofacial defect, 19 of 88 kittens expressed the deformity. The 69:19 ratio did not deviate significantly from an autosomal recessive mode of inheritance (X2 = 0.545) at the 95% confidence interval. The gene symbol, mc, was suggested by Dr. Roy Robinson to represent the meningoencephalocele syndrome (9). The association of the craniofacial deformity with the "more extreme" phenotype of the Burmese has raised a severe dichotomy amongst the Burmese breeders and a rift within fancy cat breeding societies. Conflicting results of the initial investigations, concerns of accuracy, confidentiality, and inaccurate interpretations has posed the question as to whether the "more extreme" phenotype should be propagated, and to whether "more extreme" phenotypes can be maintained without the deformity. As a result, the National Alliance of Burmese Breeders (NABB), a solely "more extreme" Burmese breeding group, has initiated a request for proposals for the Study of Craniofacial Malformations Occurring in Burmese Cats.

This proposal is in response to the request of the NABB. We propose a combination of a prospective and retrospective study to investigate the genetics of the craniofacial deformity expressed in a portion of Burmese cats. We have reviewed the previous studies and the limited available data, and have determined that a single gene is highly likely to be responsible for the defect, thus a beneficial study can be initiated. The project has four goals: 1) formally verify the mode of inheritance of the craniofacial defect and formally address the mode of inheritance of the Burmese facial morphology, 2) establish genetic linkage or non-linkage between the craniofacial defect and facial morphology in formal pedigree analysis of existing and proposed pedigrees, 3) test a panel of high resolution polymorphic genomic markers, feline microsatellite loci, for linkage to the craniofacial deformity and Burmese facial morphology, 4) provide suggestions for the eradication of the deformity from the Burmese breed.

Identification of Genetic Markers for an Inherited Craniofacial Deformity Syndrome in Burmese Cats.

L.A. Lyons, M. Menotti-Raymond, S.J. O'Brien.

Inbreeding depression, as a result of population bottlenecks and founder effects, has been shown to cause severe health and reproduction problems in cats (10,11). Bottlenecks can be a result of natural phenomenon or man-made crisises. A portion of the Burmese cats has apparently experienced a man-made bottleneck and founder effect situation. This is inherent in most all domesticated breeding programs, due to the artificial selection of particular desired phenotypes and the culling of undesired phenotypes. Unfortunately, an unforeseeable, undesirable trait has reached a critical level in portions of the Burmese breed, threatening this sub-population with extinction.

Study Design

This study will be divided into three parts in order to address: 1) the Burmese associated craniofacial deformity mode of inheritance, 2) the mode of inheritance of the dichotomized facial structure of the Burmese, 3) determine if the deformity can be separated from the "more extreme" Burmese phenotype, 4) develop genetic markers for the possible identification of deformity carriers and, 5) provide suggestions for the eradication of the deformity. The collection of the data and the analyses of the modes of inheritance, and baseline breed data should be obtainable within 12-18 months. Identification of a marker associated with the deformity/head structure can proceed once a database is established, but is not predictable. We are currently proceeding with a 5-10 year plan for a marker saturation of the feline genome.

Part 1: Pedigree Ascertainment

With the cooperation of NABB and other Burmese breeders, Burmese pedigrees will be collected to further investigate the modes of inheritance of both the craniofacial deformity and the "more extreme"/"less extreme" phenotypes. These same pedigrees will be used for the marker identification. Pedigree collection will include: blood samples (20ml or appropriate to cats weight), a tissue biopsy, phenotypic data, and breeding records. Ascertainment will be through the identification of a single deformed kitten. Retrospectively, if all parental and grandparental cats of a litter, which has previously included at least one deformed kitten, are available, then these pedigrees will be selected for the study. Kittens from litters of the exact parentage, even though a deformity may or may not have occurred in that particular litter, will be of interest. Prospectively, once a current or future breeding produces a deformity, we will collect that pedigree and any previous or future exact parentage litters, regardless of deformity presence or absence. We will request that immediately post-mortem, deformed kittens be kept frozen until shipment to our research facility. If known outcrossed breedings, implying either "less extreme" to "more extreme" or "more extreme" to a different fancy cat breed can be identified, these pedigrees would also be collected. Pedigrees of lines never associated with the deformity but exhibit a range of phenotypic expression will also be collected. Deformed kittens will be grossly examined for conformation to the defect and karyotypes will be performed on a two male and two female kittens. A formal segregation analysis using the pedigree analysis program, Pointer, will be used to evaluate the data (12) to determine modes of inheritance.

Part 2: Breed Sample Collection

In order to establish a baseline of the gene pool and to determine the inbreeding depression for the Burmese cats, we will require the collection of 10-20 blood samples (20ml each) of various fancy cat breeds. This will include non-pedigreed domestic shorthairs; and breeds associated with the Burmese, including; Siamese, Tonkinese, Dilute Burmese (Malayans), Persians, Tiffanys, Bombays, and Foreign Burmese. The genetic background of these cats will be important for future breeding recommendations, should the deformity not be separable from the "more extreme" phenotype, and to determine the "genetic" health, or inbreeding depression, of the breed. Until the genetic background of the breeds can be established with the genetic markers, test crosses will not be suggested for the study. Outbred cats should show a higher level of heterozygosity than line bred cats. The reduced heterozygosity is a measure of inbreeding depression.

Part 3: Genetic Marker Isolation and Characterization

Initially, deformed kittens will be examined for correlations with similar syndromes in humans (13). Should strong similarities be identified, then candidate genes for the defect may be suggested. If a candidate gene is suggested, this gene would be then mapped in the cat using feline interspecific backcrosses (14), thus potentially narrowing the area of the genome to scan for associated genetic markers. Micro-satellite genetic markers will be isolated and examined for polymorphism in the Burmese pedigree parents (15,16). Polymorphic markers will be typed in the pedigrees to establish the linkage of a marker with the deformity and/or head structure phenotype. Several linkage analysis programs are available, including LIPED, LINKAGE, MAPMAKER, and CRI-MAP(17-20). These programs will determine marker association with the deformity and/or head structure phenotypes and provide an estimate of distance which would be used for the accuracy of carrier detection and future breeding recommendations. Marker typing will proceed immediately in the parents, grandparents, and the associated breeds. Pedigree typing will proceed once the database is significant.

Logistics

Prior to sample collection, all Burmese breeders and breeders of the associated breeds (as listed above) will need to be informed of the study and of the required pedigree ascertainment. This will be initiated in appropriate newsletters and coordinated with the assistance of concerned volunteers. Once appropriate pedigrees and breeders are confirmed, sample collection coordination will begin. Sample collection may be most efficient and accurate at various CFA cat shows. Veterinarians will be required to assist in the sample collection, which will require anesthesia administration, blood sample collection and tissue biopsies. CFA judges will be required for phenotypic evaluation of the parental, grandparental cats, and mature offspring. Photographs, frontal and profile, will be taken of the cats for permanent record. To maintain consistency, we will minimize the number of CFA judges. CFA judges which breed Burmese cats will not be employed to avoid any possible phenotypic biasing. The phenotypic data may be quantatized be making various head measurements during the phenotypic evaluations. The measurements would at least include: muzzle breadth, distance between ears, distance between eyes, head size, as well as others. A significant database must be established prior to pedigree analyses. If pedigrees were known to be matings of two parents heterozygous for the defect (both carriers) and the genetic markers are also heterozygous, then approximately 60 offspring could detect markers of a minimum of 5cM, implying a test approximately 90% accurate for the determination of carrier status. More accuracy requires an exponential increase in offspring of the compound heterozygous matings and in marker typing. Cats which "breed true" implies homozygosity of the genes causing the selected phenotype. Markers close to the selected genes will also be homozygous and make mapping difficult at closer distances. Less informative matings, marker homozygosity, will increase the required offspring. Over 300 markers, spaced at 10cM intervals, will be required to saturate the genome, which will be selected from over 1,000 random markers.

References

1. Thompson J et al: Genetics of the Burmese cat. Heredity 34, 1943

2. Robinson R: Genetics for Cat Breeders. 3rd Ed. Pergamon Press, Oxford, 1991.

3. O'Brien SJ et al: Chromosome mapping of beta-globin and albino loci in the domestic cat reveals mammalian chromosome group. J Hered 77:374-378,1986.

4. Show Standards: The Cat Fanciers' Association, Inc. May 1,1993-April 30, 1994.

5. Zook BC et al:Encephalocele and other congenital craniofacial anomalies in Burmese cats. Vet Med/Small Anim Clin 78:695-701, 1983.

6. Searchcore: Report of the Burmese Research Group. June 14, 1984.

7. Noden DM and Evans HE: Inherited homeotic midfacial malformations in Burmese cats. J Craniofacial Genet Devel Bio (Suppl) 2:249-266, 1986.

8. Sekeles, E: Craniofacial and skeletal malformaitons in a cat. Feline Prac 11:28-31, 1981.

9. Sponenberg DP and Graf-Webster E: Heredity meningoencephalocele in Burmese cats. J Hered 77:60, 1986.

10. O'Brien SJ et al: East African cheetahs: Evidence for two population bottlenecks? Proc Natl Acad Sci USA 84:508-511, 1987.

11. Roelke ME et al: The consequences of demographic reduction and genetic depletion in the endangered Florida panther. Curr Biol 3:340-350, 1993.

12. Lalouel J-M and Morton NE: Complex segragation analysis with pointers. Hum Hered 31:312-321,1981.

13. McKusick VA: Mendelian Inheritance in Man. Johns Hopkins University Press, Baltimore, 1991.

14. Copeland NG and Jenkins NA: Developoment and apllications of a molecular genetic linkage map of the mouse genome. Trends Genet 7:113, 1991.

15. Weber JL and May PE: Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44:388-396, 1989.

16. Stallings RL et al: Evolution and distribution of (GT)n repetitive sequences in mammalian genomes. Genomics 10:807-815, 1991.

17. Ott J: A computer program for linkage analysis of general human pedigrees. Am J Hum Genet 28:528-529, 1983.

18. Linkage Analysis Package: Linkage Version 4.8, January 1989.

19. Lincoln S and Lander ES: Constructing Genetic Linkage Maps with MAPMAKER. 1987.

20. Lander E and Green P: Construction of multilocus genetic linkage maps in humans. Proc Natl Acad Sci 84:2363-2367, 1987.

Budget

Principle Investigator support: 3,000.00 (Travel to CFA shows, catteries, coordination efforts)

Transport of samples: 1,000.00

Veterinary Fees: 1,000.00

Technical Support: (Salary for 20 hrs/wk) 10,000.00

Total: 15,000.00

This genetic marker identification is a direct function of man-power and will be facilitated by this project and other projects contributing to the feline genome project. The technical support will cover sample processing and support, DNA isolation, and marker typing of the collected samples, specific to this study. Currently, nearly 100 micro-satellite markers are available for testing. An estimate of at least 12 samples will be collected for each ascertained deformity, including 4 grandparents, 2 parents, and 6 offspring of the same or other exact parentage matings. Heterozygous mating types would require at least 10 such pedigrees, minimum 120 samples, implying over 12,000 typings. Materials for processing and marker isolation and typing will be covered by the NCI. Below are estimates of actual costs: 1) Materials for collecting and processing 120 samples: 6,000.00 (vacutainers, needles, saline, lysing solutions, tissue culture media, DNA isolation buffers...) 2) Isotopes for DNA test 6,000.00 3) Equipment for microsatellite assay 15,000.00 4) Storage of sample at -70 C. 2,000.00 5) Computer software 2,000.00

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