10 April 2002
Winn Feline Health Foundation
1805 Atlantic Avenue
PO Box 1005
Manasquan, NJ 08736-0805

Dear Hilary and Tom,

This is a progress report on the project entitled "Identification of Genetic Markers for an Inherited Craniofacial Deformity Syndrome in Burmese Cats". No previous report has been made on this project as funding for the project was not received until February, 2002, at UCDavis. The Center for Companion Animal Health, CCAH, has provided matching funds of $10,000.00 per year for this two-year project. In general, funding for this project supports 50% of the salary for Dr. Heather Roberts, a post-doctoral fellow in the laboratory who also works on PKD. Recently, the Burmese head defect has been appearing in American Shorthair breedings, thus a new enthusiasm has been gained by the breeders. We have received several stillborn kittens with the defect from both Burmese and ASH breedings this past week. We plan to provide a detailed report to the breeders at the CFA annual this June. ASH breeders are experiencing several of the other anomalies that are seen in Burmese, such as "Cherry eye", which may be a result of the gene or that the shortened facial structure is becoming too extreme. My interpretation is that the facial structure is becoming too extreme. These issues will be clarified and discussed in June. The recently obtained defects are consistent with what has been observed with our previous collections. With over 30 deformed kittens, we find the condition to be a very consistent presentation, thus we need to be careful with the new observations by the ASH breeders. We also have now identified a strong candidate gene for the condition. Many genes are known to control facial and body patterning, these are termed HOX genes. HOX genes come in clusters of 13 genes and are located in at least four groups in the genome of the cat, hence a potential 52 genes are strong candidates. But, one gene that is not part of the HOX cluster family, is called Sonic Hedgehog (yes, like the Nintendo game). This gene is expressed specifically in the region of the upper jaw (the maxillary region), hence is currently our focus for the head defect in the Burmese. HOX genes are still strong candidates, but Sonic Hedgehog will be analyzed first. We are now designing genetic assays to isolate this gene in the cat. To compliment this approach, we will also continue to collect the kittens and the close relatives to perform an association study. This association study should suggest whether the candidate gene is correct and hence that we should continue to analyze and sequence that particular gene for a causative mutation. This preliminary data has been used for a grant submission to the NIH, National Institute for Dental and Craniofacial Research (NIDCR), April, 2002. The attached NIH proposal is confidential. This proposal is for two years and requests funding for $50,000.00 per year as a model for mammalian facial development.

Best regards,

Leslie A. Lyons, PhD
Assistant Professor, VM:PHR
Staff Scientist, CRPRC

Proposal to: Winn Feline Foundation
Attention: Mr. Thomas H. Dent
P.O. Box 1005 Manasquan, NJ 08736-0805

Submitting Organization: The Regents of the University California
1 Shields Avenue
Davis, CA 95616

Title of Proposed Research: Identification of genetic markers for an inherited craniofacial deformity syndrome in Burmese cats

Total Amount Requested: $30,000
Proposed Duration: 2 years
Desired Starting Date: 03/01/01 - 02/28/03
Principal Investigator: Leslie A. Lyons
Department: VM: Population Health & Reproduction
Phone Number: 530-754-5546

Matching Funds/Operating costs: UCDavis - Center for Companion Animal Health: $10,000.00 per year
UCDavis laboratory of Dr. Leslie A. Lyons: ~$10,000.00 per year

Scientific Abstract

The Burmese cat is a moderate size short-hair domesticated breed that is common to the world-wide cat fancy. The breed is distinguished by body conformation and are homozygote for the "Burmese" allele, cb, at the tyrosine locus. The contemporary line of Burmese cats carries an autosomal recessive cranial-facial defect. This defect produces a duplication of the upper maxillary region and is non-compatible with life. The presentation is congenital, non-pleiotrophic, fully penetrant and has clear and distinct features. The most extreme facial conformationsof the contemporary lines of Burmese cats have the highest likelihood of carrying the defect, but the exact demarcation as to facial conformation and carrier status is difficult to determine. Outcrossing of the contemporary lines of the Burmese cats to other breeds and Burmese populations naive to the defect have proven the mode of inheritance. Thirty deformed kittens, their first-degree relatives and controls from various lines of Burmese, other breeds, and random bred cats have been ascertained for the study. Feline-derived microsatellite are polymorphic in the Burmese breed, but do not have sufficient density for a genome-wide linkage analysis for the defect. We will attempted a focused approach to develop genetic markers near candidate genes using the feline BAC library. Known developmental genes, such as HOX 1-4@, will be isolated from the BAC library and positive clones will be screened with oligo probes of common dinucleotide repeats for the development of markers. Linkage between the markers and the defect will be determined by standard linkage analyses and homozygosity mapping.

Layman Abstract

The new feline genetics laboratory of Dr. Leslie Lyons at the UCDavis School of Veterinary Medicine is continuing the investigation of the Burmese head defect. The initial goals of the project when it was initiated in the summer of 1995 were: 1) determine the mode of inheritance of the Burmese craniofacial deformity, 2) determine the mode of inheritance of the dichotomized facial structure for 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 craniofacial defect that presents is very unique and distinct, no easily mistaken. The defect has an autosomal recessive inheritance pattern, implying two copies of the defect are required. The "more extreme" type is controlled by many genes that affect facial development, as for any cat. The defect does travel with the "more extreme" type from contemporary lines, but since the face type is a complex interaction with genes, determining the exact cut-off for extreme face type that may carry the defect is difficult. Hence, some moderate to slightly extreme cats, from contemporary lines, may not carry the defect, the correlation is difficult. Breeders should also realize that non-contemporary lines have also been selected for a "more extreme" type, but these lines are not at risk for carrying the defect. Genetic markers should help demarcate which cats carry the genetic defect. The genetic markers had never been tested in a domestic cat breed, only random bred cats, and we found that they will be affective for analysis. But, the present markers are not sufficient. Effectiveness of a marker search is determined by the amount of variation of the markers, how close the marker is to the defect and the sample size. A marker can be more distant with a larger sample size and if it has high variation. The smaller the sample size, the closer the marker needs to be. Breeders have been supportive of the project but hundreds of samples are not feasible. Over 250 samples from Burmese, Foreign Burmese, Egyptian Mau, American Shorthairs, Scottish Folds, and random bred cats have been collected for the study. But only thirty deformed kittens have been acquired, including deformed kittens from Belgium. Thus, with this sample size, markers had to be developed near candidates for continuation of the study. With the development of a large DNA (BAC) library in the fall of 1999, a focused approach to develop more effective markers for this study could now be accomplished. Many genes are strong candidates for facial development, including the HOX genes. But these genes are abundant and come in several large clusters. With the library, we can isolate specific genes of interest and develop markers near these genes. These markers can then be tested in our Burmese sample set. Thus, we have initiated a focused attempt to develop markers around candidate genes in order to include or exclude them as the gene causing the defect. Detection of a marker for the deformity will be performed by standard linkage analyses and homozygosity mapping. Until a marker is developed, breeders are cautioned as to using "extreme" cats from contemporary lines, either within their program or as outcrosses. The defect does manifest in Bombays and breeds that have used contemporary Burmese. Since this defect is autosomal recessive, it will be difficult to eradicate without a genetic test.

Progress Report

The Burmese head deformity project was initiated by Dr. Leslie Lyons, while at the Laboratory of Genomic Diversity (LGD) of the National Cancer Institute during the summer of 1995. This project was in response to a request by the National Alliance of Burmese Breeders (NABB) to the Winn Foundation. The Winn Foundation responded with a request for proposals specific to this project. Two years of breed-specific funding, totaling $30,000.00 was awarded for this research, summer 1995, 1997. The project was initially divided into three parts that would address five questions; 1) determine the mode of inheritance of the Burmese cranial-facial deformity, 2) determine the mode of inheritance of the dichotomized facial structure for 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. It was anticipated that the collection of the data and the analyses of the modes of inheritance, and baseline breed should be completed in 12-18 months, but identification of markers for the deformity or head structure would be unpredictable and would become a long-term project for the laboratory. The three parts to the project included, 1) pedigree ascertainment, 2) breed sample collection, and, 3) genetic marker characterization and isolation. The mode of inheritance for the Burmese head deformity has been confirmed to be autosomal recessive. The previous studies and literature had suggested this mode of inheritance, but confusions in nomenclature had occurred, giving the impression that different patterns had been suggested. The deformed kittens only occur when two copies of the defective gene have been inherited, one from each parent. Several catteries provided their breeding records that confirmed the autosomal recessive inheritance pattern. The most compelling data was obtained from France and Belgium. With the importation of one "more extreme" Burmese, deformed kittens were produced when the imported cat was found in both the maternal and paternal lineage. Data from outcrosses to American Shorthairs, Scottish Folds, and Bombays have supported this evidence. A detailed formal analysis using segregation analysis programs was not necessary for this aspect of the project due to this straight-forward and compelling data. The mode of inheritance of the head structure, "more extreme" or "less extreme", has been evaluated but not formally addressed. The development of measurements of the skull and facial structure were determined to be beyond the scope of project funding and would be difficult to obtain. Developmental experts at Johns Hopkins in Baltimore, MD were consulted for this aspect of the project. The coordination of specific individuals to evaluate head types and to get all the cats available at specific ages would be necessary and logistically difficult. Regardless of a formal evaluation, the correlation with the "more extreme" head type and cats that have kittens with the deformity is extremely strong. We have obtained no evidence that a "more extreme" cat has proven to be clear of producing the deformity. At this time, no conclusions can be made as to whether the gene(s) involved with the head structure is/are the exact same gene(s) producing the deformity. Normally, many genes are involved with determining the structure of the head. The deformity is caused by a single gene, acting autosomal recessively. We suggest that the gene for the deformity is physically near the genes involved with the "more extreme" head type, if not one of the genes involved with the structure of the head. If so, the use of genetic markers could identify cats that have most of the genes causing the "more extreme" head type, but have lost the gene causing the deformity. This can occur by the natural process of recombination. The frequency of the occurrence is determined by the distance between the genes involved. Since many cats have not been found to be clear of the defect but of the "more extreme" head type, the physical location of the genes involved must be extremely close. Thus, we can not determine if the deformity can be separated from the "more extreme" Burmese head type until a cat is test mated to be proven clear of carrying the deformity, or until genetic markers are found for the deformity. Over 250 samples have been collected for the study and are detailed in Table 1. Thirty deformed kittens have been collected from ten different litters, including deformities collected in Belgium. Bombays samples were received this fall at UCDavis for the study. The preliminary baseline data required 25-30 samples from cats that represent the gene pool of each breed involved in the study. The collection of samples from breeds other than Burmese and Foreign Burmese was hampered by the impression of finding Burmese genes in cats that are not Burmese. The investigators have worked hard to dismiss this misconception and have emphasized that the data is confidential. Breeders who have participated from the other breeds have had a general interest in genetics and the health of cats in the cat fancy. Many of the samples from the other breeds are from only a few catteries, thus we must be sure to not use closely related cats in the analysis, and attempt to obtain cats that represent the gene pools of each breed. A general request for samples has been published and we intend to focus heavily on Tonkinese and Bombay breeders. Buccal swabs can now be used to obtain these comparison samples, but blood samples are still required for relatives of the defective kittens. Sample collection for a related breed project has improved the sample collection and adequate samples are now available at UCDavis for the related breeds. Preliminary data has shown that the microsatellites will be powerful markers for the study and are capable of identifying a marker for the deformity. Most of the microsatellites tested in the Burmese have been heterozygous and should be informative for the study. No candidates genes for the deformity have been identified in the literature or from discussions with cranial-facial specialists at Johns Hopkins University, thus a genome wide search with all the available 300 microsatellites was considered. The samples acquired from the outcrosses to other breeds and samples collected from Europe will be particularly valuable in the search for a marker. But a small sample size and inadequate marker coverage did not suggest a high probability of detecting linkage, thus work on the project has not progressed due to the focus in the development of more sophisticated feline resources. The Burmese deformity has spread to other fancy cat breeds and to the Burmese in Europe. Personal communications with researchers at the veterinary college in Bristol have indicated that the defect has been seen in the UK but is not a present concern. We recommend that "more extreme" cats are not used in other breeding programs nor used as exports. The use of "more extreme" Burmese should be confined to "more extreme" Burmese breeding programs only.

UCDavis Burmese Project

As a new faculty member at the UCDavis School of Veterinary Medicine, Dr. Lyons' new lab will focus on feline genetics and inherited diseases, thus will reinstate the research on the Burmese. This reinstatement is possible due to the recent development of a large and robust DNA library of the cat. This library, the Feline BAC library, was coordinated by Dr. Lyons and was a collaborative effort between Dr. Lyons and Dr. Pieter DeJong at the Roswell Park Cancer Research Center in Buffalo, New York. This library became commercially available in fall, 1999, and is available at UCDavis. Burmese breeders have expressed an interest in the continuation of the project and Dr. Lyons' has a focused commitment to the project. Matching funds from the UCDavis Center for Companion Animal Health and Dr. Lyons' lab can guarantee focused attention to the research. Approximately 50% of the Burmese samples collected while at the NCI are currently at UCDavis and requests have been made for the remaining samples. Once funding has been obtained, formal requests by the Winn Foundation to the NCI will formalize the transfer of the remaining samples to UCDavis. Work can progress on the research prior to the acquisition of the outstanding samples but will be eventually required. A new call for samples will be initiated. Deformed kittens should continually be collected. Adult siblings and outstanding parental samples must be acquired. Specific breeders will be contacted for these samples. Genotyping of the cats involved with litters that have produced a deformity will recommence. We will attempt a focused approach to develop genetic markers near candidate genes using the feline BAC library. Known developmental genes, such as HOX 1-4@, will be isolated from the BAC library. The candidate genes selected will have sequence available from the mouse and humans. This sequence will allow the development of short sigments of DNA, termed primers, which can be used to amplify the feline counterpart of the gene from cat DNA. This feline specific amplified fragment of the gene will then be used to probe the cat BAC library. The library holds very large inserts and hence, a full copy of the gene can be held in one clone. These clones can then be cultured to produce many copies of the gene of interest. We will not initially analyze the gene, but the BAC library inserts are so large, DNA surrounding the genes is generally also present. This surrounding DNA will likely hold a very polymorphic marker, a microsatellite. To isolate a microsatellite from the gene of interest, short DNA segments that are composed of the microsatellite repeat sequences are used to probe the gene clones. Positive clones will be sub-cloned and sequenced to get the complete DNA sequence of the microsatellite. From this DNA sequence, short segments of DNA can be synthetically produced, termed primers. These primers are then used to amplify the microsatellite marker in the Burmese samples. Linkage between the markers and the defect will be determined by standard linkage analyses and homozygosity mapping.

Background (Original 1994 - 1995 Proposal)

UCDavis Project Budget:

Dr. Roberts will be hired as a post-graduate researcher V, which is appropriate for her degree and experience. All post-doctoral fellows have a three-year commitment upon entering the lab. This commitment includes the time to learn new laboratory techniques, collect data, analyze data, and formulation of data for publication.

Personnel PGRV - NS, Heather Roberts, PhD, 50% effort $ 18,504.00 Benefits, 18.5% of salary 3,423.00 -------------- Total salary: $ 21,927.00

Supplies Reagents: $1,000/month x 12 months $ 12,000.00
Travel Presentation of work at one scientific meeting and one breed associated meeting $ 1,500.00 ---------------
Total yearly budget: $ 35,427.00 CCAH matching funds (see attached letter): -$ 10,000.00
UCD Lyons' laboratory operating expense: -$ 10,427.00 -------------- Requested funding: $ 15,000.00