Progress 01/10/03 to 06/30/05
Outputs
Final Progress Report: NIH-NIDCR DE014965 Each of the three specific aims of this project was successful and contributed to the development of this feline model for cranial facial morphogenesis. Although the sequence analysis of sonic hedgehog (SHH) in aim 2 did not support SHH as a candidate gene, the markers developed from aim 1 assisted in the linkage analysis of specific aim 3 that implicated a genetic region controlling the Burmese craniofacial defect. The long-range goal of our research is to elucidate the gene interactions leading to development of the human craniofacial complex. Human morphology can be deciphered by analyzing complex mammalian models that have strong evolutionary conservation with humans. The objective of this R03 application was to develop a novel model of a heritable, autosomal recessive, craniofacial defect naturally found in Burmese cats that is applicable to human craniofacial morphogenesis. The central hypothesis of this proposal was that
the gene, which causes the autosomal recessive feline craniofacial abnormality, plays a vital role in human facial development. Identifying the mutation that causes the feline defect would help mammalian craniofacial development and allow the use of the cats segregating for this defect as an animal model for human facial design. The rationale for this project was that a mammalian model with a heritable craniofacial abnormality would be consistently reproducible and could be manipulated to directly address gene and protein interactions in a variety of genetic backgrounds. This feline model for craniofacial morphogenesis was developed via three specific aims. Specific aim 1: Develop highly polymorphic markers for the feline homologues of genes that are known to influence craniofacial development. The feline homologues were isolated from the feline large-DNA insert BAC library and gene-associated microsatellites were developed. Specific aim 2: Scan the feline homolog for Sonic hedgehog
(SHH) for mutations that cause the feline craniofacial defect. SHH influences maxillary development in mammals, thus SHH was a high priority candidate gene for this study. The analysis of this gene would provide comparative information between the cat and humans, and could have identified the causative mutation in the feline defect. Specific aim 3: Test the gene-associated microsatellites for linkage in the Burmese craniofacial defect pedigrees. Our working hypothesis was that by mapping gene-associated markers, genetic information, such as ESTs and other markers, could be more easily identified in other species and transferred for use in the domestic cat. This project addressed basic and clinical research regarding the processes that affect normal and abnormal development of craniofacial structures. It was innovative in that heritable single gene defects are lacking in higher mammalian species and this feline defect could become a more useful animal model for human facial
morphogenesis.
Impacts
This work confirms that the domestic cat will be a useful model for cranial facial development in all mammals, including humans. The work supports know genes involved with cranial facial development or the potential identification of a novel gene. This work also supports the possibility of identifying a mutation that can be developed into a genetic test. This genetic test will help identify carriers and will lead to the prevention of the production of the defective kittens in the breeding community.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Our feline cranial facial project investigates an inherited duplication of the upper maxillary in the Burmese breed of domestic cats. Other cat breeds that have outcrossed to Burmese, such as American Shorthairs, also have shown the presence of the same defect. The abnormality is inherited as an autosomal recessive trait, hence, carriers for the trait can not be identified. The fear of cats carrying this trait has led to the decrease of the gene pool and less exportation of cats from the USA to other countries. Several genes are known to influence the basic development of the face in all mammalian species. These genes are termed HOX clusters and SHH, sonic hedgehog. There are 4 HOX clusters of genes, each cluster has approximately 13 genes and each cluster is found on a different chromosome. Two large families of cats, one from the Burmese breed and one from the American Shorthair breed, have been ascertained from private breeders for the study. These families are
being analyzed with genetic markers that surround the genes of interest in order to further implicate or exclude the candidate HOX and SHH genes. The genetic markers have been isolated from large-insert DNA clones that contain the genes of interest, hence, the markers will be very powerful for the study. To date, one of the HOX clusters has been eliminated as a candidate. Continued research should indicate if SHH or one of the other 3 HOX clusters is responsible for the abnormality in the cat.
Impacts
We expect our investigation to identify genes that control facial development in not only the cat but all mammals. This work will help to understand the basic biology of gene interactions. Additionally, identification of markers for the cat malformation will allow the eradication of this problem from the Burmese breed.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Specific Aim 1: Isolate feline homologs of developmental genes and gene-associated microsatellites from the cat BAC library. BAC library probes have been developed for sonic hedgehog and HOXA, HOXB, HOXC, and HOXD. These probes were used to screen the RPCI-86 feline BAC library to identify large-insert clones that contain these genes. One domestic cat SHH BAC clone from was identified. \ For the HOX gene clusters, 12 clones have been identified with the HOXA cluster, 26 clones for the HOXB cluster, three for the HOXC cluster and five for the HOXD cluster. Several of clones identify with more than one cluster. Since the HOX clusters are paralogous, we anticipated that the probes used may not have been specific to a cluster. All 3 clones that match the HOXC probe also match with the HOXA cluster. Two of these clones also match to HOXB. We have not determined if the HOXC clones are specific to the HOXC cluster. Four of the 12 HOXA clones are specific to HOXA. Four of the
5 HOXD identified clones are specific to the HOXD cluster. Eighteen of 26 clones specifically identify with HOXB. Thus, 26 clones appear to be specific to a cluster by hybridization analysis. The clones that are specific to a cluster were used for the identification of gene-associated microsatellite markers Approximately 50% of the cluster-specific clones (13) have been sub-cloned and screened for microsatellites. Three of the 13 BACs have produced sub-clones with a microsatellite(s). Each microsatellite is being screened for polymorphism and polymorphic markers will be used to complete Aim 3. It is interesting that no microsatellites have been found after screening approximately 600 sub-clones of the BAC containing SHH. This sequence is being included for the completion of Aim 2 testing. Specific Aim 2: Scan Sonic hedgehog for causative mutations. Sonic hedgehog (SHH) has a strong influence on the upper maxillary region. A causative mutation in the SHH gene has not yet been
identified. The Sonic Hedgehog gene consists of three exons with large intronic sequences. Forty-one individual primers have been designed based on the human sequence for the gene as well as any consensus sequence found between human and other species such as Mouse and Rat. These primers were optimized and sequenced in a control cat (non-affected and not from the breeds that carry the disease). To date, approximately 24% of exon 1 and 75% of exon has been compared between normal and affected cats. The largest exon, exon 3, has not yet been screened. Specific Aim 3: Linkage analysis in feline pedigrees. Forty-seven new samples from cat families that have produced affected kittens have been ascertained. These samples come from Burmese, American Shorthair, and Bombay breeds. Fifty-eight of 341 samples associated with the project are affected kittens. The other samples mainly consist of relatives of these kittens along with some foreign Burmese that presumably do not have the disease in
their lines. Specific cats are being selected and prioritized for the linkage study and/or homozygosity mapping. Family pedigrees amongst these cats are being formed for the linkage analysis.
Impacts
We expect our investigation to identify genes that control facial development in not only the cat but all mammals. This work will help to understand the basic biology of gene interactions. Additionally, identification of markers for the cat malformation will allow the eradication of this problem from the Burmese breed.
Publications
- No publications reported this period
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