Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-06-18
2001-10-23
Whizanant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06306591
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the field of genetics. Specifically, the present invention relates to materials and methods used to isolate and detect a genetic defect in the fibroblast growth factor receptor 3 gene such as the defect that causes “Spider Lamb Syndrome” in sheep.
BACKGROUND
“Spider Lamb Syndrome” or “hereditary chondrodysplasia” is a semi-lethal congenital disorder in sheep causing severe skeletal abnormalities. These abnormalities can include abnormally long, spider-like legs, humped and twisted spines, deformed ribs and sternebra, facial deformities, lack of body fat, and underdevelopment of muscle. The most severe lesions progress to compression fractures from mechanical stress due to abnormal limb angulation. Vanek et al. “Comparing spider syndrome in Hampshire and Suffolk sheep”,
Vet. Med.,
82:430-437 (1987). Radiological evaluation of Spider lamb shoulders, elbows and sternum reveal multiple, irregular islands of ossification. Vanek et al. “Radiographic diagnosis of hereditary chondrodysplasia in newborn lambs”,
JAVMA,
194:244-248 (1989). Histologic examinations of the vertebrae and long bones indicate an increase in width of the zone of proliferation, as well as hypertrophy and unevenness of the growth cartilage. Chondrocytes appear vacuolated and disorganized, lining up in bent nonparallel columns. Rook et al. “Diagnosis of hereditary chondrodysplasia (spider lamb syndrome) in sheep”,
JAVMA,
188:713-718 (1988), Troyer et al. “A morphologic and biochemical evaluation of the spider syndrome in Suffolk sheep”,
Anat. Histol. Embryol.,
17:289-300 (1988). However, there are no deformities in the chondrocyte organelles (Troyer et al., 1988), suggesting that no problem exists with structural components of the cells themselves. Also, no chromosomal abnormalities can be found in Spider lambs. Vanek et al. “Comparison of G-banded chromosomes from clinically normal lambs and lambs affected with ovine hereditary chondrodysplasia (spider syndrome)”,
Am J. Vet. Res.,
49:1164-1168 (1988).
Spider lamb Syndrome was first identified in newborn black-faced lambs during the mid-1970's. The syndrome has since surfaced in several sheep breeds in the United States and Canada within the last two decades. Such sheep breeds, include, but are not limited to, North American Suffolks and Hampshires, and United States Southdowns, Oxfords and Shropshires. In addition, cases of Spider Lab Syndrome have been reported in New Zealand and Australia, after the importation of several United States Suffolk rams into Australia in the early 1990's. It is believed this disorder arose as a mutation in a Suffolk genetic line that was used heavily during the late 1960's because of desirable production and show-ring characteristics.
Breeding studies have established that the gene responsible for this disease has an autosomal recessive mode of inheritance. Thomas and Cobb, “Spider syndrome and other genetic defects'”,
Sheep Mag.
7:44-46 (1986); Berg et al. “The mode of inheritance of the ‘Spider’ Lamb Syndrome in Suffolk sheep”,
SID Res. Digest
4:1-3 (1987); Vanek et al., (1989). Thus, animals with two copies of the normal form (allele) of the gene are normal in appearance (homozygous normal or “NN”) as are, most often, animals with one copy of the normal allele and one copy of the Spider Lamb Syndrome (“SLS”) allele (heterozygous normal or “NS”) . However, the homozygous normal animal can never produce a Spider offspring whereas the heterozygous or carrier animal has about 25% Spider offspring if mated to another carrier. Those animals with two copies of the SLS allele have the Spider phenotype and are rarely used for breeding purposes. While dramatic culling of all suspected carriers would reduce the frequency of the gene, it is a long and very expensive process. Progeny testing of potential breeding rams is another method of reducing gene frequency but it is also costly.
Due to SLS's recessive nature, it would be a significant improvement in the art to have a diagnostic or genetic screening test to determine, for example, whether or not a sheep is a carrier of the gene for SLS.
DISCLOSURE OF THE INVENTION
The invention includes genetic markers for diagnosing whether a sheep carries the gene for SLS. The genetic markers are based upon the discovery of polymorphisms in the sheep (“ovine”) fibroblast growth factor receptor 3 (“FGFR3”) gene, which can be used in genetic typing of sheep for this defect. Thus, the markers can be used as selection tools for eliminating SLS carrier animals from a sheep flock. The invention also includes the isolated defective gene for SLS itself.
The invention also includes methods for screening sheep to differentiate those that possess no (“NN”), one (“NS”) , or two (“SS”) copies of the Spider Lamb Syndrome defect. In addition, methods are described for identifying other markers associated with Spider Lamb Syndrome. The markers are based upon the presence or absence of certain polymorphisms in the ovine fibroblast growth factor receptor 3 gene. Preferably, the polymorphisms are detected as polymerase chain reaction-restriction fragment length polymorphisms (“PCR-RFLP”) and/or single strand conformational polymorphisms (“SSCP”).
One aspect of the invention involves a method to screen a mammal, such as a sheep, to determine the mammal's genetics with respect to FGFR3, such as that causing Spider Lamb Syndrome in sheep. A biological sample containing genomic DNA is first obtained from the mammal. A biological sample is a sample of tissue or fluid suspected of containing an analyte polynucleotide or mutant or normal FGFR3 including, but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, external sections of skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, brain, cartilage, bone, blood cells, organs, tissue from a subject mammal and samples of in vitro cell culture constituents. The particular biological sample is then analyzed to determine whether or not a polymorphism exists in the fibroblast growth factor receptor 3 gene or the receptor itself. The presence or absence of a specific fragment or RFLP pattern or the detection of the herein described nucleotide difference may be determined by, but not limited to, polymerase chain reaction-restriction fragment length polymorphisms, direct sequencing, or single strand conformational polymorphisms.
The invention also includes a protein which includes a polypeptide comprising a mutant FGFR3, wherein the modification associated with mutation involves substituting an amino acid other than valine at, for example, position 23 of SEQ ID NO:9. Such substitution will generally be a substitution with a polar amino acid such as arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, serine, threonine, and tyrosine, especially glutamic acid.
The present invention also provides isolated antibodies, preferably monoclonal antibodies, which specifically bind to an isolated polypeptide comprised of at least amino acid residues of the mutant FGFR3.
The invention also includes kits useful for the diagnosis of mutant or wild-type FGFR3. Such kits include a kit suitable for use in the screening technique and for assaying for the presence of the FGFR3 gene by an immunoassay which comprises an antibody which specifically binds to a gene product of the FGFR3 gene, and reagent means for detecting the binding of the antibody to the gene product, the antibody and reagent means each being present in amounts effective to perform the immunoassay.
A kit according to the invention for assaying for the presence for the FGFR3 gene in a mammal by hybridization assay techniques includes oligonucleotide sequences for PCR priming of the appropriate mammalian genomic sequence; oligonucleotide probes which specifically bind to the FGFR3 gene; and reagent means for detecting the hybridization of the oligonucleotide probes to the FGFR3 gene; the probes and reagent means each being present in amounts effective to perform th
Beever Jonathan E.
Cockett Noelle E.
Clayton Howarth & Cannon, P.C.
Lu Frank
Utah State University
Whizanant Ethan
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