Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1993-08-02
2001-05-29
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C530S350000, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06238861
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the gene involved in the von Recklinghausen neurofibromatosis (NF1) disease process and, more particularly, to the identification, isolation and cloning of a nucleic add sequence corresponding to the gene. The present invention further relates to the NF1 gene product and sequence and antibodies raised thereto. The present invention also relates to methods of screening for NF1 and NF1 diagnosis, as well as conventional treatment and gene therapy utilizing recombinant technologies.
BACKGROUND OF THE INVENTION
Von Recklinghausen neurofibromatosis (NF1), often referred to as the “elephant man disease,”
1
is one of the most common autosomal dominant human disorders, affecting about 1 in 3,000 of the general population. The disease primarily involves neural crest-derived tissue and Is characterized by café-au-lait spots, neurofibromas increasing in size and number with age, learning disabilities and mental retardation, seizures, and an increased risk of malignancy. The expression of the disease is extremely variable in its symptoms and severity, and the spontaneous mutation rate is remarkably high, with about 30 to 50% of all cases representing new mutations. Clinical diagnosis of NF1 has been relatively difficult early in life, due to the variability of the symptoms and their delayed appearance.
1
1
An NIH panel, however, recently concluded that “Elephant Man” J. Merrick did not actually suffer from neurofibromatosis, but from an extremely rare disease known as the Proteus syndrome.
Direct cloning of the NF1 gene has not been possible due to the lack of a consistent abnormality in NF1 tissue which would provide sufficient information about the gene product The remaining alternative has been positional cloning of the gene, utilizing its chromosomal map position rather than its functional properties. Using this approach, genetic linkage analysis led to the assignment of the NF1 gene to the proximal long arm of chromosome 17. Subsequent collaborative multipoint mapping efforts narrowed its genetic location to about 3 centiMorgans of 17q11.2. A combination of somatic cell hybrid techniques, linking clones and pulsed field gel electrophoresis (PFGE) applied to two unrelated NF1 patients having balanced translocations t(1;17) and t(17;22), with breakpoints approximately 60 kb apart on chromosome 17, further narrowed the location of the gene to a few hundred kilobases of chromosome band 17q11.2. See Collins, F. S. et al.,
Trends in Genetics
5:217-221 (1989).
The first NF1 candidate gene was identified in mice as a site of retroviral integration in murine leukemia. See Buchberg, A. M. et al., Oncogene Research 2:149 (1988). It has now been found, however, that the human homolog EVI2A, previously named EVI2, which maps between the NF1 breakpoints, is not interrupted by the aforementioned NF1 translocations, and no abnormalities in this gene have been identified as the cause of NF1. Similarly, EVI2B, previously named NF1-c2, a gene newly identified in the course of this invention by chromosomal walking and jumping, mapped between the NF1 breakpoints, was not interrupted by the NF1 translocations and exhibited no abnormalities in NF1 patients. It thus became clear that the NF1 gene had not yet been identified.
Recently, a gene was identified by positional cloning showing mutations in individuals affected with NF1. Cawthon, R. et al.,
Cell
62:193-201 (1990); Viskochil, D. et al.,
Cell
62:187-192 (1990); Wallace, M. R. et al.,
Science
249:181-186 (1990). Further cloning and partial sequence analysis demonstrated that the gene product contains a domain showing approximately 30% similarity to the catalytic domains of yeast IRA1 and IRA2 proteins and the mammalian GTPase activating protein (GAP). Buchberg, A. et al.,
Nature
347:291-294 (1990); Xu, G. et al.,
Cell
62:599-605 (1990). GAP is a cytosolic protein that catalyzes the conversion of active GTP-bound ras p21 to the inactive GDP-bound form. Trahey M. et al.,
Science
238:542-545 (1987). It was subsequently shown that the GAP related domain of the NF1 gene product can also interact with human and yeast RAS p21 to down-regulate is activity. Ballester, R. et al.,
Cell
63:851-859 (1990); Martin, G. A. et al.,
Cell
63:343-349 (1990); Xu, G. et al.,
Cell
63:835 -841 (1990). Our previous reports of cDNA cloning of NF1 contained in parent application U.S. Ser. No. 547,090 were based on partial fragments of the transcript which is approximately 13 kb by Northern blotting. Wallace, M. R. et al.,
Science
249:181-186 (1990). The entire coding region of the NF1 gene has now been cloned and sequenced, the gene product identified and antibodies raised thereto, as described and claimed herein.
SUMMARY OF THE INVENTION
The entire coding region of the gene involved in von Recklinghausen neurofibromatosis (NF1 gene) and a ubiquitously expressed large transcript (NF1 LT) of approximately 13 kb have been isolated, cDNA cloned and sequenced as set forth in
FIGS. 16 and 19
and the Sequence Listing. Analysis of the sequences revealed an open reading frame of 2818 amino acids, although alteratively spliced products may code for different sized protein products. The gene extends for a minimum of 270 kb on chromosome 17, with its promoter in a CpG rich island. The NF1 sequence is highly conserved and shows homology to the GTPase activating proteins family (GAP). The gene is interrupted by both NF1 translocations and altered in a new mutation NF1 patient, and contains previous candidate genes EVI2A (EVI2) and EVI2B (NF1-c2) within it. Antibodies which specifically recognize the NF1 gene product hale been generated against both fusion proteins and synthetic peptides. Initial characterization of the NF1 gene product by both immunoprecipitation and Western blotting has revealed a unique protein of approximately 250 kDa. The protein has been found in a variety of human tissues and cell lines and is also present in rat and mouse tissues.
With the identification and sequencing of the gene and its corresponding gene product, nucleic acid probes and antibodies raised to the NF1 gene product can be used within the scope of the invention in a variety of hybridization and immunological assays to screen for the presence of a normal or defective NF1 gene or gene product. Functional assays to measure levels of gene function can also been employed for diagnosis or to monitor treatment. Assay kits for such screening and diagnosis in accordance with the principles of the invention can also be provided.
Patient therapy through supplementation with the normal NF1 protein, whose production can be amplified using genetic and recombinant techniques, or with its functional equivalent, is also possible. In addition, NF1 may be cured or controlled through gene therapy by correcting the gene defect in situ or using recombinant or other vehicles to deliver a DNA sequence capable of expression of the normal gene product to the patient. Treatment of non-NF1 tumors of the nervous system and growth stimulation of nervous tissue is also contemplated.
Other features and advantages of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.
REFERENCES:
patent: 4777129 (1988-10-01), Dattagupta et al.
patent: 5227292 (1993-07-01), White et al.
patent: 5532351 (1996-07-01), Stefansson
patent: 5578462 (1996-11-01), Seizinger et al.
patent: 5580955 (1996-12-01), Nur-E-Kamal et al.
Ruddle, Frank H. “The William Allan Memorial Award Address: Reverse Genetics and Beyond”Am. J. Hum. Genes36:944-53 (1984).
Sevier et al. “Monoclonal Antibodies in Clinical Immunology” (1981)J. Clin. Chem. 27(11):1797-1806.
Buchberg, A.M. et al., “Localization of Evi-2 to Chromosome 11: Linkage to Other Proto-oncogene and Growth Factor Loci Using Interspecific Backcross Mice”,Oncogene Research2:149-165 (1988).
Call, K.M. et al., “Isolation and Characterization of a Zinc Finger Polypeptide Gene at the Human Chromosome 11 Wilms' Tumor Locus”,Cel
Andersen Lone B.
Collins Francis S.
Gutmann David H.
Marchuk Douglas A.
Wallace Margaret R.
Baker & McKenzie
Konski Antoinette F.
The Regents of the University of Michigan
Zitomer Stephanie
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