Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-26
2004-03-09
Falk, Anne-Marie (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S023500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06703197
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the diagnosis and treatment of patients at risk for methionine synthase deficiency and associated altered risk for diseases such as neural tube defects, cardiovascular disease, and cancer.
BACKGROUND OF THE INVENTION
Methionine synthase (EC 2.1.1.13, 5-methyltetrahydrofolate-homocysteine methyltransferase) catalyses the remethylation of homocysteine to methionine in a reaction in which methylcobalamin serves as an intermediate methyl carrier. This occurs by transfer of the methyl group of 5-methyltetrahydrofolate to the enzyme-bound cob(I)alamin to form methylcobalamin with subsequent transfer of the methyl group to homocysteine to form methionine. Over time, cob(I)alamin may become oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of the functional enzyme occurs through the methionine synthase-mediated methylation of the cob(II)alamin in which S-adenosylmethionine is utilized as methyl donor. In
E. coli,
two flavodoxins have been implicated in the reductive activation of methionine synthase (Fujii, K. and Huennekens, F. M. (1974)
J. Biol. Chem.,
249, 6745-6753). A methionine synthase-linked reducing system has yet to be identified in mammalian cells.
Deficiency of methionine synthase activity results in hyperhomocysteinemia, homocystinuria, and megaloblastic anemia without methylmalonic aciduria (Rosenblatt, D. S. (1995)
The Metabolic and Molecular Bases of Inherited Disease.
McGraw-Hill, New York, pp. 3111-3128; Fenton, W. A. and Rosenberg, L. E. (1995)
The Metabolic and Molecular Bases of Inherited Disease.
McGraw-Hill, New York, pp. 3129-3149). Two classes of methionine synthase-associated genetic diseases have been proposed based on complementation experiments between patient fibroblast cell lines (Watkins, D. and Rosenblatt, D. S. (1988)
J. Clin. Invest.,
81, 1690-1694). One complementation group, cblE, has been postulated to be due to deficiency of the reducing system required for methionine synthesis (Rosenblatt, D. S., Cooper, B. A., Pottier, A., Lue-Shing, H., Matiaszuk, N. and Grauer, K. (1984) i J. Clin. Invest., 74, 2149-2156). Cells from patients in the cblE group fail to incorporate
14
C-methyltetrahydrofolate into methionine in whole cells but have significant methionine synthase activity in cell extracts in the presence of a potent reducing agent. The second complementation group, cblG group, is thought to result from defects of the methionine synthase apoenzyme. Mutant cells from this group show deficient methionine synthase activity in both whole cells and cell extracts (Watkins, D. and Rosenblatt, D. S. (1988)
J. Clin. Invest.,
81, 1690-1694; Watkins, D. and Rosenblatt, D. S. (1989)
Am. J. Med. Genet.,
34, 427-434). Moreover, some cblG patients show defective binding of cobalamin to methionine synthase in cells incubated with radiolabelled cyanocobalamin (Sillaots, S. L., Hall, C. A., Hurteloup, V., and Rosenblatt, D. S. (1992)
Biochem. Med. Metab. Biol.,
47, 242-249).
The cobalamin-dependent methionine synthase of
E. coli
has been crystallized and the structure of its active site determined (Luschinsky, C. L., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1992)
J. Molec. Biol.,
225, 557-560; Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994)
Science,
266, 1669-1674.). The gene encoding methionine synthase has not been cloned from mammals.
SUMMARY OF THE INVENTION
We have cloned a gene for mammalian methionine synthase from humans and discovered that mutations in this gene are associated with hyperhomocysteinemia. Hyperhomocysteinemia is a condition that has been implicated in cardiovascular disease and neural tube defects. The presence of such mutations in methionine synthase gene are, thus, associated with increased risk for cardiovascular disease, altered risk for neural tube defects, and decreased risk of colon cancer. The invention features methods for risk detection and treatment of patients with hyperhomocysteinemia, cardiovascular disease, neural tube defects, and cancer. The invention also features compounds and kits which may be used to practice the methods of the invention, methods and compounds for treating or preventing these conditions and methods of identifying therapeutics for the treatment and prevention of these conditions.
In the first aspect, the invention provides purified wild-type mammalian methionine synthase gene, and mutated and polymorphic versions of the mammalian methionine synthase gene, fragments of the wild-type, mutated, and polymorphic gene, and sense and antisense sequences which may be used in the methods of the invention. Preferably, the gene is human. The proteins encoded therefrom are also an aspect of the invention as is a methionine synthase polypeptide having conservative substitutions. Preferably, the protein is a recombinant or purified protein having a mutation conferring hyperhomocysteinemia when present in a mammal. In addition, nucleic acids, including genomic DNA, mRNA, and cDNA, and the nucleic acid set forth in SEQ ID NO: 1, or degenerate variants thereof, are provided. The shorter nucleic acid sequences are appropriate for use in cloning, characterizing mutations, the construction of mutations, and creating deletions. In one embodiment, the nucleic acid set forth in SEQ ID NO: 1 is a probe that hybridizes at high stringency to sequences found within the nucleic acid of SEQ ID NO: 1. In further embodiments, the probe has a sequence complementary to at least 50% of at least 60 nucleotides, or the sequence is complementary to at least 90% of at least 18 nucleotides. Protein fragments also are provided. The shorter peptides may be used, for example, in the generation of antibodies to the methionine synthase protein. In some embodiments of this aspect of the invention nucleic acid fragments useful for detection of mutations in the region of the methionine synthase gene which encodes the cobalamin binding domain, and for detecting those mutations which indicate an increased likelihood of hyperhomocysteinemia, are preferred. Most preferred fragments are those useful for detecting the 2756 A→G, &Dgr;bp 2640-2642, and 2758 C→G mutations/polymorphisms. Given Applicants' discovery, one skilled in the art may readily determine which nucleic acids, detection methods, and mutations are most useful. Mutant proteins encoded by these mutations, including, but not limited to, H920D, &Dgr;Ile 881, and D919G are also provided by the invention (see, for example SEQ ID NOs: 74 and 75). Such mutant and polymorphic polypeptides may have decreased or increased biological activity, relative to wild-type methionine synthase.
In a related aspect, the invention provides antibodies that specifically bind mammalian methionine synthase, and a method for generating such an antibody. The antibody may specifically bind a wild-type methionine synthase, or a mutant or polymorphic methionine synthase. A method for detecting a wild-type, mutant, or polymorphic methionine synthase using the antibody is also provided by the invention.
In a second aspect, the invention provides a method for detecting an increased or decreased risk for hyperhomocysteinemia in a fetus or individual patient. Such a fetus or patient is at increased or decreased risk for neural tube defects and/or cardiovascular disease and at a decreased risk of developing colon cancer. The method includes detection of mutations in the methionine synthase gene present in the fetus, the individual patient, and/or the blood relatives of the fetus and patient. The presence of mutations, particularly in the cobalamin binding domain, indicate an altered (e.g., increased or decreased) risk of hyperhomocysteinemia, neural tube defects, cancer, and cardiovascular disease.
In a related aspect, the invention provides kits for the detection of mutations in the human methionine synthase gene. Such kits may include, for example, nucleic acid sequences, including probes, useful for PCR, SSCP, or RFLP detection of such mutations. Antibodies specific for
Campeau Eric
Goyette Philippe
Gravel Roy A.
LeClerc Daniel
Rozen Rima
Bieker-Brady Kristina
Clark & Elbing LLP
Falk Anne-Marie
Martinex R&D Inc.
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