Note: Descriptions are shown in the official language in which they were submitted.
CA 022171~3 1998-01-06
HUMAN METHIONINE SYNTHASE: CLONING, AND METHODS
FOR EVALUATING RISK OF NEURAL TUBE DEFECTS,
CARDIOVASCULAR DISEASE, AND CANCER
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 diseasessuch as neural tube defects, cardiovascular disease, and cancer.
Background ofthe 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.,
CA 022171~3 1998-01-06
249, 6745-6753). A methionine synthase-linked reducing system has yet to be
identified in marnm~ n cells.
Deficiency of methionine synthase activity results in
hyperhomocysteinemia, homocystinuria, and megaloblastic anemia without
5 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
10 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) J. Clin. Invest., 74, 2149-2156). Cells from patients in
the cblE group fail to incorporate '4C-methyltetrahydrofolate into methionine inwhole 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
20 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
25 radiolabelled cyanocobalamin (Sillaots, S.L., Hall, C.A., Hurteloup, V., and
Rosenblatt, D.S. (1992) Biochem. Med. Metab. Biol., 47, 242-249).
CA 022171~3 1998-01-06
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 m~mm:~ls.
Summary of the Invention
We have cloned a gene for m~rnm~lian methionine synthase from
10 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
15 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
20 and methods of identifying therapeutics for the treatment and prevention of
these conditions.
In the first aspect, the invention provides purified wild-type
m~rnm~lian methionine synthase gene, and mutated and polymorphic versions
of the m~rnm~lian methionine synthase gene, fragments of the wild-type,
25 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
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methionine synthase polypeptide having conservative substitutions. Preferably,
the protein is a recombinant or purified protein having a mutation conferring
hyperhomocysteinemia when present in a m~mm~l. 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
10 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
15 embodiments of this aspect of the invention nucleic acid fragments useful fordetection 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, ~bp
20 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, AIle 881, and D919G are
also provided by the invention. Such mutant and polymorphic polypeptides
25 may have decreased or increased biological activity, relative to wild-type
methionine synthase.
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In a related aspect, the invention provides antibodies that specifically
bind m~mm~lian 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
5 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
10 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)
15 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
20 RFLP detection of such mutations. Antibodies specific for proteins having
mutations, correlated with an increased likelihood of hyperhomocysteinemia,
may also be included in the kits of the invention.
In a fourth aspect, the invention features a method for screening for
compounds which alter methionine synthase expression or ameliorate or
25 exacerbate conditions of hyperhomocysteinemia. In various embodiments, the
invention includes monitoring mutant or wild-type m~mm~lian methionine
synthase biological activity by monitoring methionine synthase enzymatic
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activity, or monitoring methionine synthase gene expression levels, by
monitoring methionine synthase gene transcription, RNA stability, RNA
translation and/or protein stability. In preferred embodiments the methionine
synthase gene or protein being monitored is a gene or protein having a mutation
5 associated with hyperhomocysteinemia, and samples are selected from purifed
or partially purified methionine synthase, cell lysate, a cell, or an animal.
Standard assay techniques known to those skilled in the art may be employed in
the various embodiments. Compounds detected using this screen can be used
to prevent or treat cardiovascular disease and neural tube defects or, in the
10 alternative, to prevent or treat colon cancer. Kits for performing the above
screens are also a part of the invention.
In a related aspect, the invention provides nucleic acids encoding
wild-type, polymorphic, and mutated methionine synthase, in which the nucleic
acid is operably linked to regulatory sequences, comprising a promoter, for the
15 expression of the encoded polypeptides. In one embodiment, the promoter is
inducible. The invention also provides cells, including prokaryotic and
eukaryotic cells, comprising the nucleic acids. The eukaryotic cells may be
yeast cells or m~mmalian cells.
In another related aspect, the invention features a transgenic m~mm~l
20 having a methionine synthase transgene. The gene may be wild-type, or may
contain a mutation or polymorphism. The m~mm~l may have a mutation
associated with hyperhomocysteinemia in its methionine synthase gene in an
expressible genetic construction or may have a deletion or knockout mutation
in one or both alleles suff1cient to abolish methionine synthase expression from25 the locus. In addition, or as a replacement, the m~mm~l may have the
methionine synthase gene from another species. For example, in one preferred
embodiment the transgenic mammal is a rodent such as a mouse and the
CA 022171~3 1998-01-06
transgene is from a human. Cells from these transgenic or knockout ~nim~l~
are also provided by the invention. Such transgenic m~mmal~ may be used to
screen for drugs for the treatment of diseases related to hyperhomocysteinemia.
In a sixth aspect, the invention features a method for treating patients
with neural tube defects, colon cancer or related cancers by the delivery of
antisense methionine synthase nucleic acid sufficient to lower the levels of
methionine synthase polypeptide biological activity.
In a related aspect, the invention provides a method for treating or
preventing cardiovascular disease, neural tube defects and cancer. The method
10 comprises detecting an altered risk of such defects by analyzing methionine
synthase nucleic acid, potential test subjects being a mammal, a potential
parent, either male or female, a pregnant mammal, or a developing embryo or
fetus, and then by exposing the subject (e.g., patient or pregnant mammal) to
metabolites or cofactors such as, but not limited to, folate, cobalamin, S-
15 adenosyl methionine, betaine, or methionine. In another related aspect, the
invention features a method of pretreating or treating colon cancer or neural
tube defects by inhibiting or activating methionine synthase biological activityin a m~mmal, pregnant mammal, embryo, or fetus. In preferred embodiments,
this inhibiting or activating may be effected by exposing the subject to nucleic20 acids, peptides or small molecule-based inhibitors or activators of methionine
synthase or substrates. The exposure is to quantities of the compound
sufficient to reduce the probability of the subject developing the disease or toconfer an increased likelihood of a decrease in the disease symptoms of the
subject.
By "methionine synthase," "methionine synthase protein," or
"methionine synthase polypeptide" is meant a polypeptide, or fragment thereof,
which has at least 50% amino acid identity to boxes 1-4 of the human
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methionine synthase polypeptide (SEQ ID NO: 2) (see Fig. 1). It is understood
that polypeptide products *om splice variants of methionine synthase gene
sequences are also included in this definition. Preferably, the methionine
synthase protein is encoded by nucleic acid having a sequence which
5 hybridizes to a nucleic acid sequence present in SEQ ID NO: 1 (human
methionine synthase cDNA) under stringent conditions. Even more preferably
the encoded polypeptide also has methionine synthase biological activity.
By "methionine synthase nucleic acid" or "methionine synthase
gene" is meant a nucleic acid, such as genomic DNA, cDNA, or mRNA, that
10 encodes methionine synthase, a methionine synthase protein, methionine
synthase polypeptide, or portion thereof, as defined above. A methionine
synthase nucleic acid also may be a methionine synthase primer or probe, or
antisense nucleic acid that is complementary to a methionine synthase nucleic
acld.
By "wild-type methionine synthase" is meant a methionine synthase
nucleic acid or methionine synthase polypeptide having the nucleic acid and/or
amino acid sequence most often observed among members of a given animal
species and not statistically associated with a disease phenotype. Wild-type
methionine synthase is biologically active methionine synthase. A wild-type
20 methionine synthase is, for example, a human methionine synthase polypeptide
having the sequence of SEQ ID NO: 1.
By "mutant methionine synthase," "methionine synthase
mutation(s)," "mutations in methionine synthase," "polymorphic methionine
synthase," "methionine synthase polymorphism(s)," "polymorphisms in
25 methionine synthase," is meant a methionine synthase polypeptide or nucleic
acid having a sequence that deviates from the wild-type sequence in a manner
sufficient to confer an altered risk for a disease phenotype, or enhanced
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protection against a disease, in at least some genetic and/or environmental
backgrounds. Such mutations may be naturally occurring or artificially
induced. They may be, without limitation, insertion, deletion, frameshift, or
missense mutations. A mutant methionine synthase protein may have one or
5 more mutations, and such mutations may affect different aspects of methionine
synthase biological activity (protein function), to various degrees.
Alternatively, a methionine synthase mutation may indirectly affect methionine
synthase biological activity by influencing, for example, the transcriptional
activity of a gene encoding methionine synthase, or the stability of methionine
10 synthase mRNA. For example, a mutant methionine synthase gene may be a
gene which expresses a mutant methionine synthase protein or may be a gene
which alters the level of methionine synthase protein in a manner sufficient to
confer a disease phenotype in at least some genetic and/or environmental
backgrounds.
By "biologically active" methionine synthase is meant a methionine
synthase protein or methionine synthase gene that provides at least one
biological function equivalent to that of the wild-type methionine synthase
polypeptide or methionine synthase gene. Biological activities of a methionine
synthase polypeptide include, and are not limited to, the ability to catalyze the
20 methylation of homocysteine to generate methionine. Preferably, a biologically
active methionine synthase will display activity equivalent to at least 35% of
wild-type activity, more preferably, a biologically active methionine synthase
will display at least 40-55% of wild-type activity, still more preferably, a
biologically active methionine synthase will display at least 60-75% of wild-
25 type activity, and most preferably, a biologically active methionine synthasewill display at least 80-90% of wild-type activity. A biologically active
methionine synthase also may display more than 100% of wild-type activity.
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Preferably, the biological activity of the wild-type methionine synthase is
determined using the methionine synthase nucleic acid of SEQ ID NO: 1 or
methionine synthase polypeptide of SEQ ID NO: 2. The degree of methionine
synthase biological activity may be intrinsic to the methionine synthase
5 polypeptide itself, or may be modulated by increasing or decreasing the numberof methionine synthase polypeptide molecules present intracellularly.
By "high stringency conditions" is meant hybridization in 2X SSC at
40~C with a DNA probe length of at least 40 nucleotides. For other definitions
of high stringency conditions, see F. Ausubel et al., Current Protocols in
Molecular Biology, pp. 6.3.1-6.3.6, John Wiley & Sons, New York, NY, 1994,
hereby incorporated by reference.
By "analyzing" or "analysis" is meant subjecting a methionine
synthase nucleic acid or methionine synthase polypeptide to a test procedure
that allows the determination of whether a methionine synthase gene is wild-
15 type or mutant. For example, one could analyze the methionine synthase genesof an animal by amplifying genomic DNA using the polymerase chain reaction,
and then determining the DNA sequence of the amplified DNA.
By "probe" or "primer" is meant a single- or double-stranded DNA
or RNA molecule of defined sequence that can base pair to a second DNA or
20 RNA molecule that contains a complementary sequence (the "target"). The
stability of the resulting hybrid depends upon the extent of the base pairing that
occurs. The extent of base-pairing is affected by parameters such as the degree
of complementarity between the probe and target molecules, and the degree of
- stringency of the hybridization conditions. The degree of hybridization
25 stringency is affected by parameters such as temperature, salt concentration,and the concentration of organic molecules such as formamide, and is
determined by methods known to one skilled in the art Probes or primers
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specific for methionine synthase nucleic acid preferably will have at least 35%
sequence identity, more preferably at least 45-55% sequence identity, still morepreferably at least 60-75% sequence identity, still more preferably at least 80-90% sequence identity, and most preferably 100% sequence identity. Probes
5 may be detectably-labelled, either radioactively, or non-radioactively, by
methods well-known to those skilled in the art. Probes are used for methods
involving nucleic acid hybridization, such as: nucleic acid sequencing, nucleic
acid amplification by the polymerase chain reaction, single stranded
conformational polymorphism (SSCP) analysis, restriction fragment
10 polymorphism (RFLP) analysis, Southern hybridization, Northern
hybridization, in situ hybridization, electrophoretic mobility shift assay
(EMSA).
By "pharmaceutically acceptable carrier" means a carrier which is
physiologically acceptable to the treated m~mm~l while retaining the
15 therapeutic properties ofthe compound with which it is ~tlmini~tered. One
exemplary pharmaceutically acceptable carrier is physiological saline. Other
physiologically acceptable carriers and their formulations are known to one
skilled in the art and described, for example, in Remington's Pharmaceutical
Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company,
20 Easton, PA.
By "substantially identical" is meant a polypeptide or nucleic acid
exhibiting at least 50%, preferably 85%, more preferably 90%, and most
preferably 95% identity to a reference amino acid or nucleic acid sequence.
For polypeptides, the length of comparison sequences will generally be at least
25 16 amino acids, preferably at least 20 amino acids, more preferably at least
25 amino acids, and most preferably 35 amino acids. For nucleic acids, the
length of comparison sequences will generally be at least 50 nucleotides,
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preferably at least 60 nucleotides, more preferably at least 75 nucleotides, andmost preferably 1 10 nucleotides.
Sequence identity is typically measured using sequence analysis
software with the default parameters specified therein (e.g., Sequence Analysis
5 Software Package of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, WI53705). This
software program matches similar sequences by assigning degrees of homology
to various substitutions, deletions, and other modifications. Conservative
nucleotide substitutions typically include substitutions which generate changes
10 within the following groups: glycine, alanine, valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine.
By "substantially pure polypeptide" is meant a polypeptide that has
been separated from the components that naturally accompany it. Typically,
15 the polypeptide is substantially pure when it is at least 60%, by weight, free
from the proteins and naturally-occurring organic molecules with which it is
naturally associated. Preferably, the polypeptide is a methionine synthase
polypeptide that is at least 75%, more preferably at least 90%, and most
preferably at least 99%, by weight, pure. A substantially pure methionine
20 synthase polypeptide may be obtained, for example, by extraction from a
natural source (e.g., a fibroblast or liver cell) by expression of a recombinantnucleic acid encoding a methionine synthase polypeptide, or by chemically
synthesizing the protein. Purity can be measured by any appropriate method,
e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC
25 analysis.
A protein is substantially free of naturally associated components
when it is separated from those cont~min;mts which accompany it in its natural
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state. Thus, a protein which is chemically synthesized or produced in a cellularsystem different from the cell from which it naturally originates will be
substantially free from its naturally associated components. Accordingly,
substantially pure polypeptides not only includes those derived from eukaryotic
organisms but also those synthesized in E. coli or other prokaryotes.
By "substantially pure DNA" is meant DNA that is free of the genes
which, in the naturally-occurring genome of the organism from which the DNA
of the invention is derived, flank the gene. The term therefore includes, for
example, a recombinant DNA which is incorporated into a vector; into an
10 autonomously replicating plasmid or virus; or into the genomic DNA of a
prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA
or a genomic or cDNA fragment produced by PCR or restriction endonuclease
digestion) independent of other sequences. Tt also includes a recombinant DNA
which is part of a hybrid gene encoding additional polypeptide sequence.
By "transgene" is meant any piece of DNA which is inserted by
artifice into a cell, and becomes part of the genome of the organism which
develops from that cell. Such a transgene may include a gene which is partly or
entirely heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism.
By "transgenic" is meant any cell which includes a DNA sequence
which is inserted by artifice into a cell and becomes part of the genome of the
organism which develops from that cell. As used herein, the transgenic
organisms are generally transgenic m~rnm~ls (e.g., rodents such as rats or mice)and the DNA (transgene) is inserted by artifice into the nuclear genome.
25 Preferably the inserted DNA encodes a protein in at least some cells of the
organism.
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By "knockout mutation" is meant an alteration in the nucleic acid
sequence that reduces the biological activity of the polypeptide normally
encoded therefrom by at least 80% relative to the unmutated gene. The
mutation may, without limitation, be an insertion, deletion, frameshift mutation,
or a missense mutation. Preferably, the mutation is an insertion or deletion, oris a frameshift mutation that creates a stop codon.
By "transformation" is meant any method for introducing foreign
molecules into a cell. Lipofection, DEAE-dextran-mediated transfection,
microinjection, protoplast fusion, calcium phosphate precipitation, retroviral
10 delivery, electroporation, and biolistic transformation are just a few of themethods known to those skilled in the art which may be used. For example,
biolistic transformation is a method for introducing foreign molecules into a
cell using velocity driven microprojectiles such as tungsten or gold particles.
Such velocity-driven methods originate from pressure bursts which include, but
15 are not limited to, helium-driven, air-driven, and gunpowder-driven techniques.
Biolistic transformation may be applied to the transformation or transfection ofa wide variety of cell types and intact tissues including, without limitation,
intracellular organelles (e.g., and mitochondria and chloroplasts), bacteria,
yeast, fungi, algae, animal tissue, and cultured cells.
By "transformed cell" is meant a cell into which (or into an ancestor
of which) has been introduced, by means of recombinant DNA techniques, a
DNA molecule encoding (as used herein) a methionine synthase polypeptide.
By "positioned for expression" is meant that the DNA molecule is
positioned adjacent to a DNA sequence which directs transcription and
25 translation of the sequence (i.e., facilitates the production of, e.g., a methionine
synthase polypeptide, a recombinant protein or a RNA molecule).
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By "promoter" is meant a minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter elements
which are sufficient to render promoter-dependent gene expression controllable
for cell type-specific, tissue-specific, temporal-specific, or inducible by
5 external signals or agents; such elements may be located in the 5' or 3' or intron
sequence regions of the native gene.
By "operably linked" is meant that a gene and one or more
regulatory sequences are connected in such a way as to permit gene expression
when the appropriate molecules (e.g., transcriptional activator proteins) are
10 bound to the regulatory sequences.
By "conserved region" is meant any stretch of six or more
contiguous amino acids exhibiting at least 30%, preferably 50%, and most
preferably 70% amino acid sequence identity between two or more of the
methionine synthase family members, (e.g., between human and bacterial
15 methionine synthase). Examples of conserved regions within methionine
synthase are Boxes 1-4 (Fig. 1).
By "detectably-labeled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide probe or primer,
a gene or fragment thereof, or a cDNA molecule. Methods for detectably-
20 labeling a molecule are well known in the art and include, without limitation,radioactive labeling (e.g., with an isotope such as 32p or 35S) and nonradioactive
labeling (e.g., chemiluminescent labeling, e.g., fluorescein labeling).
By "antisense" as used herein in reference to nucleic acids, is meant
a nucleic acid sequence that is complementary to the coding strand of a gene,
25 preferably, a methionine synthase gene. An antisense nucleic acid is capable of
preferentially lowering the activity of a mutant methionine synthase
polypeptide encoded by a mutant methionine synthase gene.
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By "purified antibody" is meant antibody which is at least 60%, by
weight, free from proteins and naturally occurring organic molecules with
which it is naturally associated. Preferably, the preparation is at least 75%,
more preferably 90%, and most preferably at least 99%, by weight, antibody,
5 e.g., a methionine synthase amino-terminus-specific antibody. A purified
antibody may be obtained, for example, by affinity chromatography using
recombinantly-produced protein or conserved motif peptides and standard
techniques.
By "specifically binds" is meant an antibody that recognizes and
10 binds a human methionine synthase polypeptide but that does not substantiallyrecognize and bind other non-methionine synthase molecules in a sample, e.g.,
a biological sample, that naturally includes protein. A preferred antibody bindsto the methionine synthase polypeptide sequence of SEQ ID NO: 2 (Fig. 3).
By "neutralizing antibodies" is meant antibodies that interfere with
15 any of the biological activities of a wild-type or mutant methionine synthasepolypeptide, for example, the ability of methionine synthase to catalyze the
transfer of a methyl group to homocysteine. The neutralizing antibody may
reduce the ability of a methionine synthase polypeptide to catalyze the transferpreferably by 10 % or more, more preferably by 25% or more, still more
20 preferably by 50% or more, yet preferably by 70% or more, and most
preferably by 90% or more. Any standard assay for the biological activity of
methionine synthase, may be used to assess potentially neutralizing antibodies
that are specific for methionine synthase.
By "expose" is meant to allow contact between an ~nim~l, cell,
25 lysate or extract derived from a cell, or molecule derived from a cell, and a test
compound.
CA 022171~3 1998-01-06
By "treat" is meant to submit or subject an animal (e.g. a human),
cell, lysate or extract derived from a cell, or molecule derived from a cell to a
test compound.
By "test compound" is meant a chemical, be it naturally-occurring or
5 artificially-derived, that is surveyed for its ability to modulate an alteration in
reporter gene activity or protein levels, by employing one of the assay methods
described herein. Test compounds may include, for example, peptides,
polypeptides, synthesized organic molecules, naturally occurring organic
molecules, nucleic acid molecules, and components thereof.
By "assaying" is meant analyzing the effect of a treatment, be it
chemical or physical, ~(lmini~tered to whole ~nim~ls or cells derived therefrom.The material being analyzed may be an ~nim~l, a cell, a lysate or extract
derived from a cell, or a molecule derived from a cell. The analysis may be for
the purpose of detecting altered protein biological activity, altered protein
15 stability, altered protein levels, altered gene expression, or altered RNA
stability. The means for analyzing may include, for example, for example, the
detection of the product of an enzymatic reaction, (e.g., the formation of
methionine as a result of methionine synthase activity), antibody labeling,
immunoprecipitation, and methods known to those skilled in the art for
20 detecting nucleic acids.
By "modulating" is meant changing, either by decrease or increase,
in biological activity.
By "a decrease" is meant a lowering in the level of biological
activity, as measured by a lowering/increasing of: a) the formation of
25 methionine as a result of methionine synthase activity; b) protein, as measured
by ELISA; c) reporter gene activity, as measured by reporter gene assay, for
example, lacZ/~-galactosidase, green fluorescent protein, luciferase, etc.; or d)
CA 022171~3 1998-01-06
mRNA, levels of at least 30%, as measured by PCR relative to an internal
control, for example, a "housekeeping" gene product such as ~-actin or
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or an externally added
nucleic acid standard. In all cases, the lowering is preferably by at least 10%
5 more preferably by at least 25%, still more preferably by at least 50%, and even
more preferably by at least 70%.
By "an increase" is meant a rise in the level of biological activity, as
measured by a lowering/increasing of: a) the formation of methionine as a
result of methionine synthase activity; b) protein, as measured by ELISA; c)
10 reporter gene activity, as measured by reporter gene assay, for example, lacZ/~-
galactosidase, green fluorescent protein, luciferase, etc.; or d) mRNA, levels of
at least 30%, as measured by PCR relative to an internal control, for example, a"housekeeping" gene product such as ~-actin or glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) or an externally added nucleic acid standard.
15 Preferably, the increase is by 10% or more, more preferably by 25% or more,
even more preferably by 2-fold, and most preferably by at least 3-fold.
By "alteration in the level of gene expression" is meant a change in
gene activity such that the amount of a product of the gene, i.e., mRNA or
polypeptide, is increased or decreased, or that the stability of the mRNA or the20 polypeptide is increased or decreased.
By "reporter gene" is meant any gene which encodes a product
whose expression is detectable and/or quantitatable by immunological,
chemical, biochemical or biological assays. A reporter gene product may, for
example, have one of the following attributes, without restriction: fluorescence25 (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ/~-galactosidase,
luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin A), or an
ability to be specifically bound by a second molecule (e.g., biotin or a
CA 022171~3 1998-01-06
-19-
detectably labelled antibody). It is understood that any engineered variants of
reporter genes, which are readily available to one skilled in the art, are also
included, without restriction, in the forgoing definition.
By "protein" or "polypeptide" or "polypeptide fragment" is meant
5 any chain of more than two amino acids, regardless of post-translational
modification (e.g., glycosylation or phosphorylation), constituting all or part of
a naturally-occurring polypeptide or peptide, or constituting a non-naturally
occurring polypeptide or peptide.
By "missense mutation" is meant the substitution of one purine or
10 pyrimidine base (i.e. A, T, G, or C) by another within a nucleic acid sequence,
such that the resulting new codon encodes an amino acid distinct from the
amino acid originally encoded by the reference (e.g. wild-type) codon.
By "deletion mutation" is meant the deletion of at least one
nucleotide within a polynucleotide coding sequence. A deletion mutation alters
15 the reading frame of a coding region unless the deletion consists of one or more
contiguous 3-nucleotide stretches (i.e. "codons"). Deletion of a codon from a
nucleotide coding region results in the deletion of an amino acid *om the
resulting polypeptide.
By "frameshift mutation" is meant the insertion or deletion of at least
20 one nucleotide within a polynucleotide coding sequence. A frameshift
mutation alters the codon reading frame at and/or downstream from the
mutation site. Such a mutation results either in the substitution of the encodedwild-type amino acid sequence by a novel amino acid sequence, or a premature
termination of the encoded polypeptide due to the creation of a stop codon, or
25 both.
Detailed Description of the Drawings
The drawings will first be briefly described.
CA 022171~3 1998-01-06
-20-
Fig. 1 is a diagram showing four homologous regions among
methionine synthases. Boxes 1 to 4 were used to design degenerate
oligonucleotides for the initial cloning experiments. Ec: Escherichia coli,
accession number J04975; Ss: Synechocystis sp., accession number D64002;
Mll and Ml2: Mycobacterium leprae, accession number U000175 (see
Drennan et al., 1994); Hi: Haemophilus influenzae, accession number U32730;
Ce: Caenorhabditis elegans, accession number Z46828; Hs: Homo sapiens, this
work. Identical residues are indicated by a star above the alignment. Amino
acid position for each protein is shown at left.
Fig. 2 is a diagram showing overlapping PCR fragments generated to
clone human methionine synthase. Oligonucleotides are described in Table 1.
Primers in parentheses designate misp~ lhlg outcomes that generated valid
internal sequence. iPCRc: inverse PCR on cDNA, iPCRg: inverse PCR on
genomic DNA.
Fig. 3 is a diagram showing nucleotide sequence (SEQ ID NO: 1)
and deduced amino acid sequence (SEQ ID NO: 2) of human methionine
synthase. The nucleotide residue numbering is shown in the left margin, and
the amino acid residue numbering is shown in the right margin.
Fig. 4 is a photograph showing mapping of the human methionine
20 synthase gene using FISH. Signals are clearly visible at lq43 (arrows).
Figs. 5A-5C is a series of photographs showing diagnostic tests for
mutations in the methionine synthase gene. Numbers above the gel lanes
correspond to patients cell lines whereas the letter "c" identifies wild-type
controls. Fig. 5A: HaeIII restriction analysis of genomic DNA PCR products
25 using primers #1796 and #305A. The 2756A~G change creates a HaeIII site.
Expected fragments, 2756A allele: 189 bp, 2756G allele: 159 and 30 bp (the 30
bp fragment was run off the gel). Fig. 5B: Heteroduplex analysis of PCR
CA 022171~3 1998-01-06
products amplified from RT reactions of patient 1892 and 3 controls. RT-PCR
was done with primers #1772 and #1773. Expected PCR product: 338bp,
heteroduplexes can be seen above this band in patient 1892 (heterozygous for
~2640-2642). C. Sau96I restriction analysis of genomic DNA PCR products.
PCR was done as in (A). The 2758C G mutation abolishes a Sau96I
restriction endonuclease site in patient 2290. Expected fragments, control
allele: 159, 30 bp, mutant allele: 189 bp (the 30 bp fragment has been run off
the gel).
Fig 6. shows an amino acid sequence comparison among methionine
10 synthases in the Box 2 region. Identical residues are indicated by a star above
the alignment. Dots show partially conserved residues, for which at least 6/7
identical or similar residues can be aligned (A,G,S,T; D,E,N,Q; V,L,I,M; K,R;
and F,W,Y (Bordo,D. and Argos,P. (1991) J. Molec. Biol., 217, 721-729)).
Mutations identified in this work are shown below the alignment. For
15 abbreviations, see Fig.1; Mm: Mus musculus. The seven amino acids
conserved in cobalamin-binding proteins (Drennan, C.L., Huang, S.,
Drummond, J.T., Matthews, R.G., and Ludwig, M.L. (1994) Science, 266,
1669-1674) are underlined.
Detailed Description
We used specific regions of homology within the methionine
synthase sequences of several lower organisms to clone a human methionine
synthase cDNA (SEQ ID NO: 1) by a combination of RT-PCR and inverse
PCR. The enzyme (SEQ ID NO:2) is 1265 amino acids in length and contains
the seven residue structure-based sequence fingerprint identified for cobalamin-25 containing enzymes. The gene was localized to chromosome 1 q43 by the FISH
technique. We have identified one missense mutation and a 3 base pair
CA 022171~3 1998-01-06
deletion in patients of the cblG complementation group of inherited
homocysteine/folate disorders by SSCP and sequence analysis, as well as an
amino acid substitution present in high frequency in the general population.
We conclude that the cDNA that we have identified corresponds to
human methionine synthase on the basis of homology to known methionine
synthases and by the identification of mutations in patients with a deficiency of
enzyme activity. The most striking sequence conservation was found in four
boxes of 9- 13 amino acids. Box 2 has been proposed to correspond to part of
the cobalamin binding domain (Drennan, C.L., Huang, S., Drummond, J.T.,
10 Matthews, R.G., and Ludwig, M.L. (1994) Science, 266, 1669-1674). It
contains 13 consecutive residues that are identical in all known methionine
synthases. Three amino acids within box 2 and four others C-terminal to it
correspond to residues proposed by Drennan et al. (Drennan, C . L., Huang, S .,
Drum~rlond, J.T., Matthews, R.G., and Ludwig, M.L. (1994) Science, 266,
15 1669-1674) as a structure-based sequence fingerprint for cobalamin binding.
The three amino acids appear to make direct contact with the lower face of the
corrin ring and dimethylbenzimidazole tail of cobalamin, determined from the
crystal structure of the E. coli enzyme at 3 A resolution (Drennan, C . L.,
Huang, S., Drumnlond, J.T., Matthews, R.G., and Ludwig, M.L. (1994)
20 Science, 266, 1669-1674). All seven residues are identical in the human
sequence (Fig. 6).
A survey of the NCBI databases for homology to the human
methionine synthase using BLASTP yielded the various methionine synthases
listed in Fig. 1, as well as the glut~ te mutase (S41332, Q05488) and
25 methylmalonyl-CoA mutase (P11653)(adenosyl-cobalamin dependent mutases)
used to deduce the sequence fingerprint for cobalamin binding (Drennan,
C.L., Huang, S., Drummond, J.T., Matthews, R.G., and Ludwig, M.L.
CA 022171~3 1998-01-06
-23 -
(1994) Science, 266, 1669-1674). Homology was also found with the
cobalamin binding region of the corrinoid: coenzyme M methyltransferase of
Methanosarcina barkeri (U36337), the 5-methyltetrahydrofolate corrinoid/iron
sulfur protein methyltransferase of Clostridium thermoaceticum (L34780) and
5 the B12-dependent 2-methyleneglutarate mutase of Clostridium barkeri
(S43552,S43237). Further,homologywasfoundwiththeB12-bindingsite
domain of the recently identified putative methionine synthase of
Agrobacterium tumefaciens (U48718; partial N-terminal sequence is given, up
to region of box 4). Significantly, homology with the B 12-binding site domain
was also found in the Hg resistance protein of Myxococcus xanthus (Z21955).
This protein has not been described as having a cobalamin prosthetic group.
The two mutations we have identified as candidates for causing cblG
disease are located in the vicinity of the cobalamin binding domain by
comparison with E. coli methionine synthase (Fig. 6). Ile881 corresponds by
sequence alignment to Val855 in the E. coli enzyme. Val855 is within a beta
sheet strand that is part of an oc/~3 domain that is a variant of the Rossmann
nucleotide binding fold. The H920D substitution is found in a region which, in
the E. coli enzyme, is in an oc helix at the C-terminal end of the o~/13 domain. It
is interesting that the polymorphism we have identified is at the adjacent
20 residue (D919G). The functional role of the polymorphism and deleterious
mutations will have to be examined in expression experiments to confirm their
precise effect on the protein.
Through the cloning of a cDNA for human methionine synthase and
mutations therein, we can now determine the properties of the human enzyme
25 and complete the characterization of mutations in patients with severe synthase
deficiency. This analysis has allowed us to tie mutations in the gene to
disturbances in homocysteine metabolism which are known to result in
CA 022171~3 1998-01-06
-24-
hyperhomocysteinemia is a risk factor for cardiovascular disease (Boushey,
C.J., Beresford, S.A., Omenn, G.S., and Motulsky, A.G. (1995) JAMA,
274, 1049-1057) and neural tube defects (Steegers- Theunissen, R.P., Boers,
G.H., Trijbels, F.J., Finkelstein, J.D., Blom, H.J., Thomas, C.M., Borm,
G.F., Wouters, M.G., and Eskes, T.K. (1994) Metab. Clin. Exp., 43,
1475-1480; and Mills, J.L., McPartlin, J.M., Kirke, P.N., Lee, Y.J.,
Conley, M.R., Weir, D.G. and Scott, J.M. (1995) Lancet, 345, 149-151).
Our observations indicate the importance of methionine synthase as
one of several genes involved in homocysteine metabolism. Results with other
10 pathway genes underscores the significance of our findings. For example, a
recently-identified mutation in methylenetetrahydrofolate reductase, the
enzyme that synthesizes the 5-methyltetrahydrofolate substrate for the
methionine synthase reaction, results in mild hyperhomocysteinemia
(Frosst,P., Blom,H.J., Milos,R., Goyette,P., Sheppard,C.A.,
15 Matthews,R.G., Boers,G.J., den Heijer,M., Kluijtmans,L.A., van den
Heuvel,L.P., et al. (1995) Nat. Genet., 10, 111-113). Evidence is
accumulating that this mutation, present in 35-40% of alleles, is a risk factor in
both cardiovascular disease and neural tube defects (Rozen,R. (1996) Clin.
Invest. Med., 19, 171-178). We believe that genetic variants of methionine
20 synthase similarly lead to mild hyperhomocysteinemia with consequent impact
on these two multifactorial disorders.
We used specific regions of homology within the methionine
synthase sequences, including a portion of the cobalamin binding site
determined from the E. coli enzyme, to design degenerate oligonucleotides for
25 RT-PCR-dependent cloning of human methionine synthase. We confirmed the
identification of the cDNA sequences for human methionine synthase by the
high degree of homology to the enzymes in other species and the identification
CA 022171~3 1998-01-06
-25 -
of mutations in patients from the cblG complementation group. We also
mapped the gene to human chromosome 1.
The assays described herein can be used to test for compounds that
modulate methione synthase activity and hence may have therapeutic value in
5 the prevention of neural tube defects, prevention and/or treatment of colon
cancer, cardiovascular disease, hyperhomocysteinemia, and megaloblastic
anemia without methylmalonic aciduria.
Screens for compounds that modulate methionine synthase enzymatic activity
Screens for potentially useful therapeutic compounds that modulate
methionine synthase activity may be readily performed, for example, by assays
that measure the incorporation of [14C]5-methyltetrahydrofolate into
methionine or protein, or assays that measure the conversion of [14C]-
homocysteine into methionine or protein. Examples of such assays, which
15 employ whole cells or cell lysates, are well-known to those skilled in the art
(see, e.g., Schuh, S., et al., N. Engl. J. Med. 1984, 310:686-69; Rosenblatt, D.S., et al., J. Clin. Invest. 1984, 74:2149-2156; Watkins, D., and Rosenblatt, D.S., J. Clin. Invest. 1988, 81: 1690- 1694; and Watkins, D., and Rosenblatt, D. S.,
Am. J. Med. Genet. 1989, 34:427-434), and may be readily adapted for high
20 throughput screening.
ELISA for the detection of compounds that modulate methionine synthase
expression
Enzyme-linked immunosorbant assays (ELISAs) are easily
incorporated into high-throughput screens designed to test large numbers of
25 compounds for their ability to modulate levels of a given protein. When used in
the methods of the invention, changes in a given protein level of a sample,
CA 022171~3 1998-01-06
-26-
relative to a control, reflect changes in the methionine synthase expression
status of the cells within the sample. Protocols for ELISA may be found, for
example, in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley
& Sons, New York, NY, 1997. Lysates from cells treated with potential
5 modulators of methionine synthase expression are prepared (see, for example,
Ausubel et al., supra), and are loaded onto the wells of microtiter plates coated
with "capture" antibodies specific for methionine synthase. Unbound antigen
is washed out, and a methionine synthase-specific antibody, coupled to an
agent to allow for detection, is added. Agents allowing detection include
10 alkaline phosphatase (which can be detected following addition of colorimetric
substrates such as p-nitrophenolphosphate), horseradish peroxidase (which can
be detected by chemiluminescent substrates such as ECL, commercially
available from Amersham) or fluorescent compounds, such as FITC (which can
be detected by fluorescence polarization or time-resolved fluorescence). The
15 amount of antibody binding, and hence the level of a methionine synthase
polypeptide within a lysate sample, is easily quantitated on a microtiter plate
reader.
As a baseline control for methionine synthase expression, a sample
that is not exposed to test compound is included. Housekeeping proteins are
20 used as internal standards for absolute protein levels. A positive assay result,
for example, identification of a compound that decreases methionine synthase
expression, is indicated by a decrease in methionine synthase polypeptide
within a sample, relative to the methionine synthase level observed in cells
which are not treated with a test compound.
CA 022171~3 1998-01-06
Reporter ~ene assays for compounds that modulate methionine synthase
expression
Assays employing the detection of reporter gene products are
extremely sensitive and readily amenable to automation, hence making them
5 ideal for the design of high-throughput screens. Assays for reporter genes mayemploy, for example, colorimetric, chemiluminescent, or fluorometric detection
of reporter gene products. Many varieties of plasmid and viral vectors
containing reporter gene cassettes are easily obtained. Such vectors contain
cassettes encoding reporter genes such as lacZ/~-galactosidase, green
10 fluorescent protein, and luciferase, among others. Cloned DNA fragments
encoding transcriptional control regions of interest (e.g. that of the mammalianmethionine synthase gene) are easily inserted, by DNA subcloning, into such
reporter vectors, thereby placing a vector-encoded reporter gene under the
transcriptional control of any gene promoter of interest. The transcriptional
15 activity of a promoter operatively linked to a reporter gene can then be directly
observed and quantitated as a function of reporter gene activity in a reporter
gene assay.
Cells are transiently- or stably-transfected with methionine synthase
control region/reporter gene constructs by methods that are well known to those
20 skilled in the art. Transgenic mice containing methionine synthase control
region/reporter gene constructs are used for late-stage screens in vzvo. Cells
containing methionine synthase/reporter gene constructs are exposed to
compounds to be tested for their potential ability to modulate methionine
synthase expression. At appropriate timepoints, cells are lysed and subjected to25 the appropriate reporter assays, for example, a colorimetric or
chemiluminescent enzymatic assay for lacZ/~-galactosidase activity, or
fluorescent detection of GFP. Changes in reporter gene activity of samples
CA 022171~3 1998-01-06
-28-
treated with test compounds, relative to reporter gene activity of apl~lopliate
control samples, indicate the presence of a compound that modulates
methionine synthase expression.
Quantitative PCR of methionine synthase mRNA as an assay for compounds
5 that modulate methionine synthase expression
The polymerase chain reaction (PCR), when coupled to a preceding
reverse transcription step (rtPCR), is a commonly used method for detecting
vanishingly small quantities of a target mRNA. When performed within the
linear range, with an appropriate internal control target (employing, for
10 example, a housekeeping gene such as actin), such quantitative PCR provides
an extremely precise and sensitive means of detecting slight modulations in
mRNA levels. Moreover, this assay is easily performed in a 96-well format,
and hence is easily incorporated into a high-throughput screening assay. Cells
are treated with test compounds for the appropriate time course, lysed, the
15 mRNA is reverse-transcribed, and the PCR is performed according to
commonly used methods, (such as those described in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1997),
using oligonucleotide primers that specifically hybridize with methionine
synthase nucleic acid. Changes in product levels of samples exposed to test
20 compounds, relative to control samples, indicate test compounds that modulate methionine synthase expression.
Secondary screens of test compounds that appear to modulate methionine
synthase activity
After test compounds that appear to have methionine synthase-
25 modulating activity are identified, it may be necessary or desirable to subject
CA 022171~3 1998-01-06
-29-
these compounds to further testing. At late stages testing will be performed in
vivo to confirm that the compounds initially identified to affect methionine
synthase activity will have the predicted effect in vivo.
Test Compounds
In general, novel drugs for prevention of neural tube defects, or
prevention and/or treatment of colon cancer or cardiovascular disease are
identified from large libraries of both natural product or synthetic (or semi-
synthetic) extracts or chemical libraries according to methods known in the art.Those skilled in the field of drug discovery and development will understand
10 that the precise source of test extracts or compounds is not critical to the
screening procedure(s) of the invention. Accordingly, virtually any number of
chemical extracts or compounds can be screened using the exemplary methods
described herein. Examples of such extracts or compounds include, but are not
limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation15 broths, and synthetic compounds, as well as modification of existing
compounds. Numerous methods are also available for generating random or
directed synthesis (e.g., semi-synthesis or total synthesis) of any number of
chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-,
and nucleic acid-based compounds. Synthetic compound libraries are
20 commercially available from Brandon Associates (Merrimack, NH) and
Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant, and animal extracts are
commercially available from a number of sources, including Biotics (Sussex,
UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce,
25 FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and
synthetically produced libraries are produced, if desired, according to methods
CA 022171~3 1998-01-06
-30-
known in the art, e.g., by standard extraction and fractionation methods.
Furthermore, if desired, any library or compound is readily modified using
standard chemical, physical, or biochemical methods.
In addition, those skilled in the art of drug discovery and
5 development readily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, or any
combination thereof) or the elimination of replicates or repeats of materials
already known for their therapeutic activities for homocysteinemia,
megaloblastic anemia without methylmalonic aciduria, cardiovasular disease,
10 colon cancer, and neural tube defects should be employed whenever possible.
When a crude extract is found to modulate methionine synthase
biological activity, further fractionation of the positive lead extract is necessary
to isolate chemical constituents responsible for the observed effect. Thus, the
goal of the extraction, fractionation, and purification process is the careful
15 characterization and identification of a chemical entity within the crude extract
that modulates methionine synthase biological activity. The same assays
described herein for the detection of activities in mixtures of compounds can beused to purify the active component and to test derivatives thereof. Methods of
fractionation and purification of such heterogenous extracts are known in the
20 art. If desired, compounds shown to be useful agents for treatment are
chemically modified according to methods known in the art. Compounds
identified as being of therapeutic value may be subsequently analyzed using
mammalian models of homocysteinemia, megaloblastic anemia without
methylmalonic aciduria, cardiovasular disease, colon cancer, and neural tube
25 defects.
CA 022171~3 1998-01-06
Therapy
Compounds identif1ed using any of the methods disclosed herein,
may be ~flministered to patients or experimental animals with a
pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.5 Conventional pharmaceutical practice may be employed to provide suitable
formulations or compositions to a(lminister such compositions to patients or
experimental ~nim~l.s. Although intravenous a(lministration is preferred, any
appropriate route of a~lmini~stration may be employed, for example, parenteral,
subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,
10 intraventncular, intracapsular, intraspinal, intracisternal, intraperitoneal,intranasal, aerosol, or oral a-lministration. Therapeutic formulations may be inthe form of liquid solutions or suspensions; for oral a(lministration,
formulations may be in the form of tablets or capsules; and for intranasal
formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found in,
for example, "Remington's Pharmaceutical Sciences." Formulations for
parenteral aflministration may, for example, contain excipients, sterile water, or
saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide
20 polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene
copolymers may be used to control the release of the compounds. Other
potentially useful parenteral delivery systems for antagonists or agonists of the
invention include ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for inhalation may
25 contain excipients, for example, lactose, or may be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and
CA 022171~3 1998-01-06
deoxycholate, or may be oily solutions for ~(1ministration in the form of nasal
drops, or as a gel.
Examples
The following examples are to illustrate, not limit the invention.
l~ )le 1: Clonin~ human methionine svnthase cDNA.
An initial survey of the NCBI databases yielded several sequences
corresponding to methionine synthase from different organisms. Comparison
of these sequences generated four very conserved regions identified as Boxes
1-4 in Fig. 1 (SEQ ID Nos:3-25). Degenerate oligonucleotides (SEQ ID
10 Nos:26-66) were synthesized corresponding to these conserved sequences
(Table 1). These were used as primers for RT-PCR with human and mouse
mRNA. These experiments yielded PCR products which were subcloned,
sequenced and aligned as shown in Fig. 2. In subsequent experiments,
oligonucleotide primers were specified *om the non-degenerate internal
15 sequences of the subclones and additional PCR products encompassing the
conserved boxes were obtained. In later experiments, additional sequences
were obtained by inverse PCR ("PCR", Fig. 2) to obtain upstream or
downstream sequences from those already determined. At the 3' end, a mouse
sequence was obtained from the dbEST database (Accession Number
20 W33307). This sequence was used as the source of primers for additional PCR
experiments. Throughout the experiments, the sequences of the PCR products
were considered provisionally authentic if they were homologous to the
methionine synthase sequences obtained from the databases. The sequences
were taken as error *ee by comparison of the sequences of at least two, and
25 usually three, independent PCR reactions. Sequences were linked into a
common sequence if RT-PCRs bridging independently isolated sequences were
CA 022171~3 1998-01-06
-33-
successful. Through this approach the complete coding sequence was
determined through exclusive use of PCR reactions.
The coding sequence of human methionine synthase contains 3795
bp (SEQ ID NO: 1) encoding a polypeptide of 1265 amino acids in length (SEQ
5 ID NO:2) (Fig. 3), exceeding the length of published methionine synthases by
11-29 residues. The putative initiation codon is in a sequence of good context
for the initiation of translation in eukaryotic cells (_ACAACATGT, underlined
nucleotides matching Kozak consensus (Kozak,M. (1991) J. Biol. Chem.,
266, 19867-19870)). The predicted MW of methionine synthase is 141,000,
comparing favorably with the published size of 151,000 based on SDS-
polyacrylamide electrophoresis of the pig enzyme (Chen, Z., Crippen, K.,
Gulati, S., and Banerjee, R. (1994) J. Biol. Chem., 269, 27193-27197). It
shares 58% identity with the E. coli and 65% identity with the C. elegans
enzyme.
15 ~ml le 2: Chromosomal location.
Using FISH, the gene encoding methionine synthase was mapped to
chromosome band 1 q43, close to the telomeric region of the long arm (Fig. 4).
A total of 50 cells with at least one signal were observed. A signal was seen on1 chromatid in 26 cells, on two chromatids in 14 cells, on 3 chromatids in 7
20 cells, and on 4 chromatids in 3 cells. These results confirm the previous
assignment of the gene to chromosome 1 by Mellman et al. (Mellman, I.S., Lin,
P.F., Ruddle, F.H., and Rosenberg, L.E. (1979) Proc. Natl. Acad. Sci. USA, 76,
405-409), who used cobalamin binding as a marker for the enzyme in human-
hamster hybrids.
CA 022171~3 1998-01-06
-34-
~x~mple 3: Mutations in the cblG complementation ~roup.
Patients with deficiency of methionine synthase activity have been
grouped into the cblG complementation group in cell fusion experiments
(Watkins, D. and Rosenblatt, D.S. (1988) J. Clin. Invest., 81, 1690-1694).
5 Fibroblast cultures from patients assigned to cblG were examined by RT-PCR
based SSCP analysis. Three mutations were identified by sequencing PCR
fragments showing band shifts by SSCP (Fig. 5). In each case, the change was
confirmed by an independent diagnostic test on genomic DNA or a separate
preparation of cDNA from patient fibroblasts. One of the mutations, 2756A~G
10 (D919G), was confirmed by a diagnostic test that monitored the presence of a
HaeIII site created by the mutation (Fig. 5A). Using this test, it was identified
as a polymorphism since it was seen in 8 of 52 control alleles (15%). In t~,vo
other cases, candidate deleterious mutations were identified. One is a 3 bp
deletion, bp 2640-2642, that results in the deletion of an isoleucine codon
15 (~Ile881). It was confirmed by heteroduplex analysis of cDNA generated by
RT-PCR (Fig. 5B). The second is a point mutation, 2758C~G. It results in the
amino acid substitution H920D. It was confirmed in genomic DNA by the loss
of a Sau96I site (Fig. 5C). The latter two mutations were heterozygous in the
patient cell lines. Their second mutation has not been identified. The
20 candidate deleterious mutations were not seen in panels of 68 or 52 control
alleles, respectively.
li'x~mple 4: Additional Roles for Methionine Svnthase Polvmorphism
(Asp919Glv or D919G) in Disease.
The following data suggest that the D919G polymorphism
25 contributes to altered metabolism of homocysteine, methionine, folates, Vit.
B12, and S-adenosylmethionine.
CA 022171~3 1998-01-06
First in a Montreal study (n=303), in which mother-child pairs (cases
and controls) were examined, we observed that infants who were homozygous
for the polymorphism (Gly/Gly; Table 2) were at decreased risk for NTD.
Measurements of serum folate, RBC folate, plasma homocysteine and serum
5 cobalamin did not give any statistically significant differences, except the trend
was toward low folate levels in Gly/Gly individuals (cases and controls).
A second study (n=255) in California also examined the methionine
synthase polymorphism as a risk factor for neural tube defects (Table 3). This
study shows a similar decreased risk of neural tube defects in children
10 homozygous for Gly/Gly. Since the study encompassed a mix of whites and
Hispanics, the data were reexamined stratified according to ethnic origin. Both
groups showed a protective effect of Gly/Gly.
In summary, two independent studies suggest a protective effect of
Gly/Gly against the risk of neural tube defects. This is likely to be mediated by
15 a mild reduction in methionine synthase activity.
Next, in a study of colon cancer, (212 cases and 345 controls), we
observed a decreased risk for colon cancer in the individuals who were
homozygous for the polymorphism (relative risk = 0.62); see Table 4. In the
same study, we observed significantly decreased levels of plasma folate in
20 individuals who were homozygous for the polymorphism; see Table S.
The Boston study described in Tables 4 and S is presented again in
Table 6 with the data stratified according to alcohol intake. As shown in the
table, Gly/Gly individuals with a low to medium alcohol intake had a relative
risk associated with colon cancer of 0.1 l. The combined data (low + high
25 alcohol) gave a risk level of 0.62 (Table 6).
In summary, drug therapy targeted to a reduction in methionine
synthase activity may be protective in individuals at risk for colon cancer or at
CA 022171~3 1998-01-06
-36-
risk for neural tube defects. Additional polymorphisms or mutations may also
exert a protective effect against the risk of neural tube defects or colon cancer.
Conversely, it is understood that some polymorphisms and/or mutations may
enhance the risk of neural tube defects or colon cancer, for example, by
5 increasing methionine synthase activity.
Example 5: Role of Polymorphism on Homocysteine and Folate Levels.
Third, in a study of individuals participating in the U.S. NHLBI
Family Heart Study, we observed both an increase in plasma homocysteine
following a methionine load and a decrease in plasma folate in individuals who
10 were homozygous for this polymorphism; see Table 7.
F~mple 6: Methionine Svnthase Assavs for the Detection of Compounds
that Modulate Methionine Synthase Activity and Expression
Potentially useful therapeutic compounds that modulate (e.g.
increase or decrease) methionine synthase activity or expression may be
15 isolated by various screens that are well-known to those skilled in the art. Such
compounds may modulate methionine synthase expression at the pre- or post-
transcriptional level, or at the pre- or post-translational level.
Fx~mple 7: Materials and Methods
Cell lines. The skin fibroblast lines are from patients with
20 methionine synthase deficiency. They were assigned to the cblG
complementation group in cell fusion experiments assayed by 14C-
methyltetrahydrofolate incorporation into cellular macromolecules (Watkins,
D. and Rosenblatt, D.S. (1988) J. Clin. Invest., 81, 1690-1694). Control
fibroblasts were from other laboratory stocks or the Montreal Children's
CA 022171~3 1998-01-06
Hospital Cell Repository for Mutant Human Cell Strains. Of the patients for
which non-polymorphic mutations were found, WG 1892, a Caucasian male,
was diagnosed at the age of 4 years with developmental delay, tremors, gait
instability, megaloblastic anemia and homocystinuria; and WG2290, also a
Caucasian male, was diagnosed at age 3 months with failure to thrive, severe
eczema, megaloblastic anemia and surprisingly both homocystinuria and
methylmalonic aciduria.
Materials. The T/A cloning kit was from Invitrogen. The Geneclean
III Kit was obtained from Bio 101 Inc. and the Wizard Mini-Preps were from
10 Promega. The random-primed DNA labelling kit was from Boehringer-
Mannheim. Taq polymerase, Superscript II reverse transcriptase, AMV reverse
transcriptase, Trizol reagent, DNAzol reagent, T4 DNA ligase, and restriction
enzymes were purchased from Gibco BRL. The Sequenase kit for manual
sequencing was from United States Biochemicals. The o~-[3sS]dATP (12.5
15 Ci/mole) was from Dupont or ICN. The oligonucleotide primers were
synthesized by R. Clarizio of the Montreal Children's Hospital Research
Institute Oligonucleotide Synthesis Facility or the Sheldon Biotechnology
Centre, McGill University.
Homology matches. Comparisons were made between the
20 published E. coli cobalamin-dependent methionine synthase sequence and
sequences in the NCBI databases (dbEST and GenBank) using the BLAST
programs.
PCR cloning and DNA sequencing. DNA was prepared from
fibroblast pellets by the method of Hoar et al. (Hoar,D.I., Haslam,D.B., and
25 Starozik,D.M. (1984) Prenat. Diag., 4, 241-247). Total cellular RNA was
isolated by the method of Chirgwin et al. (Chirgwin,J.M., Przybyla,A.E.,
MacDonald,R.J., and Rutter,W.J. (1979) Biochemistry, 18, 5294-5299) and
CA 022171~3 1998-01-06
-38-
reverse-transcribed using oligo-dTls as primer. PCR was conducted using
degenerate oligonucleotides as primers, paired so as to link the sequences of
different homology boxes. The PCRs were conducted as described previously
(Triggs-Raine,B.L., Akerman,B.R., Clarke,J.T., and Gravel,R.A. (1991)
5 Am. J. Hum. Genet., 49, 1041-1054) except that the temperature of incubation
was modified to accommodate the use of reduced temperatures in the annealing
step or by step-down PCR (Hecker,K.H. and Roux,K.H. (1996)
Biotechniques, 20, 478-485. (Abstract)). In some experiments, inverse PCR
was used to determine sequence upstream or downstream of known sequence
10 (Ochrnan,H., Medhora,M.M., Garza,D., and Hartl,D.L. (1990) PCR
Protocols: A Guide to Methods and Applications, Academic Press, San
Diego, pp. 219-227). In these instances, genomic DNA or cDNA prepared by
reverse transcription of RNA was digested with different four base restriction
endonucleases, ligated with T4 DNA ligase, and amplified by PCR using
1~ adjacent oligonucleotides priming in opposite directions. Templates for inverse
PCR at the cDNA level were generated with 12.5 ~lg RNA reversed transcribed
using AMV-RT. Second strand synthesis was carried out using the random-
primed DNA labelling kit adding 1 ~ll of each dNTP. Samples were incubated
30 min. at 37~C. Template was then treated as genomic DNA for digestion and
20 ligation. Inverse PCR was used to obtain the 5' and 3' ends of the cDNA and to
define an intron sequence adjacent to a splice junction for the design of a
mutation diagnostic test. The PCR products were purified with Geneclean and
were subcloned in the pCR2.1 vector and transformed into E. coli as per the
supplier's protocol (TA Cloning Kit). The candidate clones were sequenced
25 manually or by the DNA Core Facility of the Canadian Genetic Diseases
Network or the McGill University Sheldon Biotechnology Centre.
CA 022171~3 1998-01-06
-39-
Mutation analysis. Genomic DNA and RNA were isolated from
control or patient fibroblast pellets using the DNAzol or Trizol reagents,
respectively, as per the manufacturer. The cDNA template for PCR was
prepared by reverse transcription of 3-5 ug total RNA in reactions containing
400 U of Superscript II reverse transcriptase and 100ng random hexamers in a
total reaction volume of 20 ul. SSCP analysis was performed as described
previously (Triggs-Raine,B.L., Akerman,B.R., Clarke,J.T., and Gravel,R.A.
(1991) Am. J. Hum. Genet.,49,1041-1054) in reactions containing 4 111 of
template, 1 !11 of each dTTP, dCTP, dGTP (0.625 mM), 0.5 ~l of dATP (0.625
10 mM), 1 ~ [35S]-dATP (12.5 Ci/mole). The radio labelled PCR products
mixed with sequencing stop solution were heat denatured and quick chilled on
ice prior to loading (Triggs-Raine,B .L., Akerman,B .R., Clarke,J.T ., and
Gravel,R.A. (1991) Am. J. Hum. Genet., 49,1041-1054). As well, an aliquot
of each sample was run without prior heating to identify the duplex product.
15 The fragments were subjected to electrophoresis in a 6% acrylamide/10%
glycerol gel in lX TBE for 18 hrs at 8 watts at room temperature. The gel was
dried and exposed to Biomax film (Kodak). Fragments that displayed band
shifts were sequenced directly.
Two mutations were confirmed directly in PCR amplification
20 products from genomic DNA and one mutation was confirmed in reversed
transcribed mRNA. The PCR reactions for mutation confirmation were
performed using 4 ~l of cDNA template or 500 ng genomic DNA, 500 ng of
specific primers, 2.5 U Taq polymerase and 1.5 mM MgCl2 in a 50 111 volume.
Heteroduplex analysis was accomplished by preheating PCR products to 95~C
25 for five minutes and subjecting the samples to electrophoresis in a 9%
polyacrylamide gel (Triggs-Raine,B.L., Akerman,B.R., Clarke,J.T., and
Gravel,R.A. (1991) Am. J. Hum. Genet., 49, 1041-1054). Other diagnostic
CA 022171~3 1998-01-06
-40 -
assays were accomplished by digesting a 15 Ill sample of the PCR products
with the indicated restriction endonuclease prior to electrophoresis.
Chromosomal loc~ tion. Human metaphase spreads were
obtained from short-term cultures of phytohemaglutinin-stimulated peripheral
5 blood lymphocytes. The cells were synchronized with thymidine and treated
with BrdU during the late S-phase before harvesting for simultaneous
observation of the hybridized sites and chromosome banding. The protocol for
FISH was essentially as described previously (Lemieux, N., Malfoy, B., and
Forrest, G.L. (1993) Genomics, 15, 169-172; Zhang, X.X., Rozen, R.,
lO Hediger, M.A., Goodyer, P., and Eydoux, P. (1994) Genomics, 24,
413-414). Briefly, a 5 kb DNA fragment ofthe methionine synthase genomic
DNA (generated by PCR using oligonucleotides #1782 and #1780) was
labelled by nick translation with biotin-16-dUTP (Boehringer-Mannheim),
ethanol precipitated and dissolved in hybridization buffer at a final
15 concentration of 8 ng/~ll. The slides were denatured in 70% forrnamide, 2 x
SSC at 70~C for 2 min. The biotinylated probe was denatured in the
hybridization buffer at 95~C for lO min, quickly cooled on ice, then applied on
slides. Post-washing was done by rinsing in 50% formamide, 2 x SSC at 37~C.
The slides were incubated with rabbit antibiotin antibody (Enzo Biochemicals),
20 biotinylated goat anti-rabbit antibodies (BRL) and streptavidin-FITC. They
were stained with propidium iodide and mounted in p-phenylenediamine, pH
11. Cells were observed under the microscope (Zeiss), then captured through a
CCD camera and processed using a FISH software (Applied Imaging).
CA 022l7l~3 l998-0l-06
-41 -
Table 1. Oligonucleotides used for cDNA cloning, chromosome mapping and mutation detection.
Oligonucleotidesa Sequence Location
b
D1729 (SEQ ID NO:26) 5'-GAYGGNGCNATGGGNACNATGATHCA 100-125
D1730 (SEQ ID NO:27) 5'-GCNACNGTNAARGGNGAYGTNCAYGAYAT 2332-
1 0 2360
D1731 (SEQ ID NO:28) 5'-RTTYTTNCCDATRTCRTGNACRTCNCCYTT 2370-
2341
D1733 (SEQ ID NO:29) 5'-RTGNAGRTAYTCNGCRAANGCYTCNGC 3426-
3400
D1754 (SEQ ID NO:30) 5'-ATRTGRTCNGGNGTNGTNCCRCARCANCCNCC 992-961
D1755 (SEQ ID NO:31) 5'-GGNGGNTGYTGYGGNACNACNCCNGAYCAYAT 961-992
M1806A (SEQ ID NO:32) 5'-GTCTGTGTCATAGCCCAGAATGGG 3795-
3772
M1806B (SEQ ID NO:33) 5'-TCAGTCTGTGTCATAGCCCAGAAT 3798-
3775
305A (SEQ ID NO:34) 5'-GAACTAGAAGACAGAAATTCTCTA
(intronic)
407A (SEQ ID NO:35) 5'-TTCCGAGGTCAGGAATTTAAAGATCA 151-176
407B (SEC ID NO:36) 5'-GTGTTCTTCGTTTAGCTTCTCCCG 150-127
407D (SEQ ID NO:37) 5'-CCCCAGCCAGCAAGTATTCCTTAT 268-245
1107A (SEQ ID NO:38) 5'-CTAGGTTGTATTTCCTTGAGGATC 3856-
3833
1406D (SEQ ID NO:39) 5'-GGAGCTGGAAAAATGTTTCTACCTC 2170-
2194
1406E (SEQ ID NO:40) 5'-ACAGGAGGGAAGAAAGTCATTCAG 1963-
1986
1706A (SEQ ID NO:41) 5'-CCTTCAATTATATTGAGAGGTCGGG 2129-
2105
1707A (SEQ ID NO:42) 5'-CAACCCGAAGGTCTGAAGAAAACC 28-51
1707B (SEC ID NO:43) 5'-CCCGCGCTCCAAGACCTGTCG 7-27
1707C (SEC ID NO:44) 5'-CGACAGGTCTTGGAGCGCGGG 27-7
1758 (SEC~ ID NO:45) 5'-GGAGTCATGACTCCTAAATCAATAACTC 2432-
2405
1760 (SEQ ID NO:46) 5'-GACGACTACAGCAGCATCATGGT 3355-
3377
1766 (SEQ ID NO:47) 5'-AAAAATCATTTCATCCAGGGAA 2526-
2505
1772 (SEQ ID NO:48) 5'-ATAGGCAAGAACATAGTTGGAGTAGT 2359-
2384
1773 (SEQ ID NO:49) 5'-TTTCATCTAACAGCTGGGAACACAC 2698-
2674
1774 (SEQ ID NO:50) 5'-TGCCTCTCAGACTTCATCGCTCCC 3241-
3264
1780 (SEQ ID NO:51) 5'-TGCAGCCTGGGGCACAGCAGC 3168-
3148
1782 (SEQ ID NO:52) 5'-ATGGATTGGCTGTCTGAACCTCAC 2824-
2847
1796 (SEQ ID NO:53) 5'-CATGGAAGAATATGAAGATATTAGAC 2727-
2752
1803 (SEQ ID NO:54) 5'-ACCATCATCCTCATAGGCCTTGCT 3354-
3331
1806C (SEQ ID NO:55) 5'-CAGACCTGCGAAGGTTGCGGTAC 3482-
3504
1806F (SEQ ID NO:56) 5'-GAAGTGGTTGCTCCTCCAATCAAC 2591-
2568
CA 022171~3 1998-01-06
-42 -
1808 (SEQ ID NO:57) 5'-GAGCAGCTTTCAGTATCTTATCACAT 2458-
2433
1827 (SEQ ID NO:58) 5'-ACAAGTTGTGTTCCTCCATTCCAGT 1657-
1633
1828 (SEQ ID NO:59) 5'-AGAGCGCTGTAATGTTGCAGGATCA 1125-
1149
1907B (SEQ ID NO:60) 5'-T(~'l''l"l"l l'CAATGCCCTTCACAAGGG 2057-
2033
1907C (SEQ ID NO:61) 5'-TAAAAAGTATGGAGCTGCTATGGTG 1464-
1 0 1488
2606A (SEQ ID NO:62) 5'-GACCAGACAGTAACATATGTCCTTC 1078-
1054
2606B (SEQ ID NO:63) 5'-ACATTACAGCGCTCTCCAATGTTAAC 1139-
1114
2706A (SEQ ID NO:64) 5'-TGAGGTTGAGAAATGGCTTGGACC 3750-
3773
2706B (SEQ ID NO:65) 5'-GCCACAGATATGTTCTTCCTCAATG 3749-
3725
3107A (SEQ ID NO:66) 5'-TGTGGAGAGCACGTCTTCTCTGCC -55 - -32
a Numbers with the prefix "D" refer to oligonucleotides with degenerate bases shown as N (any
base), H (A, C, or T), D (A, G, or T), Y (T or C), or R (A or G); those with the prefix "M" refer
to mouse sequences (see Fig. 3).
b From the first methionine codon, see Fig. 3.
CA 022171~3 1998-01-06
-43 -
Table 2. MS Polymorphism in Neural Tube Defects - Montreal Study
Case Control
Cases mothers Controls mothers Odds
Genotype N % N % N % N %
ratio* 95% C.I.
Asp/Asp 38 69 40 66 59 6155 61
Asp/Gly 17 31 20 33 28 2934 38
Gly/Gly 0 0.9 1 2 10 10 1 1 0.07 0.004-
1.29
N 55 61 97 90
* Odds ratio calculated for genotypes Asp/Asp vs Gly/Gly
(to permit the calculation, the 0 cell was increased to 0.5)
Table 3. MS Polymorphisms in Neural Tube Defects - California Study
Genotype Cases Controls Odds
Ethnic Group 2756A-G N % N % ratio* 95% C.I.
Overall Asp/Asp 64 67104 64 1.0
Asp/Gly 30 32 49 30 0.99 0.56- 1.72
Gly/Gly 1 1 7 4 0.23 0.05- 1.92
White only Asp/Asp 21 66 38 66 2.0
Asp/Gly 10 31 16 28 1.1 0.44- 2.9
Gly/Gly 1 3 3 5 0.60 0.11 - 5.6
Hispanic only Asp/Asp 43 68 66 63 1.0
Asp/Gly 20 32 33 31 0.9 0.45- 1.8
Gly/Gly 0 0 4 4 0
* Odds ration calculated for genotypes Asp/Asp vs Gly/Gly
CA 022171~3 1998-01-06
-44-
Table 4. Frequency of MS genotype and relative risk (RR) of colorectal cancer
by MS genotype
MS Genotype Cases Controls
n % n % RR95%CI
Asp/Asp 145 (68) 234 (68) 1.0
Asp/Gly 61 (29) 95 (28) 1.020.69 - 1.50
Gly/Gly 6 (3) 16 (5) 0.620.24- 1.64
Total 212 345
Table 5. Mean of homocysteine and folate (geometric) by case control status
and MS Genotype in a colon cancer study
MS genotype Cases ControlsCases & Controls
n mean n mean n mean
Folate (Bio-Kit) ng/ml
Asp/Asp 115 3.8 201 3.9 * 3163.9 **
Asp/Gly 49 4.1 * 80 3.8 * 1293.9 **
Gly/Gly 6 2.1 12 2.3 18 2.2
Homocysteine (~M)
Asp/Asp 66 12.5 160 12.1 226 12.3
Asp/Gly 30 10.8 50 11.6 80 11.2
Gly/Gly 4 13.4 9 11.7 13 12.5
* = p < 0.05
** =p<O.Ol
Table 6. Age Adjusted Relative Risk of Colon Cancer According to MS
Polymorphism and Alcohol Intake Status Among US Physicians
CA 022171~3 1998-01-06
-45 -
Genotype
2756A->G Case Control
Alcohol intake Asp919Gly s s Odds 95% C.I.
N N ratio
Low - Medium Asp/Asp 1013 2e+09 1.0
0 - 0.8 drinks/day Asp/Gly 7113 0.87 0.54 - 1.4
Gly/Gly 9 0.11 0.01 - 0.82
N
High Asp/Asp 3721 7e+06 0.74 0.46 - 1.19
51 - 2+ drinks/day Asp/Gly 563 1.15 0.60 - 2.18
Gly/Gly 3.83 0.72-20.47
N
CA 022171~3 1998-01-06
-46 -
Table 7. Mean Homocysteine and Folate Status by MS Genotype
(Date of Analysis: February 18, 1997)
Methionine Synthase Genotype
Asp/As Asp/Gly Gly/Gly
p
Result Result P Result P
N 252 111 17
Fasting Hcy (,uM) 8.5 8.4 0.76 8.7
post-methionine load Hcy 17.9 19.6 0.74
(~M) 7.4 0.05* 6.9 22.3
Folate (microb test) 0.37 0.03*
6.3
0.37
