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WO2001062969A2 - Variants de recepteurs oestrogeniques alpha et procede de detection desdits variants - Google Patents

Variants de recepteurs oestrogeniques alpha et procede de detection desdits variants Download PDF

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Publication number
WO2001062969A2
WO2001062969A2 PCT/US2001/005358 US0105358W WO0162969A2 WO 2001062969 A2 WO2001062969 A2 WO 2001062969A2 US 0105358 W US0105358 W US 0105358W WO 0162969 A2 WO0162969 A2 WO 0162969A2
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ofthe
nucleic acid
estrogen receptor
protein
sequence
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WO2001062969A3 (fr
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Francis Kalush
Michael J. Cassel
Stuart Soo-In Hwang
Emily S. Winn-Deen
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Applied Biosystems Inc
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PE Corp
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Priority to CA002400915A priority Critical patent/CA2400915A1/fr
Priority to JP2001561777A priority patent/JP2004537959A/ja
Priority to AU2001243196A priority patent/AU2001243196A1/en
Priority to EP01916135A priority patent/EP1259650A2/fr
Publication of WO2001062969A2 publication Critical patent/WO2001062969A2/fr
Publication of WO2001062969A3 publication Critical patent/WO2001062969A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • Figure 6. The distribution and frequency of many ofthe SNPs ofthe present invention.
  • Figure 7. A graphic representation of the human ESR1 locus.
  • the present invention provides isolated nucleic acid molecules that contain one or more SNPs disclosed by the present invention.
  • the present invention further provides isolated nucleic acid molecules that encode the variant protein.
  • Such nucleic acid molecules will consist of, consist essentially of, or comprise one or more SNPs ofthe present invention.
  • the nucleic acid molecule can have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences.
  • nucleic acid is isolated from remote and unimportant flanking sequences and is of appropriate length such that it can be subjected to the specific manipulations or uses described herein such as recombinant expression, preparation of probes and primers for the SNP position, and other uses specific to the SNP-containing nucleic acid sequences.
  • an "isolated" nucleic acid molecule such as a cDNA molecule containing a
  • the present invention further provides a protein that is comprised ofthe amino acid sequences summarized in Figure 1, including one or more ofthe sequence polymorphisms provided in Figure 2.
  • a protein is comprised of an amino acid sequence when the amino acid sequence is at least part ofthe final amino acid sequence ofthe protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. A brief description of how various types of these proteins can be made/isolated is provided below.
  • the estrogen receptor protein ofthe present invention can be attached to heterologous sequences to form chimeric or fusion proteins.
  • Such chimeric and fusion proteins comprise a estrogen receptor protein operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the estrogen receptor protein. "Operatively linked” indicates that the estrogen receptor protein and the heterologous protein are fused in-frame.
  • the heterologous protein can be fused to the N-terminus or C-terminus ofthe estrogen receptor protein.
  • the fusion protein does not affect the activity ofthe estrogen receptor protein per se.
  • the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, Hi-tagged and Ig fusions.
  • enzymatic fusion proteins for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, Hi-tagged and Ig fusions.
  • Such fusion proteins, particularly poly-His fusions can facilitate the purification of recombinant estrogen receptor protein.
  • expression and/or secretion of a protein can be increased by using a heterologous signal sequence.
  • polypeptides also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification ofthe mature polypeptide or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included
  • the mature polypeptide is fused with another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification ofthe mature polypeptide or a pro-protein sequence.
  • Known modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures.
  • Predicted domains and functional sites are readily identifiable by computer programs well-known and readily available to those of skill in the art (e.g., PROSITE analysis).
  • the proteins ofthe present invention can be used in assays to determine the biological activity ofthe protein, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels ofthe protein (or its binding partner or receptor) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products. Methods for performing the uses listed above are well known to those skilled in the art.
  • the estrogen receptor proteins ofthe present invention are useful for biological assay. Such assays involve any ofthe known estrogen receptor functions or activities or properties useful for the diagnosis and treatment of estrogen receptor-related conditions.
  • these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness.
  • Compounds can be identified that activate (agonist) or inactivate (antagonist) the receptor to a desired degree. Such compounds can be selected for the ability to act on one or more ofthe variant estrogen receptor proteins of the present invention.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase/15625 fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., S- labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • Agents that modulate the protein ofthe present invention can be identified using one or more ofthe above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.
  • the estrogen receptor proteins ofthe present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the estrogen receptor. Accordingly, the invention provides methods for detecting the presence, or levels of, the estrogen receptor variants ofthe present invention (or encoding mRNA) in a cell, tissue, or organism. The method involves contacting a biological sample with a compound capable of interacting with the receptor protein (or gene or mRNA encoding the receptor) such that the interaction can be detected.
  • One agent for detecting a protein in a sample is an antibody capable of selectively binding to a variant form ofthe estrogen receptor protein.
  • samples include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the estrogen receptor proteins ofthe present invention also provide targets for diagnosing active disease, or predisposition to disease, in a patient having a variant estrogen receptor, particularly a disease involving the estrogen pathway, such as bone growth, cell differentiation, etc.
  • the receptor can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant receptor activity. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification as provided in Figure 2.
  • Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered receptor activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other ofthe known assay techniques useful for detecting mutations in a protein. Particularly useful are the variation provided in Figure 2.
  • In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • the peptide can be detected in vivo in a subject by introducing into the subject a labeled antipeptide antibody.
  • the peptides are also useful in pharmacogenomic analysis.
  • Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11) :983-985 (1996)), and Linder, M.W. (Clin. Chem. 43(2):254-266 (1997)).
  • the clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism.
  • the genotype ofthe individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound.
  • an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge.
  • the antibodies ofthe present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab') 2 , and Fv fragments.
  • Antibodies are preferably prepared from regions or discrete fragments ofthe estrogen receptor protein. Antibodies can be prepared from any region ofthe peptide as described herein. However, preferred regions will include those involved in function/activity and/or receptor/binding partner interaction.
  • An antigenic fragment will typically comprise at least 10 contiguous amino acid residues.
  • the antigenic peptide can comprise, however, at least 12, 14, 20 or more amino acid residues.
  • Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface ofthe protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness.
  • the antibodies can be used to isolate the estrogen receptor protein ofthe present invention by standard techniques, such as affinity chromatography or immunoprecipitation.
  • the antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced protein expressed in host cells.
  • such antibodies are useful to detect the presence ofthe estrogen receptor protein ofthe present invention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course of normal development.
  • such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression.
  • such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development. Antibody detection of circulating fragments ofthe full length estrogen receptor protein can be used to identify turnover.
  • the antibodies can be used to assess expression in disease states such as in active stages ofthe disease or in an individual with a predisposition toward disease related to the protein's function, particularly diseases involving bone growth/formation/degeneration.
  • disease states such as in active stages ofthe disease or in an individual with a predisposition toward disease related to the protein's function, particularly diseases involving bone growth/formation/degeneration.
  • the antibody can be prepared against the normal protein. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence ofthe specific mutant protein.
  • the antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism.
  • the diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting the expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.
  • antibodies are useful in pharmacogenomic analysis.
  • antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities.
  • the antibodies are also useful as diagnostic tools as an immunological marker for aberrant estrogen receptor protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.
  • nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.
  • nucleic acid molecules that consist ofthe nucleotide sequences shown in Figure 1, including one or more ofthe sequence polymorphisms provided in Figure 2.
  • a nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence ofthe nucleic acid molecule.
  • a fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length ofthe fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions ofthe peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.
  • a probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 50-55% homologous to each other typically remain hybridized to each other.
  • the conditions can be such that sequences at least about 65%, at least about 70%, or at least about 75% or more homologous to each other typically remain hybridized to each other.
  • the nucleic acid molecules are also useful for expressing antigenic portions ofthe proteins.
  • the nucleic acid molecules are also useful for constructing transgenic animals expressing all, or apart, ofthe nucleic acid molecules and peptides.
  • nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.
  • In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.
  • Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a estrogen receptor proteins ofthe present invention, such as by measuring a level of a receptor-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a receptor gene has been mutated.
  • the nucleic acid molecules are also useful in diagnostic assays for qualitative changes in estrogen receptor nucleic acid, and particularly in qualitative changes that lead to pathology.
  • the nucleic acid molecules can be used to detect mutations in estrogen receptor genes and gene expression products such as mRNA.
  • the nucleic acid molecules can be used as hybridization probes to detect naturally-occurring genetic mutations in the estrogen receptor gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form ofthe estrogen receptor gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a estrogen receptor protein.
  • RNA/RNA or RNA/DNA duplexes Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al, Science 230:1242 (1985)); Cotton et al, PNAS 85:4391 (1988); Saleeba et al, Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al, PNAS 86:2166 (1989); Cotton et al, Mutat. Res. 255:125-144 (1993); and Hayashi et al, Genet. Anal. Tech. Appl.
  • a probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.
  • the consecutive nucleotides can either include the target SNP position, or be a specific region in close enough proximity 5' and/or 3' to the SNP position to carry out the desired assay.
  • primer and probe sequences can readily be determined using the sequences provided in Figures 1, 2, and 9. It will be apparent to one of skill in the art that such primers and probes are useful as diagnostic probes or amplification primers for genotyping SNPs of the present invention, and can be incorporated into a kit format. For analyzing SNPs, it may be appropriate to use oligonucleotides specific to alternative primers.
  • allele-specific oligonucleotides referred to as “allele-specific oligonucleotides”, “allele-specific probes”, or “allele- specific primers”
  • the design and use of allele-specific probes for analyzing polymorphisms is described by e.g., Saiki et al, Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548.
  • allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals.
  • Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one ofthe alleles.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16- mer, at either the 8 or 9 position) ofthe probe. This design of probe achieves good discrimination in hybridization between different allelic forms.
  • Allele-specific probes are often used in pairs, the "pairs" may be identical except for a one nucleotide mismatch that represents the allelic variants at the SNP position.
  • One member of a pair perfectly matches a reference form of a target sequence and the other member perfectly matches a variant form.
  • several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence.
  • an allele-specific primer hybridizes to a site on target DNA overlapping the SNP position and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17 2427-2448 (1989). This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two-primers, resulting in a detectable product that indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3 '-most position ofthe oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • This PCR-based assay can be utilized as part ofthe TaqMan assay, described below.
  • Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers may include a container which will accept the test sample, a container which contains the SNP probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe.
  • the kit can further comprise reagents for PCR or other enzymatic reactions, and instractions for using the kit.
  • the previously unidentified SNPs ofthe present invention can be routinely identified using the sequence information disclosed herein and can be readily incorporated into one ofthe established kit formats which are well known in the art.
  • Arrays or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • the microarray is prepared and used according to the methods described in US Patent 5,837,832, Chee et al, PCT application W095/11995 (Chee et al), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci.
  • arrays are produced by the methods described by Brown et al., US Patent No. 5,807,522. Arrays or microarrays are commonly referred to as "DNA chips".
  • oligonucleotide probes ⁇ such as allele-specific oligonucleotides, may be implemented in an array, wherein each probe or pair of probes corresponds to a different SNP position.
  • the oligonucleotides are synthesized at designated areas on a substrate using a light- directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides probes to perfectly matched and mismatched target sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (e.g., a chip) at selected positions.
  • a solid support e.g., a chip
  • Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime, each corresponding to a particular SNP position or allelic variant.
  • probes are attached to a solid support in an ordered, addressable array.
  • the array/chip technology has already been applied with success in numerous cases.
  • arrays may generally be "tiled” for a large number of specific polymorphisms.
  • “Tiling” refers to the synthesis of a defined set of oligonucleotide probes that are made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members ofthe basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995. In a particular aspect, arrays are tiled for a number of specific SNPs.
  • the array is tiled to include a number of detection blocks, each detection block being specific for a specific SNP or a set of SNPs.
  • a detection block may be tiled to include a number of probes that span the sequence segment that includes a specific SNP.
  • the probes are synthesized in pairs differing at the SNP position.
  • monosubstituted probes are also generally tiled within the detection block. Such methods can readily be applied to the SNP information disclosed herein.
  • the chips may comprise an array of nucleic acid sequences of fragments of about 1 nucleotides in length.
  • the chip may comprise an array including at least one ofthe sequences selected from the group consisting of those disclosed in the Figures 1, 2, 8, 9, and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymorphic base.
  • the polymorphic base is within 5, 4, 3, 2, or 1 nucleotides from the center ofthe polynucleotide, more preferably at the center of said polynucleotide.
  • the chip may comprise an array containing any number of polynucleotides ofthe present invention.
  • An oligonucleotide may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference.
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number which lends itself to the efficient use of commercially available instrumentation.
  • test samples ofthe present invention include, but are not limited to, nucleic acid extracts, cells, and protein or membrane extracts from cells, which may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • the test sample used in the above-described methods will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as the sample to be assayed. Methods of preparing nucleic acid, protein, or cell extracts are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.
  • Multicomponent integrated systems may also be used to analyze SNPs. Such systems miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.
  • An example of such technique is disclosed in US patent 5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.
  • Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements ofthe samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas ofthe microchip to create functional microscopic valves and pumps with no moving parts. Varying the voltage controls the liquid flow at intersections between the micro-machined channels and changes the liquid flow rate for pumping across different sections ofthe microchip.
  • the vector may integrate into the host cell genome and produce additional copies ofthe nucleic acid molecules when the host cell replicates.
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) ofthe nucleic acid molecules.
  • the vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).
  • the regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage ⁇ , the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
  • expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers.
  • regions that modulate transcription include the SV40 enhancer, the cytomegaloviras immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation.
  • Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals.
  • the person of ordinary skill in the art would be aware ofthe numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989).
  • a variety of expression vectors can be used to express a nucleic acid molecule.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retrovirases. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, eg. cosmids and phagemids.
  • the regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or otlier ligand.
  • host cells i.e. tissue specific
  • inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or otlier ligand.
  • a variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
  • Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium.
  • Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
  • the invention provides fusion vectors that allow for the production ofthe peptides.
  • Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.
  • a proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety.
  • Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase.
  • Typical fusion expression vectors include pGEX (Smith et al, Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, Gene ⁇ P:301-315 (1988)) and pET lid (Studier et al, Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).
  • Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein.
  • the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).
  • the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors.
  • mammalian expression vectors include pCDM8 (Seed, B. Nature 52 :840(1987)) and pMT2PC (Kaufman et al, EMBOJ. tf:187-195 (1987)).
  • the expression vectors listed herein are provided by way of example only ofthe well- known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules.
  • the person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression ofthe nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA.
  • an antisense transcript can be produced to all, or to a portion, ofthe nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each ofthe parameters described above in relation to expression ofthe sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
  • the invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • bacteriophage and viral vectors these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction.
  • Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.
  • RNA derived from the DNA constracts described herein can also be used to produce these proteins using RNA derived from the DNA constracts described herein.
  • Host cells are also useful for conducting cell-based assays involving the estrogen receptor protein or estrogen receptor protein fragments, such as those described above as well as other formats known in the art.
  • a recombinant host cell expressing a native estrogen receptor protein is useful for assaying compounds that stimulate or inhibit estrogen receptor protein function.
  • a transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Any ofthe estrogen receptor protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.
  • any ofthe regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression ofthe estrogen receptor protein to particular cells.
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression ofthe transgene.
  • a system is the cre/loxP recombinase system of bacteriophage Pl .
  • the cre/loxP recombinase system see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).
  • Another example of a recombinase system is the FLP recombinase system of 5. cerevisiae (O'Gorman et al. Science 257:1351-1355 (1991).
  • mice containing transgenes encoding both the Cre recombinase and a selected protein is required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 555:810-813 (1997) and PCT hitemational Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal.
  • the offspring born of this female foster animal will be a clone ofthe animal from which the cell, e.g., the somatic cell, is isolated.
  • Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, estrogen receptor protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo estrogen receptor protein function, including ligand interaction, the effect of specific mutant estrogen receptor protein on estrogen receptor protein function and ligand interaction, and the effect of chimeric estrogen receptor protein. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more estrogen receptor protein functions.
  • primer set in Figure 2(c) and Ml 3 primers were used for overlapping PCR and clone sequencing.
  • Table 2 Summary of changes in heterozygosity in clinical samples. SNPs had a frequency greater than two and a quality score greater than 20 (See Figure 8).
  • the method developed for SNP discovery was designed to recover haplotype data. SNPs could be associated into a specific haplotype.
  • the sample cDNA was from a random population present in unknown proportions. SNPs coming from a specific clone were clustered and built into haplotyes.
  • Figure 4 (b) is the non-singleton haplotype data that were fitted to a neighbor-joining tree. If a tree were cut at the arrow, the clade including 3L, 10-4, ... 73L would be partitioned from the rest ofthe tree, as "Clade X".
  • the following table will illustrate a difference in the incidence of tumors in haplotypes on Clade X vs. the rest ofthe tree. The incidence of each haplotype was first counted by adding the numbers after the dashes, wherein L represented the tumorous Liverpool samples and the non-L represented Coriell controls.
  • a chi-square was calculated based on the 2 x2 table as of 21.29, which, with one degree of freedom that has a probability less than 0.0001. Therefore, the Clade X ofthe ER1 gene has a much greater chance of being associated with a tumor. This entire clade is so rare elsewhere in the world. Even among Europeans, it was present only once out of 20 haplotypes.
  • Figure 4 (c) was a reconstructed haplotypes cladogram which indicated a subset of SNPs in ER1 that preserve the property of having clades highly enriched in the tumor samples or the control.
  • the topmost clade (38L-3 and above) has a count(frequency) of 27 in the Liverpool sample and 2 in the controls.
  • the remainder ofthe tree has a count of 44 in Liverpool and 54 in Coriell.
  • This 2 x 2 table has a chi-square of 21.095 and P ⁇ 0.0005.
  • these 10 SNPs capture most ofthe informative discriminating alleles in the original 46 SNPs.
  • ESR1 Genomic Sequencing- The Complete Genomic Structure of Estrogen Receptor alpha Estrogen receptor (ER) is a member ofthe nuclear hormone receptor gene superfamily. This family of genes is characterized by a modular structure with three distinct domains: a variable (N)-terminal domain, a highly conserved DNA binding domain, and a conserved (C)-terminal domain (reviewed in 1, 2). Functionally, the (N)-terminus domain regulates transactivation, the DNA binding domain regulates dimerization and DNA binding, and the (C)-terminus domain regulates transactivation, dimerization, ligand binding, nuclear translocation, silencing, and Heat Shock Protein binding.
  • N variable
  • C conserved
  • the domain modularity in the nuclear hormone receptor gene superfamily exists because the major subfamilies of these genes evolved through a simple gene duplication early in evolution (6).
  • the nuclear hormone receptor gene family can be separated according to two different classification schemes, one based on hormone binding, the other based on dimerization and how the receptors bind to their respective DNA response elements (for a review, see 2).
  • the cDNA for ER ⁇ was first cloned and sequenced from the MCF-7 breast cancer cell line and was found to have 27% identity and 41% conservation to the v-erb-A gene (7).
  • ER ⁇ was mapped to chromosome 6q25.1 using Fluorescence In Situ Hybridization (FISH) and chromosome banding (8).
  • FISH Fluorescence In Situ Hybridization
  • ER ⁇ a novel estrogen receptor
  • ER ⁇ and ER ⁇ were shown to have 96% sequence identity in the DNA binding domain, 58% identity in the ligand-binding domain, and low similarity in the 5' and 3' ends as well as in the hinge (domain D).
  • a variety of ER ⁇ and ER ⁇ variants have since been described, including single and multiple exon deletions, truncated transcripts, and transcripts containing insertions (11,12,13).
  • BAC DNA was isolated from verified clones using QIAGEN columns (QIAGEN, Inc., Valencia, CA) according to the manufacturer's specifications. Shotgun libraries were prepared following standard protocols (17). Briefly, isolated BAC DNA was sonicated, polished, and size fractionated. Size selected DNA fragments were then subcloned into pUC19 using standard ligation techniques. Ligated DNA was transformed into Electrocompetent cells (Life Technologies, Rockville, MD) and grown overnight. 3) DNA Sequencing and Annotation
  • Sequencing reactions were performed using Big Dye Terminator chemistry (Applied Biosystems, Foster City, CA) and run on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems). Phred (18), Phrap and Consed (19) were used for base calling, assembly, and finishing, respectively. Exon locations were determined using Cross_Match to compare the published gene sequences to the genomic contig.
  • exon IG is located approximately 45 kb upstream of exon IE and conforms to the GT/AG splice site consensus sequence ( Figure 1, table 1).
  • Table 3 Exon-Intron Boundaries and Locations in the Human Estrogen Receptor: Exon sequences are shown in upper case and intron sequences are shown in lower case. Splice sites are shown in bold.
  • Table 4 Exon-Intron Boundaries and Locations in the Human Synaptic Nuclei Expressed Gene 2. Exon sequences are shown in upper case and intron sequences are shown in lower case. Splice sites are shown in bold.

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Abstract

La présente invention repose sur le séquençage d'ADN génomique du chromosome 6 humain et d'ADNc pour définir la structure génomique de gènes de récepteurs oestrogéniques alpha et de nouveaux polymorphismes/haplotypes dans le gène ou la protéine de récepteur oestrogénique. Ces polymorphismes/haplotypes peuvent conduire à toute une gamme de troubles qui sont induits/modulés par un récepteur oestrogénique variant, tels qu'une susceptibilité au cancer, à l'ostéoporose, aux maladies cardio-vasculaires, etc. Sur la base de cette approche par séquençage, la présente invention concerne des séquences nucléotidiques génomiques, des séquences d'ADNc, des séquences d'acides aminés et des polymorphismes de séquences/haplotypes dans les gènes de récepteur oestrogénique alpha, des méthodes de détection de ces séquences/polymorphismes/haplotypes dans un échantillon, des méthodes de détermination du risque pour un patient d'être atteint ou de développer un trouble induit par un récepteur oestrogénique variant et des méthodes de criblage de composés destinés à être utilisés pour traiter des troubles induits par un récepteur oestrogénique variant.
PCT/US2001/005358 2000-02-22 2001-02-20 Variants de recepteurs oestrogeniques alpha et procede de detection desdits variants Ceased WO2001062969A2 (fr)

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JP2001561777A JP2004537959A (ja) 2000-02-22 2001-02-20 エストロゲン受容体α変異体及びその検出方法
AU2001243196A AU2001243196A1 (en) 2000-02-22 2001-02-20 Estrogen receptor alpha variants and methods of detection thereof
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US6617162B2 (en) 2001-12-18 2003-09-09 Isis Pharmaceuticals, Inc. Antisense modulation of estrogen receptor alpha expression
JP2006504392A (ja) * 2002-01-07 2006-02-09 パーレジェン サイエンス インク． 遺伝分析の系および方法
EP1427807A4 (fr) * 2001-08-21 2006-02-22 Applera Corp Variantes alpha du recepteur d'oestrogenes et methodes de detection associees
WO2005087811A3 (fr) * 2004-03-10 2006-03-30 Univ Creighton Recepteurs des oestrogenes et procedes d'utilisation associes
WO2013036201A1 (fr) * 2011-09-06 2013-03-14 Agency For Science, Technology And Research Vaccin polypeptidique

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GREEN S ET AL.: "Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A" NATURE, vol. 320, 1986, pages 134-139, XP001009860 cited in the application *
JAZAERI O ET AL.: "Expression of estrogen receptor alpha mRNA and protein variants in human endometrial carcinoma" GYNECOLOGIC ONCOLOGY, vol. 74, July 1999 (1999-07), pages 38-47, XP001011870 *
LEMIEUX P AND FUQUA S: "The role of the estrogen receptor in tumor progression" THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, vol. 56, no. 1-6, 1996, pages 87-91, XP001010876 *
LORENTZON M ET AL.: "Estrogen receptor gene polymorphism, but not estradiol levels, is related to bone density in healthy adolescent boys: A cross-sectional and longitudinal study" THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, vol. 84, no. 12, 1999, pages 4597-4601, XP001011802 *
MURPHY L C ET AL.: "Estrogen receptor variants and mutations" JOURNAL OF STEROID BIOCHEMISTRY & MOLECULAR BIOLOGY, vol. 62, no. 5-6, 1997, pages 363-372, XP001010877 cited in the application *
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1427807A4 (fr) * 2001-08-21 2006-02-22 Applera Corp Variantes alpha du recepteur d'oestrogenes et methodes de detection associees
US6617162B2 (en) 2001-12-18 2003-09-09 Isis Pharmaceuticals, Inc. Antisense modulation of estrogen receptor alpha expression
JP2006504392A (ja) * 2002-01-07 2006-02-09 パーレジェン サイエンス インク． 遺伝分析の系および方法
JP2009005708A (ja) * 2002-01-07 2009-01-15 Perlegen Sciences Inc 遺伝分析の系および方法
WO2005087811A3 (fr) * 2004-03-10 2006-03-30 Univ Creighton Recepteurs des oestrogenes et procedes d'utilisation associes
US7745230B2 (en) 2004-03-10 2010-06-29 Creighton University Estrogen receptors and methods of use
US8512952B2 (en) * 2004-03-10 2013-08-20 Creighton University Estrogen receptors and methods of use
CN102504025B (zh) * 2004-03-10 2015-10-21 克赖顿大学 雌激素受体和使用方法
WO2013036201A1 (fr) * 2011-09-06 2013-03-14 Agency For Science, Technology And Research Vaccin polypeptidique
US10258676B2 (en) 2011-09-06 2019-04-16 Agency For Science, Technology And Research Polypeptide vaccine

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