WO2003014387A2 - Polymorphisms in the human gene for cyp1a2 and their use in diagnostic and therapeutic applications - Google Patents

Polymorphisms in the human gene for cyp1a2 and their use in diagnostic and therapeutic applications Download PDF

Info

Publication number
WO2003014387A2
WO2003014387A2 PCT/EP2002/008893 EP0208893W WO03014387A2 WO 2003014387 A2 WO2003014387 A2 WO 2003014387A2 EP 0208893 W EP0208893 W EP 0208893W WO 03014387 A2 WO03014387 A2 WO 03014387A2
Authority
WO
WIPO (PCT)
Prior art keywords
gene
polynucleotide
cyp1a2
polypeptide
drug
Prior art date
Application number
PCT/EP2002/008893
Other languages
French (fr)
Other versions
WO2003014387A3 (en
Inventor
Leszek Wojnowski
Elena Presecan-Siedel
Original Assignee
Epidauros Biotechnologie Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Epidauros Biotechnologie Ag filed Critical Epidauros Biotechnologie Ag
Publication of WO2003014387A2 publication Critical patent/WO2003014387A2/en
Publication of WO2003014387A3 publication Critical patent/WO2003014387A3/en

Links

Classifications

    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a polymorphic CYP1A2 polynucleotide. Moreover, the invention relates to genes or vectors comprising the polynucleotides of the invention and to a host cell genetically engineered with the polynucleotide or gene of the invention. Further, the invention relates to methods for producing molecular variant polypeptides or fragments thereof, methods for producing cells capable of expressing a molecular variant polypeptide and to a polypeptide or fragment thereof encoded by the polynucleotide or the gene of the invention or which is obtainable by the method or from the cells produced by the method of the invention. Furthermore, the invention relates to an antibody which binds specifically the polypeptide of the invention.
  • the invention relates to a transgenic non-human animal.
  • the invention also relates to a solid support comprising one or a plurality of the above mentioned polynucleotides, genes, vectors, polypeptides, antibodies or host cells.
  • methods of identifying a polymorphism, identifying and obtaining a pro- drug or drug or an inhibitor are also encompassed by the present invention.
  • the invention relates to methods for producing of a pharmaceutical composition and to methods of diagnosing a disease.
  • the invention relates to a method of detection of the polynucleotide of the invention.
  • comprised by the present invention are a diagnostic and a pharmaceutical composition. Even more, the invention relates to uses of the polynucleotides, genes, vectors, polypeptides or antibodies of the invention.
  • the invention relates to a diagnostic kit.
  • Human CYP1A2 protein is a member of the cytochrome P450 superfamily (CYPs) and is involved in the metabolic activation of carcinogens (aromatic amines, heterocyclic amines, nitrosamines, nitroaromatics), mycotoxins (aflatoxin B-i, ipomeanol, sterigmatocystin), estrogens (17 ⁇ -estradiol) and metabolization of several drugs (caffeine, theophylline, phenacetine, acetaminophen, nicotine, tacrine, imipramine, antipyrine, aminopyrine, clozapine) (Landi, IARC Sci Publ 148 (1999), 173-95).
  • carcinogens aromatic amines, heterocyclic amines, nitrosamines, nitroaromatics
  • mycotoxins aflatoxin B-i, ipomeanol, sterigmatocystin
  • estrogens 17. ⁇ -estradiol
  • CYP1A2 has been detected mainly in the liver where it is constitutively expressed and it can be induced (by cigarette smoking, dietary factors, several drugs, chronic hepatitis, exposure to polybrominated biphenyls, 3- methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin) (Eaton,
  • CYP1A2 activity has been described and several factors (gender, race, genetic polymorphisms, exposure to inducers) have been discussed as an underlying reason (Ikeya, Mol Endocrinol 3 (1989), 1399-408).
  • Slow and intermediate CYP1A2 metabolizers represent about 50% of Caucasians, while their frequency in Japanese seems to be much lower (9-14% (Nakajima, Cancer Epidemiol Biomarkers Prev 3 (1994), 413-21 ; Yokoi, Pharmacogenetics (1995), S65- 9)).
  • -3858G>A and -164C>A may affect CYP1A2 inducibility and one - 3595G>T (position 31341 in the GenBank sequence with Accession No: AF253322, GI: 13430063) the gene's constitutive expression.
  • Table 1 in the Examples shows a summary of all alleles discovered to date and their localisation on GenBank sequence with Accession No: AF253322, GI: 13430063. None of the above cited documents, however, suggests that the disclosed SNPs may be suitable as diagnostic or prognostic markers in human therapy. In other words, the available SNPs do not explain the CYP1A2 variability and are not sufficient as predictive markers for the expression level and/or activity of CYP1 A2 nor are they sufficient for reliable diagnosis and/or treatment of diseases related to CYP1A2.
  • the present invention relates to a polynucleotide comprising a polynucleotide selected from the group consisting of:
  • a polynucleotide capable of hybridizing to a CYP1A2 gene wherein said polynucleotide is having at a position corresponding to position -1045, -881 , 182, 615, 1253, 1352, 1471 , 1513, 1877, 1953, 2045, 2159, 2321 , 3482, 3606, 3614, 5113, or 5371 of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1), a nucleotide exchange of at least one nucleotide;
  • a polynucleotide capable of hybridizing to a CYP1A2 gene wherein said polynucleotide is having at a position corresponding to position -1045, 1352, 1471 , 1513, 1877 or 5371 , of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1) an A, at a position corresponding to position 2321 , 3482 or 3614 of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1) a C, at a position corresponding to position - 881 , 1253, 1953, 2159 or 3606 of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1) a G, at a position corresponding to position 18
  • polynucleotides or the term “polypeptides” refers to different variants of a polynucleotide or polypeptide. Said variants comprise a reference or wild type sequence of the polynucleotides or polypeptides of the invention as well as variants which differ therefrom in structure or composition.
  • Reference sequence for the polynucleotide is GenBank Accession No: AF253322, Gl:13430063.
  • Reference sequence for the polypeptide of the invention is GenBank Accession No: NM_000761 , Gl:13325061.
  • the differences in structure or composition usually occur by way of nucleotide or amino acid substitutions.
  • said nucleotide substitutions comprised by the present invention result in one or more changes of the corresponding amino acids of the polypeptide of the invention.
  • the variant polynucleotides and polypeptides also comprise fragments of the polynucleotides or polypeptides specified herein.
  • the term "polynucleotides” as used herein preferably encompasses the nucleic acid sequences specifically referred to by SEQ ID NOS and in the tables below as well as polynucleotides comprising the reverse complementary nucleic acid sequence thereto.
  • the polynucleotides and polypeptides as well as the aforementioned fragments thereof are characterized in accordance with the present invention as being associated with a CYP1A2 dysfunction or dysregulation comprising, e.g., insufficient, altered drug metabolism, altered expression level, altered protein level and/or altered activity level.
  • insufficient drug metabolism means reduced or loss of metabolic activity of the CYP1A2 enzyme.
  • altered drug metabolism means reduced or enhanced metabolic activity of CYP1A2 or altered metabolic profile.
  • expression level as referred to in the context of the present invention means the detectable amount of transcripts of the CYP1A2 gene relative to the amount of transcripts for a housekeeping gene, such as PLA2, actin or GAPDH. The amount of transcripts can be determined by standard molecular biology techniques including Northern analysis, RNAse protection assays, PCR based techniques encompassing Taq-Man analysis.
  • protein level refers to the detectable amount of CYP1A2 relative to the amount of a protein encoded by a housekeeping gene, such as PLA2.
  • the amount of proteins can be determined by standard biochemical techniques, such as Western analysis, ELISA, RIA or other antibody based techniques known in the art.
  • activity level means the detectable biological activity of CYP1A2 relative to the activity of an encoded by the allellic variants of these genes as disclosed in the present invention relative to the activity of the protein encoded by the corresponding wild-type allele of the gene.
  • preferable proteins are obtained from cells or tissues of a subject having the aforementioned wild-type alleles of the respective genes in their genomes.
  • said dysfunctions or dysreguiations cause a disease or disorder or a prevalence for said disease or disorder.
  • said disease is cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia or any other disease caused by a dysfunction or dysregulation due to a polynucleotide or polypeptides of the invention, also referred to as CYP1A2 gene associated diseases in the following.
  • hybridizing refers to polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with a CYP1A2 dysfunction or dysregulation.
  • said hybridizing polynucleotides are also associated with said dysfunctions and dysreguiations.
  • said polynucleotides capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with CYP1A2 dysfunctions or dysreguiations are at least 70%, at least 80%, at least 95% or at least 100% identical to the polynucleotides of the invention or parts thereof which are associated with CYP1A2 dysfunctions or dysreguiations.
  • said polynucleotides may be useful as probes in Northern or Southern Blot analysis of RNA or DNA preparations, respectively, or can be used as oligonucleotide primers in PCR analysis dependent on their respective size.
  • hybridizing polynucleotides which are useful for analysing DNA-Protein interactions via, e.g., electrophoretic mobility shift analysis (EMSA).
  • said hybridizing polynucleotides comprise at least 10, more preferably at least 15 nucleotides in length while a hybridizing polynucleotide of the present invention to be used as a probe preferably comprises at least 100, more preferably at least 200, or most preferably at least 500 nucleotides in length.
  • hybridization conditions are referred to in standard text books such as Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
  • Preferred in accordance with the present invention are polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with a CYP1A2 dysfunction or dysregulation under stringent hybridization conditions, i.e.
  • stringent hybridization conditions refere to an overnight incubation at 42°C in a solution comprising 50% formamide hybridization solution, followed by at least two washing steps at 60°C in 0.2 x SSC with 1% SDS.
  • nucleotides or amino acids may differ in the indicated number but may still have similar neighboring nucleotides or amino acids.
  • Said nucleotides or amino acids which may be exchanged, deleted or comprise additional nucleotides or amino acids are also comprised by the term "corresponding position".
  • Said nucleotides or amino acids may for instance together with their neighbors form sequences which may be involved in the regulation of gene expression, stability of the corresponding RNA or RNA editing, as well as encode functional domains or motifs of the protein of the invention.
  • the mode and population distribution of genetic variations in the CYP1A2 gene has been analyzed by sequence analysis of relevant regions of the human said gene from many different individuals. It is a well known fact that genomic DNA of individuals, which harbor the individual genetic makeup of all genes, including the CYP1A2 gene, can easily be purified from individual blood samples.
  • pharmacogenomics has been proposed as a tool useful in the identification and selection of patients which can respond to a particular drug without side effects.
  • This identification/selection can be based upon molecular diagnosis of genetic polymorphisms by genotyping DNA from leukocytes in the blood of patient, for example, and characterization of disease (Bertz, Clin. Pharmacokinet. 32 (1997), 210-256; Engel, J. Chromatogra. B. Biomed. Appl. 678 (1996), 93-103).
  • the mutations in the variant genes of the invention sometime result in amino acid substitutions either alone or in combination. It is of course also possible to genetically engineer such mutations in wild type genes or other mutant forms. Methods for introducing such modifications in the DNA sequence of said genes are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
  • said amino acid substitutions in the amino acid sequence of the protein encoded by the polynucleotide of the invention is due to one or more nucleotide substitutions.
  • said nucleotide substitutions may result in an amino acid substitution of P to L at position corresponding to position 61 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061), S to R at position corresponding to position 298 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061), I to T at position corresponding to position 401 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , GM3325061) or T to I at position corresponding to position 438 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , GM3325061).
  • the mutations in the CYP1A2 gene detected in accordance with the present invention are listed in Table 2.
  • the methods of the mutation analysis followed standard protocols and are described in detail in the Examples. In general such methods are to be used in accordance with the present invention for evaluating the phenotypic spectrum as well as the overlapping clinical characteristics of diseases or conditions related to dysfunctions or dysreguiations and diseases related to the metabolism of drugs (e.g. clozapine clearence) or the metabolic activation pathways of several chemical carcinogens, environmental and dietary compounds known to induce CYP1A2, such as: aromatic or heterocyclic amines, nitroaromatic compounds, mycotoxins and estrogens, cigarette smoke, charbroiled or high-temperature cooked meat, cruciferous vegetables.
  • the characterization of said mutants may form the basis of the development of improved methods for evaluation of several cancer risks (e.g. lung, liver, breast, prostata, endometrial, colorectal, urinary bladder), or the development of improved drugs, such as drugs which are used e.g in cancer therapy, as non-narcotic antipyretic and analgesic drugs (phenacetin) (Devonshire, Br J Clin Pharmacol 16 (1983), 157-66.), and in the treatment of diseases including congenital jaundice (Zaccaro, Biochem Pharmacol 61 (2001), 843-9.), porphyria cutanea tarda (Christiansen, Hum Genet 107 (2000), 612-4), tardive dyskinesia in schizophrenia (Basile, Mol Psychiatry 5 (2000), 410-7.).
  • drugs which are used e.g in cancer therapy, as non-narcotic antipyretic and analgesic drugs (phenacetin) (Devonshire, Br J Clin Pharmacol 16 (1983), 157-
  • Said methods encompass for example haplotype analysis, single-strand conformation polymorphism analysis (SSCA), denaturating gradient gel electrophoresis (DGGE), PCR, direct sequencing, HPLC-based techniques, invasive cleavage assay, mass spectroscopy, microarray, a rolling circle extension assay, primer extension assay, a molecular beacon assay and a ligase chain reaction assay.
  • SSCA single-strand conformation polymorphism analysis
  • DGGE denaturating gradient gel electrophoresis
  • PCR direct sequencing
  • HPLC-based techniques invasive cleavage assay
  • mass spectroscopy mass spectroscopy
  • microarray microarray
  • primer extension assay primer extension assay
  • a molecular beacon assay a molecular beacon assay
  • ligase chain reaction assay ligase chain reaction assay.
  • polynucleotides which comprise at least two of the polynucleotides specified hereinabove, i.e. polynucleotides having a nucleotide sequence which contains at least two of the mutations comprised by the above polynucleotides or listed in Figure 1 below.
  • the haplotype determined in accordance with the present invention can be characterized by at least two of said mutations in the CYP1 A2 locus.
  • the polynucleotide of the invention may further comprise at least one nucleotide deletion, addition and/or substitution other than those specified hereinabove, for example those described in the prior art; e.g. listed in Table 1.
  • the genetic knowledge deduced from the present invention can now be used to exactly and reliably characterize the genotype of a patient.
  • diseases or a prevalence for a disease which are associated with CYP1A2 dysfunction or dysregulation such as cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia referred to herein can be predicted and preventive or therapeutical measures can be applied accordingly.
  • a suitable individual therapy can be designed based on the knowledge of the individual genetic makeup of a subject with respect to the polynucleotides of the invention and improved therapeutics can be developed as will be further discussed below.
  • unusual effect is an undesirable or insufficient response to the administration of a therapeutic compound, i.e. an effect that is not directed to alleviating the symptoms or cause of the disease beeing treated. Unusual effects range from minor inconveniences to more serious events.
  • the CYP1A2 "status”, defined by the expression level and activity of the CYP1A2 protein, can be not only altered in many disease or disorders including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia (see above), but can also be variable in normal tissue, due to genetic variations/polymorphisms.
  • the identification of polymorphisms associated with altered CYP1A2 expression and/or activity is important for the prediction of drug uptake and subsequently for the prediction of therapy outcome, including side effects of medications. Therefore, analysis of CYP1A2 variations indicative of CYP1A2 function, is a valuable tool for therapy with drugs, which are substrates of CYP1A2 and has, thanks to the present invention, now become possible.
  • the polynucleotide of the present invention is associated with cancer, congenital jaundice, porphyria cutanea tarda, or tardive dyskinesia in schizophrenia.
  • cancer congenital jaundice
  • proteosarcoma a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a a.
  • the present invention relates to a polynucleotide which is DNA or RNA.
  • the polynucleotide of the invention may be, e.g., DNA, cDNA, genomic DNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.
  • said polynucleotide is part of a vector, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide of the invention.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the invention furthermore relates to a gene comprising the polynucleotide of the invention.
  • genes comprise structural elements which encode an amino acid sequence as well as regulatory elements which are involved in the regulation of the expression of said genes.
  • Structural elements are represented by exons which may either encode an amino acid sequence or which may encode for RNA which is not encoding an amino acid sequence but is nevertheless involved in RNA function, e.g. by regulating the stability of the RNA or the nuclear export of the RNA.
  • Regulatory elements of a gene may comprise promoter elements or enhancer elements both of which could be involved in transcriptional control of gene expression. It is very well known in the art that a promoter is to be found upstream of the structural elements of a gene. Regulatory elements such as enhancer elements, however, can be found distributed over the entire locus of a gene. Said elements could be reside, e.g., in introns, regions of genomic DNA which separate the exons of a gene. Promoter or enhancer elements correspond to polynucleotide fragments which are capable of attracting or binding polypeptides involved in the regulation of the gene comprising said promoter or enhancer elements. For example, polypeptides involved in regulation of said gene comprise the so called transcription factors.
  • Said introns may comprise further regulatory elements which are required for proper gene expression.
  • Introns are usually transcribed together with the exons of a gene resulting in a nascent RNA transcript which contains both, exon and intron sequences.
  • the intron encoded RNA sequences are usually removed by a process known as RNA splicing. However, said process also requires regulatory sequences present on a RNA transcript said regulatory sequences may be encoded by the introns.
  • regulatory elements of a gene could be also involved in the control of genetic stability of a gene locus. Said elements control, e.g., recombination events or serve to maintain a certain structure of the DNA or the arrangement of DNA in a chromosome.
  • single nucleotide polymorphisms can occur in exons of a gene which encode an amino acid sequence as discussed supra as well as in regulatory regions which are involved in the above discussed process.
  • the analysis of the nucleotide sequence of a gene locus in its entirety including, e.g., introns is in light of the above desirable.
  • the polymorphisms comprised by the polynucleotides of the present invention can influence the expression level of CYP1A2 protein via mechanisms involving enhanced or reduced transcription of the CYP1A2 gene, stabilization of the gene's RNA transcripts and alteration of the processing of the primary RNA transcripts.
  • a nucleotide substitution results in altered expression of the variant gene compared to the corresponding wild type gene.
  • the present invention relates to a vector comprising the polynucleotide of the invention or the gene of the invention.
  • Said vector may be, for example, a phage, plasmid, viral or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.
  • the polynucleotides or genes of the invention may be joined to a vector containing selectable markers for propagation in a host.
  • a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate, or in a complex with a charged lipid or in carbon-based clusters. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.
  • the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.
  • Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E.
  • regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poIy-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM ⁇ , pRc/CMV, pcDNAI, pcDNA3 (Invitrogen), pSPORTI (GIBCO BRL), pFastBac (Invitrogen), pYES (Invitrogen).
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus may be used for delivery of the polynucleotides or vector of the invention into targeted cell population.
  • viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus.
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).
  • the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • isolated fractions thereof refers to fractions of eukaryotic or prokaryotic cells or tissues which are capable of transcribing or transcribing and translating RNA from the vector of the invention. Said fractions comprise proteins which are required for transcription of RNA or transcription of RNA and translation of said RNA into a polypeptide. Said isolated fractions may be, e.g., nuclear and cytoplasmic fractions of eukaryotic cells such as of reticulocytes.
  • the present invention furthermore relates to a host cell genetically engineered with the polynucleotide of the invention, the gene of the invention or the vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell; see supra.
  • the polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • the recombinant DNA molecule of the invention can be used for "gene targeting” and/or “gene replacement", for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, mammalian or, preferably, human cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • the term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of a variant polypeptide of the invention.
  • Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
  • a polynucleotide coding for a mutant form of variant polypeptides of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Methods for preparing fused, operably linked genes and expressing them in bacteria or animal cells are well-known in the art (Sambrook, supra). The genetic constructs and methods described therein can be utilized for expression of variant polypeptides of the invention in, e.g., prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host.
  • the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
  • the transformed prokaryotic hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the proteins of the invention can then be isolated from the grown medium, cellular lysates, or cellular membrane fractions.
  • the isolation and purification of the microbially or otherwise expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
  • the invention relates to a method for producing a molecular variant CYP1A2 polypeptide or fragment thereof comprising culturing the above described host cell; and recovering said protein or fragment from the culture.
  • the present invention relates to a method for producing cells capable of expressing a molecular variant CYP1A2 polypeptide comprising genetically engineering cells with the polynucleotide of the invention, the gene of the invention or the vector of the invention.
  • the cells obtainable by the method of the invention can be used, for example, to test drugs according to the methods described in D. L. Spector, R. D. Goldman, L. A. Leinwand, Cells, a Lab manual, CSH Press 1998. Furthermore, the cells can be used to study known drugs and unknown derivatives thereof for their ability to complement the deficiency caused by mutations in the CYP1A2 gene.
  • the host cells preferably lack a wild type allele, preferably both alleles of the CYP1A2 gene and/or have at least one mutated from thereof.
  • the gene comprising an allele as comprised by the polynucleotides of the invention could be introduced into the wild type locus by homologous replacement.
  • the invention relates to a polypeptide or fragment thereof encoded by the polynucleotide of the invention, the gene of the invention or obtainable by the method described above or from cells produced by the method described above.
  • the variant polypeptide of the invention can be further modified by conventional methods known in the art.
  • By providing said variant proteins according to the present invention it is also possible to determine the portions relevant for their biological activity or inhibition of the same.
  • the terms "polypeptide” and "protein” as used herein are exchangeable. Moreover, what is comprised by said terms is standard textbook knowledge.
  • the present invention furthermore relates to an antibody which binds specifically to the polypeptide of the invention.
  • the antibody specifically recognizes or binds an epitope containing one or more amino acid substitution(s) as defined above.
  • Antibodies against the variant polypeptides of the invention can be prepared by well known methods using a purified protein according to the invention or a (synthetic) fragment derived therefrom as an antigen.
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals.
  • said antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, human or humanized antibody, primatized, chimerized or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these.
  • antibodies or fragments thereof to the aforementioned polypeptides can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
  • antibodies can be used, for example, for the immunoprecipitation and immunolocalization of the variant polypeptides of the invention as well as for the monitoring of the presence of said variant polypeptides, for example, in recombinant organisms, and for the identification of compounds interacting with the proteins according to the invention.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the antibody of the present invention specifically recognizes an epitope containing one or more amino acid substitution(s) resulting from a nucleotide exchange as defined supra.
  • Antibodies which specifically recognize modified amino acids such as phospho- Tyrosine residues are well known in the art. Similarly, in accordance with the present invention antibodies which specifically recognize even a single amino acid exchange in an epitope may be generated by the well known methods described supra.
  • the antibody of the present invention is monoclonal or polyclonal.
  • the invention also relates to a transgenic non-human animal comprising at least one polynucleotide of the invention, the gene of the invention or the vector of the invention as described supra.
  • the present invention also encompasses a method for the production of a transgenic non-human animal comprising introduction of a polynucleotide or vector of the invention into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom.
  • the non-human animal can be used in accordance with the method of the invention described below and may be a non-transgenic healthy animal, or may have a disease or disorder, preferably a disease caused by at least one mutation in the gene of the invention.
  • transgenic animals are well suited for, e.g., pharmacological studies of drugs in connection with variant forms of the above described variant polypeptides since these polypeptides or at least their functional domains are conserved between species in higher eukaryotes, particularly in mammals.
  • Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.
  • the DNA of the embryos can be analyzed using, e.g., Southern blots with an appropriate probe or based on PCR techniques.
  • a transgenic non-human animal in accordance with the invention may be a transgenic mouse, rat, hamster, dog, monkey, rabbit, pig, frog, nematode such as Caenorhabditis elegans, fruitfly such as Drosophila melanogaster or fish such as torpedo fish or zebrafish comprising a polynucleotide or vector of the invention or obtained by the method described above, preferably wherein said polynucleotide or vector is stably integrated into the genome of said non-human animal, preferably such that the presence of said polynucleotide or vector leads to the expression of the variant polypeptide of the invention.
  • the mammal is preferably a laboratory animal such as a mouse or rat.
  • the transgenic non-human animal of the invention is a mouse, a rat or a zebrafish.
  • the invention also relates to a solid support comprising one or a plurality of the polynucleotide, the gene, the vector, the polypeptide, the antibody or the host cell of the invention in immobilized form.
  • solid support refers to a flexible or non-flexible support that is suitable for carrying said immobilized targets.
  • Said solid support may be homogenous or inhomogeneous.
  • said solid support may consist of different materials having the same or different properties with respect to flexibility and immobilization, for instance, or said solid support may consist of one material exhibiting a plurality of properties also comprising flexibility and immobilization properties.
  • Said solid support may comprise glass-, polypropylene- or silicon-chips, membranes oligonucleotide-conjugated beads or bead arrays.
  • immobilized means that the molecular species of interest is fixed to a solid support, preferably covalently linked thereto.
  • This covalent linkage can be achieved by different means depending on the molecular nature of the molecular species.
  • the molecular species may be also fixed on the solid support by electrostatic forces, hydrophobic or hydrophilic interactions or Van-der-Waals forces.
  • the above described physico-chemical interactions typically occur in interactions between molecules.
  • biotinylated polypeptides may be fixed on a avidin- coated solid support due to interactions of the above described types.
  • polypeptides such as antibodies, may be fixed on an antibody coated solid support.
  • the immobilization is dependent on the chemical properties of the solid support.
  • the nucleic acid molecules can be immobilized on a membrane by standard techniques such as UV-crosslinking or heat.
  • said solid support is a membrane, a glass- or poylpropylene- or silicon-chip, are membranes oligonucleotide-conjugated beads or a bead array, which is assembled on an optical filter substrate.
  • the present invention relates to an in vitro method for identifying a polymorphism said method comprising the steps of:
  • prevalence means that individuals are susceptible for one or more disease(s) which are associated with CYP1A2 dysfuntion or dysregulation or could already have one or more of said disease(s).
  • one CYP1A2 associated disease can be used to determine the susceptibility for another CYP1A2 associated disease, e.g. impaired drug metabolism may be indicative for a prevalence for, e.g. cancer.
  • symptoms which are indicative for a prevalence for developing said diseases are very well known in the art and have been sufficiently described in standard textbooks such as Pschyrembel.
  • polymorphisms according to the present invention which are associated with CYP1A2 dysfunction or dysregulation or one or more disease(s) based thereon should be enriched in subgroups of individuals which have a prevalence for said diseases versus subgroups which have no prevalence for said diseases.
  • the above described method allows the rapid and reliable detection of polymorphism which are indicative for one or more CYP1A2 associated disease(s) or a susceptibility therefor.
  • a large number of individuals having no prevalence might be screened for polymorphisms in general.
  • a reference sequences comprising polymorphisms which do not correlate to one or more CYP1A2 associated disease(s) can be obtained. Based on said reference sequences it is possible to efficiently and reliably determine the relevant polymorphisms.
  • the present invention relates to a method for identifying and obtaining a pro-drug or a drug capable of modulating the activity of a molecular variant of a CYP1 A2 polypeptide comprising the steps of:
  • compound in a method of the invention includes a single substance or a plurality of substances which may or may not be identical.
  • Said compound(s) may be chemically synthesized or produced via microbial fermentation but can also be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compounds may be known in the art but hitherto not known to be useful as an inhibitor, respectively.
  • the plurality of compounds may be, e.g., added to the culture medium or injected into a cell or non-human animal of the invention.
  • a sample containing (a) compound(s) is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound, in question or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample. It can then be determined whether said sample or compound displays the desired properties, for example, by the methods described herein or in the literature (Spector et al., Cells manual; see supra). Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s).
  • said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical.
  • the methods of the present invention can be easily performed and designed by the person skilled in the art, for example in accordance with other cell based assays described in the prior art or by using and modifying the methods as described herein.
  • the person skilled in the art will readily recognize which further compounds may be used in order to perform the methods of the invention, for example, enzymes, if necessary, that convert a certain compound into a precursor.
  • Such adaptation of the method of the invention is well within the skill of the person skilled in the art and can be performed without undue experimentation.
  • Compounds which can be used in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like. Said compounds may act as agonists or antagonists of the inveniton. Said compounds can also be functional derivatives or analogues of known drugs. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA.
  • derivatives and analogues can be tested for their effects according to methods known in the art or as described.
  • peptide mimetics and/or computer aided design of appropriate drug derivatives and analogues can be used, for example, according to the methods described below.
  • Such analogs comprise molecules may have as the basis structure of known CYP1 A2 substrates and/or inhibitors and/or modulators; see infra.
  • Appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptides of the invention by computer assistant searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120).
  • the present invention provides methods for identifying and obtaining compounds which can be used in specific doses for the treatment of specific forms of CYP1 A2 associated diseases, e.g. dysfunctions or dysreguiations of the drug metabolism such as cancer.
  • the present invention relates to a method for identifying and obtaining an inhibitor of the activity of a molecular variant of a CYP1A2 polypeptide comprising the steps of:
  • said cell is a cell, obtained by the method of the invention or can be obtained from the transgenic non-human animal as described supra.
  • the present invention relates to a method of identifying and obtaining a pro-drug or drug capable of modulating the activity of a molecular variant of a CYP1 A2 polypeptide comprising the steps of:
  • said measuring step comprises measuring the formation of a second complex of said protein and said inhibitor candidate.
  • said measuring step comprises measuring the amount of said first molecule that is not bound to said protein.
  • said first molecule is a agonist or antagonist or a substrate and/or a inhibitor and/or a modulator of the polypeptide of the invention, e.g., with a radioactive or fluorescent label.
  • the present invention relates to a method of identifying and obtaining an inhibitor capable of modulating the activity of a molecular variant of a CYP1A2 polypeptide comprising the steps of:
  • said measuring step comprises measuring the formation of a second complex of said protein and said compound.
  • said measuring step comprises measuring the amount of said first molecule that is not bound to said protein.
  • said first molecule is labeled.
  • the invention furthermore relates to a method for the production of a pharmaceutical composition comprising the steps of the method as described supra; and the further step of formulating the compound identified and obtained or a derivative thereof in a pharmaceutically acceptable form.
  • therapeutically useful compounds identified according to the methods of the invention can be formulated and administered to a patient as discussed above.
  • therapeutic doses determined to be appropriate by one skilled in the art and for definitions of the term "pharmaceutical composition” see infra.
  • the present invention encompasses a method for the preparation of a pharmaceutical composition comprising the steps of the above-described methods; and formulating a drug or pro-drug in the form suitable for therapeutic application and preventing or ameliorating the disorder of the subject diagnosed in the method of the invention.
  • Drugs or pro-drugs after their in vivo administration are metabolized in order to be eliminated either by excretion or by metabolism to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459).
  • a corresponding formulation as a pro-drug can be used which is converted into its active in the patient.
  • Precautionary measures that may be taken for the application of pro-drugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329).
  • said drug or prodrug is a derivative of a medicament as defined hereinafter.
  • the present invention also relates to a method of diagnosing a disorder related to the presence of a molecular variant of the CYP1A2 gene or susceptibility to such a disorder comprising determining the presence of a polynucleotide or the gene of the invention in a sample from a subject.
  • the method of testing the status of a disorder or susceptibility to such a disorder can be effected by using a polynucleotide gene or nucleic acid of the invention, e.g., in the form of a Southern or Northern blot or in situ analysis.
  • Said nucleic acid sequence may hybridize to a coding region of either of the genes or to a non-coding region, e.g. intron.
  • said nucleic acid molecule can again be used in Northern blots.
  • said testing can be done in conjunction with an actual blocking, e.g., of the transcription of the gene and thus is expected to have therapeutic relevance.
  • a primer or oligonucleotide can also be used for hybridizing to one of the above mentioned CYP1A2 gene or corresponding mRNAs.
  • the nucleic acids used for hybridization can, of course, be conveniently labeled by incorporating or attaching, e.g., a radioactive or other marker. Such markers are well known in the art.
  • the labeling of said nucleic acid molecules can be effected by conventional methods.
  • the presence or expression of variant CYP1A2 gene can be monitored by using a primer pair that specifically hybridizes to either of the corresponding nucleic acid sequences and by carrying out a PCR reaction according to standard procedures. Specific hybridization of the above mentioned probes or primers preferably occurs at stringent hybridization conditions.
  • mRNA, cRNA, cDNA or genomic DNA obtained from the subject may be sequenced to identify mutations which may be characteristic fingerprints of mutations in the polynucleotide or the gene of the invention.
  • the present invention further comprises methods wherein such a fingerprint may be generated by RFLPs of DNA or RNA obtained from the subject, optionally the DNA or RNA may be amplified prior to analysis, the methods of which are well known in the art.
  • RNA fingerprints may be performed by, for example, digesting an RNA sample obtained from the subject with a suitable RNA-Enzyme, for example RNase Ti, RNase T 2 or the like or a ribozyme and, for example, electrophoretically separating and detecting the RNA fragments as described above.
  • RNA-Enzyme for example RNase Ti, RNase T 2 or the like or a ribozyme
  • An additional embodiment of the present invention relates to a method wherein said determination is effected by employing an antibody of the invention or fragment thereof.
  • the antibody used in the method of the invention may be labeled with detectable tags such as a histidine flags or a biotin molecule.
  • the invention relates to a method of diagnosing a disorder related to the presence of a molecular variant of a CYP1A2 gene or susceptibility to such a disorder comprising determining the presence of a polypeptide or the antibody of the invention in a sample from a subject.
  • said disorder is cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia.
  • the above described method is comprising PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques, single-strand conformation polymorphism analysis (SSCA), denaturating gradient gel electrophoresis (DGGE), direct sequencing, HPLC-based techniques, invasive cleavage assay, mass spectroscopy, microarray, a rolling circle extension assay, a primer extension assay and a molecular beacon assay, hybridization techniques or immunoassays. Said techniques are very well known in the art.
  • the invention relates to a method of detection of the polynucleotide or the gene of the invention in a sample comprising the steps of
  • the invention also relates to an in vitro method for diagnosing a disease comprising the steps of the method described supra, wherein binding of said polynucleotide or gene to said immobilized targets on said solid support is indicative for the presence or the absence of said disease or a prevalence for said disease.
  • the invention furthermore relates to a diagnostic composition comprising the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention.
  • These pharmaceutical compositions comprising, e.g., the antibody may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation.
  • Acceptable salts comprise acetate, methylester, HCI, sulfate, chloride and the like.
  • the compounds may be administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
  • the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like.
  • the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
  • the dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment.
  • compositions which comprise antisense- oligonucleotides which specifically hybridize to RNA encoding mutated versions of the polynucleotitde or gene according to the invention or which comprise antibodies specifically recognizing a mutated polypeptide of the invention but not or not substantially the functional wild-type form is conceivable in cases in which the concentration of the mutated form in the cells should be reduced.
  • the particular drug selection, dosage regimen and corresponding patients to be treated can be determined in accordance with the present invention.
  • the dosing recommendations will be indicated in product labeling by allowing the prescriber to anticipate dose adjustments depending on the considered patient group, with information that avoids prescribing the wrong drug to the wrong patients at the wrong dose.
  • the present invention relates to the use of the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention for the preparation of a diagnostic composition for diagnosing a disease.
  • a gene encoding a functional and expressible polypeptide of the invention can be introduced into the cells which in turn produce the protein of interest.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res.
  • the gene may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell.
  • said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell.
  • the nucleic acid sequence is operatively linked to regulatory elements allowing for the expression and/or targeting of the polypeptides of the invention to specific cells.
  • Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acids to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729).
  • Gene therapy may be carried out by directly administering the recombinant DNA molecule or vector of the invention to a patient or by transfecting cells with the polynucleotide or vector of the invention ex vivo and infusing the transfected cells into the patient.
  • the present invention relates to the use of the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention for the preparation of a pharmaceutical composition for treating a disease.
  • said disease is cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia.
  • the present invention also encompasses an in vitro method for identifying a single nucleotide polymorphism said method comprising the steps of
  • the present invention relates to an in vitro method for identifying a single nucleotide polymorphism said method comprising the steps of (a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low CYP1A2 protein level and at least one or more further subgroup(s) do have a higher CYP1 A2 protein level, and (b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low CYP1A2 protein level with said at least one or more further subgroup(s) having a higher CYP1A2 protein level.
  • the present invention encompasses an in vitro method for identifying a single nucleotide polymorphism said method comprising the steps of
  • an in vitro method for identifying a single nucleotide polymorphism comprising the steps of
  • an in vitro method for identifying a single nucleotide polymorphism comprising the steps of (a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low plasma concentration of a drug and at least one or more further subgroup(s) do have a higher plasma concentration of a drug, and (b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low plasma concentration of a drug with said at least one or more further subgroup(s) having a higher plasma concentration of a drug.
  • said drug is a substrate of CYP1A2.
  • said substrate is caffeine, theophylline, phenacetine, acetaminophen, nicotine, tacrine, imipramine, antipyrine, aminopyrine, clozapine or olanzapine.
  • the present invention relates to a diagnostic kit for detection of a single nucleotide polymorphism comprising the polynucleotide, the gene, the vector, the polypeptide, the antibody, the host cell, the transgenic non-human animal or the solid support of the invention.
  • the kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic cells and animals.
  • the kit of the invention can be used for carrying out a method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as research tool.
  • the parts of the kit of the invention can be packaged individually in vials or other appropriate means depending on the respective ingredient or in combination in suitable containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art.
  • the kit may be used for methods for detecting expression of a mutant form of the polypeptides, genes or polynucleotides in accordance with any one of the above-described methods of the invention, employing, for example, immunoassay techniques such as radioimmunoassay or enzymeimmunoassay or preferably nucleic acid hybridization and/or amplification techniques such as those described herein before and in the Examples as well as pharmacokinetic studies when using non-human transgenic animals of the invention.
  • immunoassay techniques such as radioimmunoassay or enzymeimmunoassay or preferably nucleic acid hybridization and/or amplification techniques such as those described herein before and in the Examples as well as pharmacokinetic studies when using non-human transgenic animals of the invention.
  • the figures illustrate the invention:
  • Figure 1 Polymorphic genomic DNA sequences and the resulting changes in the protein sequence. Sequences are listed in 5' — > 3' orientation. Letters in lowercase indicate non-coding sequences, letters in uppercase indicate coding sequences. Primer regions are underlined. Variant sites are shown framed, wherein the mutated nucleotide or amino acid is depicted. Sequences are listed according to their localisation in the CYP1A2 gene (5' ⁇ 3').
  • CYP1A2 protein variants referred to in the present invention are located on the surface of the CYP protein as shown by comparative protein modelling (Guex, Electrophoresis (1997) 18: 2714-2723).
  • a protein model of CYP1A2 was generated by using SWISS-MODEL which is a fully automated protein structure homology- modeling server, accessible via the Expasy web server (http://www.expasv.ch/swissmod/SWISS-MODEL.html) and further processing with the Deep View (Swiss-PdbViewer) software (http://www.expasy.ch/spdbv/). This alterations will change protein protein interactions and will thereby alter the activity of the CYP 1A2 enzyme.
  • Example 1 Isolation of genomic DNA from human blood, generation and purification of CYP1A2 gene fragments
  • Genomic DNA was isolated from blood samples using Qiagen blood DNA isolation kits. Oligonucleotides used in the screen were designed based on the recently determined sequence and organisation of the human CYP1A locus (Corchero, Pharmacognetics (2001), 1-6). Primer sequences and PCR fragment lengths are given in Table 2. Amplified fragments were processed through PCR purification columns (Qiagen) and sequenced on PE ABI 3700 DNA Analysers using the same primers as for PCR. The sequences were analysed for the presence of polymorphisms using the PHRED/PHRAP/POLYPHRED/CONSED software package (University of Washington, Seattle, WA, USA).
  • Example 2 Determination of genetic variations within the CYP1A2 locus
  • Sequence diversity within the CYP1A locus in African-Americans, Japanese, Chinese and Koreans was determined by PCR amplification and sequencing of 45, 50, 47 and 50 DNA samples, respectively.
  • the PCR fragments encompass the entire protein- coding region of CYP1A2 and the non-coding exon 1 , a portion of the 3'-UTR in exon 7, as well as a portion of the 5'-flanking region.
  • the already known CYP1A2*1 E (cb-v-027) and CYP1A2*1B (cb-v-003) alleles were also found in the screen.
  • the CYP1A2*1 E allele had a frequency of 5% in Japanese (i.e. similar to the 8.2% reported previously, see Tablel), 8.8% in African-Americans and Chinese samples, and 11.6% in Korean samples.
  • the CYP1A2*1 B allele had a frequency of 77.9% in Japanese, 89.7% in African-Americans, 86.9% in Chinese and 86% in Korean samples.
  • the allele frequency of CYP1A2*1B allele in all mentioned ethnic groups is much higher than in Caucasians (33%, see Table 1).
  • the homozygous T/T is the major variant
  • Japanese, African- Americans, Chinese and Koreans the homozygous C/C is the major variant.
  • a total of 18 new variants were detected in the screen and their allelic frequencies in the four ethnic groups ranged between 0 % and 90 % (Table 3 and Table 4).
  • One variant is located within the 5' flanking sequence upstream the transcriptional start site of CYP1A2, one variant is located in the untranslated exon 1 , one variant is located in the 3' untranslated region of exon 7, eight variants are located in introns, whereas seven have been found in the protein-coding sequence. Among the latter ones, four variants result in amino acid substitutions of the CYP1A2 protein.
  • the frequencies of the four protein variants which were not found in 100 analysed Caucasian samples were determined in the Japanese and African-Americans samples (see Table 3) and in Chinese and Korean samples (see Table 4).
  • the g.182C>T variant results in a P61L amino acid exchange in exon 2 and was found only in African-Americans samples in 1 out of 35 samples.
  • the g.1513C>A variant leads to a S298R amino acid exchange in exon 3 and was found only in African-Americans samples in 7 out of 44 samples.
  • the g.3482T>C variant results in a 1401 T amino acid exchange in exon 6 and was found only in the Japanese samples in 1 out of 40 samples.
  • the fourth g.5113C>T variant results in a T438I aminoacid exchange in exon 7 and was found only in the Korean samples in 3 out of 43 samples.
  • the silent g.2045C>T variant (cb-v-011 , Table 3) has been found only in Japanese in 1 out of 50 samples; the silent g.615C>T variant (cb-v-018, Table 4) has been found only in Chinese in 1 out of 46 samples, while the silent g.1471G>A variant (cb-v-019, Table 4) has been found only in Koreans in 1 out of 46 samples.
  • the first nucleotide A of the first ATG of the nucleotide sequence of CYP1 A2 protein has been taken as position +1
  • Table 3 New polymorphisms detected in the CYP1A2 gene in Japanese and African-American ethnic groups.
  • Variants are listed according to their localisation along the gene. Polymorphism nomenclature is based on Antonarakis et al. (Antonarakis, Hum Mutat 11 (1998), 1-3) using the sequence AF253322, Gl:13430063 as genomic reference sequence wherein the A of the ATG at position 34942 is +1. Sequence context, local alignment at the polymorphic site with the major allele sequence given at the top and the variant sequence given below wherein in the variant sequence nucleotides which are identical to those at the top sequence are represented by a dot. N: number of chromosomes analysed (calculated by doubling the number of individuals).
  • variants were detected in the heterozygous state except for 1 homozygous individual for variant cb-v-013 and a mix of homo/heterozygous individuals for variant cb-v-012 (the major allele in Japanese and African-American ethnic groups).
  • Variants cb-v-004 to cb-v-010 and cb-v-015 were found only in African-Americans.
  • Variants cb-v-011 and cb-v-014 were found only in Japanese.
  • Table 4 New polymorphisms detected in the CYP1A2 gene in Chinese and Korean ethnic groups.
  • variants were detected in the heterozygous state except for 1 homozygous individual for variant cb-v-013 and a f homo/heterozygous individuals for variant cb-v-012 (the major allele in Chinese and Korean ethnic groups).
  • Variants cb-v- and cb-v-021 was found only in Chinese.
  • Variants cb-v-017, cb-v-019 and cb-v-020 were found only in Koreans.
  • Kiyohara C. Genetic polymorphism of enzymes involved in xenobiotic metabolism and the risk of colorectal cancer. J Epidemiol 10, 349-60. (2000).
  • Cytochrome P4501A2 enzyme induction and genetic control in determining 4-aminobiphenyl-hemogIobin adduct levels. Cancer Epidemiol Biomarkers Prev 5, 693-8. (1996).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention relates to a polymorphic CYP1 A2 polynucleotide. Moreover, the invention relates to genes or vectors comprising the polynucleotides of the invention and to a host cell genetically engineered with the polynucleotide or gene of the invention. Further, the invention relates to methods for producing molecular variant polypeptides or fragments thereof, methods for producing cells capable of expressing a molecular variant polypeptide and to a polypeptide or fragment thereof encoded by the polynucleotide or the gene of the invention or which is obtainable by the method or from the cells produced by the method of the invention. Furthermore, the invention relates to an antibody which binds specifically the polypeptide of the invention. Moreover, the invention relates to a transgenic non-human animal. The invention also relates to a solid support comprising one or a plurality of the above mentioned polynucleotides, genes, vectors, polypeptides, antibodies or host cells. Furthermore, methods of identifying a polymorphism, identifying and obtaining a pro- drug or drug or an inhibitor are also encompassed by the present invention. In addition, the invention relates to methods for producing of a pharmaceutical composition and to methods of diagnosing a disease. Further, the invention relates to a method of detection of the polynucleotide of the invention. Furthermore, comprised by the present invention are a diagnostic and a pharmaceutical composition. Even more, the invention relates to uses of the polynucleotides, genes, vectors, polypeptides or antibodies of the invention. Finally, the invention relates to a diagnostic kit.

Description

Polymorphisms in the human gene for CYP1A2 and their use in diagnostic and therapeutic applications
The present invention relates to a polymorphic CYP1A2 polynucleotide. Moreover, the invention relates to genes or vectors comprising the polynucleotides of the invention and to a host cell genetically engineered with the polynucleotide or gene of the invention. Further, the invention relates to methods for producing molecular variant polypeptides or fragments thereof, methods for producing cells capable of expressing a molecular variant polypeptide and to a polypeptide or fragment thereof encoded by the polynucleotide or the gene of the invention or which is obtainable by the method or from the cells produced by the method of the invention. Furthermore, the invention relates to an antibody which binds specifically the polypeptide of the invention. Moreover, the invention relates to a transgenic non-human animal. The invention also relates to a solid support comprising one or a plurality of the above mentioned polynucleotides, genes, vectors, polypeptides, antibodies or host cells. Furthermore, methods of identifying a polymorphism, identifying and obtaining a pro- drug or drug or an inhibitor are also encompassed by the present invention. In addition, the invention relates to methods for producing of a pharmaceutical composition and to methods of diagnosing a disease. Further, the invention relates to a method of detection of the polynucleotide of the invention. Furthermore, comprised by the present invention are a diagnostic and a pharmaceutical composition. Even more, the invention relates to uses of the polynucleotides, genes, vectors, polypeptides or antibodies of the invention. Finally, the invention relates to a diagnostic kit.
Human CYP1A2 protein is a member of the cytochrome P450 superfamily (CYPs) and is involved in the metabolic activation of carcinogens (aromatic amines, heterocyclic amines, nitrosamines, nitroaromatics), mycotoxins (aflatoxin B-i, ipomeanol, sterigmatocystin), estrogens (17β-estradiol) and metabolization of several drugs (caffeine, theophylline, phenacetine, acetaminophen, nicotine, tacrine, imipramine, antipyrine, aminopyrine, clozapine) (Landi, IARC Sci Publ 148 (1999), 173-95).
In humans, CYP1A2 has been detected mainly in the liver where it is constitutively expressed and it can be induced (by cigarette smoking, dietary factors, several drugs, chronic hepatitis, exposure to polybrominated biphenyls, 3- methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin) (Eaton,
Pharmacogenetics 5 (1995), 259-74.) by transcriptional mechanisms. Currently, caffeine metabolites are used in epidemiological studies as an index for CYP1A2 activity (Catteau, Eur J Clin Pharmacol 47 (1995), 423-30; Tantcheva-Poor, Pharmacogenetics 9 (1999), 131-44.).
Wide interindividual differences in CYP1A2 activity have been described and several factors (gender, race, genetic polymorphisms, exposure to inducers) have been discussed as an underlying reason (Ikeya, Mol Endocrinol 3 (1989), 1399-408). Slow and intermediate CYP1A2 metabolizers represent about 50% of Caucasians, while their frequency in Japanese seems to be much lower (9-14% (Nakajima, Cancer Epidemiol Biomarkers Prev 3 (1994), 413-21 ; Yokoi, Pharmacogenetics (1995), S65- 9)). Epidemiological studies suggest an increased risk of colon and bladder cancer in subjects with high CYP1A2 activity (Kiyohara, J Epidemiol 10 (2000), 349-60.), and a higher level of 4-aminobiphenyl-haemoglobin adducts in moderate smokers with CYP1A2 rapid metabolizing phenotype (Landi, Cancer Epidemiol Biomarkers Prev 5 (1996), 693-8.).
Until now, no nucleotide differences that could explain the phenotypic variability of the CYP1A2 gene have been found in any exon or exon-intron junctions (Nakajima, Cancer Epidemiol Biomarkers Prev 3 (1994), 413-21). Also, no systematic investigation of SNPs in different ethnic groups has been made. Several independent efforts have been undertaken to identify genetic polymorphisms in the 5'-flanking region of the CYP1A2 gene: Ikeya et al (Ikeya, Mol Endocrinol 3 (1989), 1399-408), screened the promoter region to -1907 bp from transcription start (position -1907 in GenBank sequence with Accession No: AF253322, GI: 13430063 wherein position 34048 has been numbered +1), Quattrochi et al (Quattrochi, Mol Pharmacol 36 (1989), 66-71), screened the promoter region to -3205 bp from transcriptional start (position -3208 in GenBank sequence with Accession No: AF253322, GI: 13430063 wherein position 34048 has been numbered +1) and Aitchison et al (Aitchison, Pharmacogenetics 10 (2000), 695-704), extended the search from -3166 to -3735 bp from transcriptional start (positions -3169 to -3738 in GenBank sequence with Accession No: AF253322, GI: 13430063 wherein position 34048 has been numbered +1). Recently, the whole intergenic region between CYP1A1 and CYP1A2, which is 23 kb long (GenBank Accession number AF253322, Gl:13430063), was sequenced by Corchero et al (Corchero, Pharmacogneetics 2001 1-6 (2001), ). Among the promoter SNPs found to date, three promoter variants with functional significance have been identified. Two of them: -3858G>A and -164C>A (position 31079 and position 34779, respectively, in the GenBank sequence with Accession No: AF253322, Gl:13430063) may affect CYP1A2 inducibility and one - 3595G>T (position 31341 in the GenBank sequence with Accession No: AF253322, GI: 13430063) the gene's constitutive expression. Table 1 in the Examples shows a summary of all alleles discovered to date and their localisation on GenBank sequence with Accession No: AF253322, GI: 13430063. None of the above cited documents, however, suggests that the disclosed SNPs may be suitable as diagnostic or prognostic markers in human therapy. In other words, the available SNPs do not explain the CYP1A2 variability and are not sufficient as predictive markers for the expression level and/or activity of CYP1 A2 nor are they sufficient for reliable diagnosis and/or treatment of diseases related to CYP1A2.
Thus, improved means and methods for diagnosing and treating a variety of diseases and disorders based on dysfunctions or dysregulations of drug metabolism were not available yet but are nevertheless highly desirable. Thus, the technical problem underlying the present invention is to comply with the above specified needs.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a polynucleotide comprising a polynucleotide selected from the group consisting of:
(a) a polynucleotide having the nucleic acid sequence of any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 53, 54, 55, 57, 58, 59, 60, 61 , 63, 64, 65, 66, 67, 68, 70, 71 , 72, 74, or 75; (b) a polynucleotide encoding a polypeptide having the amino acid sequence of any one of SEQ ID NO: 56, 62, 69, or 73;
(c) a polynucleotide capable of hybridizing to a CYP1A2 gene, wherein said polynucleotide is having at a position corresponding to position -1045, -881 , 182, 615, 1253, 1352, 1471 , 1513, 1877, 1953, 2045, 2159, 2321 , 3482, 3606, 3614, 5113, or 5371 of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1), a nucleotide exchange of at least one nucleotide;
(d) a polynucleotide capable of hybridizing to a CYP1A2 gene, wherein said polynucleotide is having at a position corresponding to position -1045, 1352, 1471 , 1513, 1877 or 5371 , of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1) an A, at a position corresponding to position 2321 , 3482 or 3614 of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1) a C, at a position corresponding to position - 881 , 1253, 1953, 2159 or 3606 of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1) a G, at a position corresponding to position 182, 615, 2045 or 5113 of the CYP1A2 gene (GenBank Accession No: AF253322, Gl:13430063 wherein position 34942 has been numbered +1) a T;
(e) a polynucleotide encoding a CYP1A2 polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution at position 61 , 298, 401 or 438 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061); and
(f) a polynucleotide encoding a CYP1A2 polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution of P to L at position corresponding to position 61 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , GI: 13325061), S to R at position corresponding to position 298 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , GI: 13325061), I to T at position corresponding to position 401 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061) or T to I at position corresponding to position 438 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061). In the context of the present invention the term "polynucleotides" or the term "polypeptides" refers to different variants of a polynucleotide or polypeptide. Said variants comprise a reference or wild type sequence of the polynucleotides or polypeptides of the invention as well as variants which differ therefrom in structure or composition. Reference sequence for the polynucleotide is GenBank Accession No: AF253322, Gl:13430063. Reference sequence for the polypeptide of the invention is GenBank Accession No: NM_000761 , Gl:13325061. The differences in structure or composition usually occur by way of nucleotide or amino acid substitutions. Preferably, said nucleotide substitutions comprised by the present invention result in one or more changes of the corresponding amino acids of the polypeptide of the invention.
The variant polynucleotides and polypeptides also comprise fragments of the polynucleotides or polypeptides specified herein. The term "polynucleotides" as used herein preferably encompasses the nucleic acid sequences specifically referred to by SEQ ID NOS and in the tables below as well as polynucleotides comprising the reverse complementary nucleic acid sequence thereto. The polynucleotides and polypeptides as well as the aforementioned fragments thereof are characterized in accordance with the present invention as being associated with a CYP1A2 dysfunction or dysregulation comprising, e.g., insufficient, altered drug metabolism, altered expression level, altered protein level and/or altered activity level. The term "insufficient drug metabolism" as referred to in the present invention means reduced or loss of metabolic activity of the CYP1A2 enzyme. The term "altered drug metabolism" as referred to in the present invention means reduced or enhanced metabolic activity of CYP1A2 or altered metabolic profile. The term "expression level" as referred to in the context of the present invention means the detectable amount of transcripts of the CYP1A2 gene relative to the amount of transcripts for a housekeeping gene, such as PLA2, actin or GAPDH. The amount of transcripts can be determined by standard molecular biology techniques including Northern analysis, RNAse protection assays, PCR based techniques encompassing Taq-Man analysis. The term "protein level" refers to the detectable amount of CYP1A2 relative to the amount of a protein encoded by a housekeeping gene, such as PLA2. The amount of proteins can be determined by standard biochemical techniques, such as Western analysis, ELISA, RIA or other antibody based techniques known in the art. The term "activity level" means the detectable biological activity of CYP1A2 relative to the activity of an encoded by the allellic variants of these genes as disclosed in the present invention relative to the activity of the protein encoded by the corresponding wild-type allele of the gene. Biological assays for the aforementioned protein are well known in the art and described in Butler, Pharmacogenetics 2 (1992), 116-127; Fuhr, Pharmacogenetics 4 (1994), 109-116; McQuilkin, Cancer Epidemiol. Biomarkers Prev. 4 (1995), 139-146; Campbell, Clin. Pharmacol. Ther. 42 (1987), 157-165; Sachse, Br. J. Clin. Pharmacol. 47 (1999), 445-449; Tatcheva-Poor, Pharmacogenetics 9 (1999), 131-144; Sesardic, Br. J. Clin. Pharmacol. 26 (1988), 363-372; Robson, Biochem. Pharmacol. 37 (1988), 1651-1659. As a reference standard, preferable proteins are obtained from cells or tissues of a subject having the aforementioned wild-type alleles of the respective genes in their genomes. A further aspect of the present invention is that said dysfunctions or dysreguiations cause a disease or disorder or a prevalence for said disease or disorder. Preferably, as will be discussed below in detail, said disease is cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia or any other disease caused by a dysfunction or dysregulation due to a polynucleotide or polypeptides of the invention, also referred to as CYP1A2 gene associated diseases in the following.
The term "hybridizing" as used herein refers to polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with a CYP1A2 dysfunction or dysregulation. Thus, said hybridizing polynucleotides are also associated with said dysfunctions and dysreguiations. Preferably, said polynucleotides capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with CYP1A2 dysfunctions or dysreguiations are at least 70%, at least 80%, at least 95% or at least 100% identical to the polynucleotides of the invention or parts thereof which are associated with CYP1A2 dysfunctions or dysreguiations. Therefore, said polynucleotides may be useful as probes in Northern or Southern Blot analysis of RNA or DNA preparations, respectively, or can be used as oligonucleotide primers in PCR analysis dependent on their respective size. Also comprised by the invention are hybridizing polynucleotides which are useful for analysing DNA-Protein interactions via, e.g., electrophoretic mobility shift analysis (EMSA). Preferably, said hybridizing polynucleotides comprise at least 10, more preferably at least 15 nucleotides in length while a hybridizing polynucleotide of the present invention to be used as a probe preferably comprises at least 100, more preferably at least 200, or most preferably at least 500 nucleotides in length.
It is well known in the art how to perform hybridization experiments with nucleic acid molecules, i.e. the person skilled in the art knows what hybridization conditions s/he has to use in accordance with the present invention. Such hybridization conditions are referred to in standard text books such as Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. Preferred in accordance with the present invention are polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with a CYP1A2 dysfunction or dysregulation under stringent hybridization conditions, i.e. those which do not cross hybridize to unrelated polynucleotides such as polynucleotides encoding a polypeptide different from the CYP1A2 polypeptides of the invention. Preferably, stringent hybridization conditions refere to an overnight incubation at 42°C in a solution comprising 50% formamide hybridization solution, followed by at least two washing steps at 60°C in 0.2 x SSC with 1% SDS.
The term "corresponding" as used herein means that a position is not only determined by the number of the preceding nucleotides and amino acids, respectively. The position of a given nucleotide or amino acid in accordance with the present invention which may be deleted, substituted or comprise one or more additional nucleotide(s) may vary due to deletions or additional nucleotides or amino acids elsewhere in the gene or the polypeptide. Thus, under a "corresponding position" in accordance with the present invention it is to be understood that nucleotides or amino acids may differ in the indicated number but may still have similar neighboring nucleotides or amino acids. Said nucleotides or amino acids which may be exchanged, deleted or comprise additional nucleotides or amino acids are also comprised by the term "corresponding position". Said nucleotides or amino acids may for instance together with their neighbors form sequences which may be involved in the regulation of gene expression, stability of the corresponding RNA or RNA editing, as well as encode functional domains or motifs of the protein of the invention. In accordance with the present invention, the mode and population distribution of genetic variations in the CYP1A2 gene has been analyzed by sequence analysis of relevant regions of the human said gene from many different individuals. It is a well known fact that genomic DNA of individuals, which harbor the individual genetic makeup of all genes, including the CYP1A2 gene, can easily be purified from individual blood samples. These individual DNA samples are then used for the analysis of the sequence composition of the alleles of the CYP1A2 gene that are present in the individual which provided the blood sample. The sequence analysis was carried out by PCR amplification of relevant regions of said genes, subsequent purification of the PCR products, followed by automated DNA sequencing with established methods (e.g. ABI dyeterminator cycle sequencing). One important parameter that had to be considered in the attempt to determine the individual genotypes and identify novel variants of the CYP1A2 gene by direct DNA- sequencing of PCR-products from human blood genomic DNA is the fact that each human harbors (usually, with very few abnormal exceptions) two gene copies of each autosomal gene (diploidy). Because of that, great care had to be taken in the evaluation of the sequences to be able to identify unambiguously not only homozygous sequence variations but also heterozygous variations. The details of the different steps in the identification and characterization of novel polymorphisms in the CYP1A2 gene (homozygous and heterozygous) are described in the Examples below.
Over the past 20 years, genetic heterogeneity has been increasingly recognized as a significant source of variation in drug response. Many scientific communications (Meyer, Ann. Rev. Pharmacol. Toxicol. 37 (1997), 269-296 and West, J. Clin. Pharmacol. 37 (1997), 635-648) have clearly shown that some drugs work better or may even be highly toxic in some patients than in others and that these variations in patient's responses to drugs can be related to molecular basis. This "pharmacogenomic" concept spots correlations between responses to drugs and genetic profiles of patient's (Marshall, Nature Biotechnology, 15 (1997), 954-957; Marshall, Nature Biotechnology, 15 (1997), 1249-1252). In this context of population variability with regard to drug therapy, pharmacogenomics has been proposed as a tool useful in the identification and selection of patients which can respond to a particular drug without side effects. This identification/selection can be based upon molecular diagnosis of genetic polymorphisms by genotyping DNA from leukocytes in the blood of patient, for example, and characterization of disease (Bertz, Clin. Pharmacokinet. 32 (1997), 210-256; Engel, J. Chromatogra. B. Biomed. Appl. 678 (1996), 93-103). For the founders of health care, such as health maintenance organizations in the US and government public health services in many European countries, this pharmacogenomics approach can represent a way of both improving health care and reducing overheads because there is a large cost to unnecessary drugs, ineffective drugs and drugs with side effects.
The mutations in the variant genes of the invention sometime result in amino acid substitutions either alone or in combination. It is of course also possible to genetically engineer such mutations in wild type genes or other mutant forms. Methods for introducing such modifications in the DNA sequence of said genes are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
For the investigation of the nature of the alterations in the amino acid sequence of the polypeptides of the invention may be used such as BRASMOL that are obtainable from the Internet. Furthermore, folding simulations and computer redesign of structural motifs can be performed using other appropriate computer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computers can be used for the conformational and energetic analysis of detailed protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). These analysis can be used for the identification of the influence of a particular mutation on binding and/or processing of drugs.
Usually, said amino acid substitutions in the amino acid sequence of the protein encoded by the polynucleotide of the invention is due to one or more nucleotide substitutions. Preferably said nucleotide substitutions, may result in an amino acid substitution of P to L at position corresponding to position 61 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061), S to R at position corresponding to position 298 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061), I to T at position corresponding to position 401 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , GM3325061) or T to I at position corresponding to position 438 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , GM3325061).
The mutations in the CYP1A2 gene detected in accordance with the present invention are listed in Table 2. The methods of the mutation analysis followed standard protocols and are described in detail in the Examples. In general such methods are to be used in accordance with the present invention for evaluating the phenotypic spectrum as well as the overlapping clinical characteristics of diseases or conditions related to dysfunctions or dysreguiations and diseases related to the metabolism of drugs (e.g. clozapine clearence) or the metabolic activation pathways of several chemical carcinogens, environmental and dietary compounds known to induce CYP1A2, such as: aromatic or heterocyclic amines, nitroaromatic compounds, mycotoxins and estrogens, cigarette smoke, charbroiled or high-temperature cooked meat, cruciferous vegetables. Advantageously, the characterization of said mutants may form the basis of the development of improved methods for evaluation of several cancer risks (e.g. lung, liver, breast, prostata, endometrial, colorectal, urinary bladder), or the development of improved drugs, such as drugs which are used e.g in cancer therapy, as non-narcotic antipyretic and analgesic drugs (phenacetin) (Devonshire, Br J Clin Pharmacol 16 (1983), 157-66.), and in the treatment of diseases including congenital jaundice (Zaccaro, Biochem Pharmacol 61 (2001), 843-9.), porphyria cutanea tarda (Christiansen, Hum Genet 107 (2000), 612-4), tardive dyskinesia in schizophrenia (Basile, Mol Psychiatry 5 (2000), 410-7.). Said methods encompass for example haplotype analysis, single-strand conformation polymorphism analysis (SSCA), denaturating gradient gel electrophoresis (DGGE), PCR, direct sequencing, HPLC-based techniques, invasive cleavage assay, mass spectroscopy, microarray, a rolling circle extension assay, primer extension assay, a molecular beacon assay and a ligase chain reaction assay. On the basis of thorough clinical characterization of many patients the phenotypes can then be correlated to these mutations.
Also comprised by the polynucleotides referred to in the present invention are polynucleotides which comprise at least two of the polynucleotides specified hereinabove, i.e. polynucleotides having a nucleotide sequence which contains at least two of the mutations comprised by the above polynucleotides or listed in Figure 1 below. Thus, the haplotype determined in accordance with the present invention can be characterized by at least two of said mutations in the CYP1 A2 locus. Further, the polynucleotide of the invention may further comprise at least one nucleotide deletion, addition and/or substitution other than those specified hereinabove, for example those described in the prior art; e.g. listed in Table 1. This allows the study of synergistic effects of said mutations in the CYP1A2 gene and/or a polypeptide encoded by said polynucleotide on the pharmacological profile of drugs in patients who bear such mutant forms of the gene or similar mutant forms that can be mimicked by the above described proteins. It is expected that the analysis of said synergistic effects provides deeper insights into the onset of CYP1A2 dysfunctions or dysreguiations or diseases related to altered drug metabolism as described supra. From said deeper insight the development of diagnostic and pharmaceutical compositions related to CYP1A2 dysfunctions or dysreguiations or diseases related to drug metabolism will greatly benefit.
As is evident to the person skilled in the art, the genetic knowledge deduced from the present invention can now be used to exactly and reliably characterize the genotype of a patient. Advantageously, diseases or a prevalence for a disease which are associated with CYP1A2 dysfunction or dysregulation, such as cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia referred to herein can be predicted and preventive or therapeutical measures can be applied accordingly. Moreover in accordance with the foregoing, in cases where a given drug takes an unusual effect, a suitable individual therapy can be designed based on the knowledge of the individual genetic makeup of a subject with respect to the polynucleotides of the invention and improved therapeutics can be developed as will be further discussed below.
The term "unusual effect" is an undesirable or insufficient response to the administration of a therapeutic compound, i.e. an effect that is not directed to alleviating the symptoms or cause of the disease beeing treated. Unusual effects range from minor inconveniences to more serious events.
In general, the CYP1A2 "status", defined by the expression level and activity of the CYP1A2 protein, can be not only altered in many disease or disorders including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia (see above), but can also be variable in normal tissue, due to genetic variations/polymorphisms. The identification of polymorphisms associated with altered CYP1A2 expression and/or activity is important for the prediction of drug uptake and subsequently for the prediction of therapy outcome, including side effects of medications. Therefore, analysis of CYP1A2 variations indicative of CYP1A2 function, is a valuable tool for therapy with drugs, which are substrates of CYP1A2 and has, thanks to the present invention, now become possible.
In line with the foregoing, preferably, the polynucleotide of the present invention is associated with cancer, congenital jaundice, porphyria cutanea tarda, or tardive dyskinesia in schizophrenia.
The term "cancer", "congenital jaundice", "porphyria cutanea", or "tardive dyskinesia in schizophrenia" used herein are very well known and characterized in the art. For example, several variants of cancer exist and are comprised by said term as meant in accordance with the invention. For a detailed list of symptoms which are indicative for cancer and the other aforementioned diseases it is referred to text book knowledge, e.g. Pschyrembel or Stedman.
In a further embodiment the present invention relates to a polynucleotide which is DNA or RNA.
The polynucleotide of the invention may be, e.g., DNA, cDNA, genomic DNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. Preferably said polynucleotide is part of a vector, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide of the invention. Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
The invention furthermore relates to a gene comprising the polynucleotide of the invention.
It is well known in the art that genes comprise structural elements which encode an amino acid sequence as well as regulatory elements which are involved in the regulation of the expression of said genes. Structural elements are represented by exons which may either encode an amino acid sequence or which may encode for RNA which is not encoding an amino acid sequence but is nevertheless involved in RNA function, e.g. by regulating the stability of the RNA or the nuclear export of the RNA.
Regulatory elements of a gene may comprise promoter elements or enhancer elements both of which could be involved in transcriptional control of gene expression. It is very well known in the art that a promoter is to be found upstream of the structural elements of a gene. Regulatory elements such as enhancer elements, however, can be found distributed over the entire locus of a gene. Said elements could be reside, e.g., in introns, regions of genomic DNA which separate the exons of a gene. Promoter or enhancer elements correspond to polynucleotide fragments which are capable of attracting or binding polypeptides involved in the regulation of the gene comprising said promoter or enhancer elements. For example, polypeptides involved in regulation of said gene comprise the so called transcription factors. Said introns may comprise further regulatory elements which are required for proper gene expression. Introns are usually transcribed together with the exons of a gene resulting in a nascent RNA transcript which contains both, exon and intron sequences. The intron encoded RNA sequences are usually removed by a process known as RNA splicing. However, said process also requires regulatory sequences present on a RNA transcript said regulatory sequences may be encoded by the introns.
In addition, besides their function in transcriptional control and control of proper RNA processing and/or stability, regulatory elements of a gene could be also involved in the control of genetic stability of a gene locus. Said elements control, e.g., recombination events or serve to maintain a certain structure of the DNA or the arrangement of DNA in a chromosome.
Therefore, single nucleotide polymorphisms can occur in exons of a gene which encode an amino acid sequence as discussed supra as well as in regulatory regions which are involved in the above discussed process. The analysis of the nucleotide sequence of a gene locus in its entirety including, e.g., introns is in light of the above desirable. The polymorphisms comprised by the polynucleotides of the present invention can influence the expression level of CYP1A2 protein via mechanisms involving enhanced or reduced transcription of the CYP1A2 gene, stabilization of the gene's RNA transcripts and alteration of the processing of the primary RNA transcripts.
Therefore, in a furthermore preferred embodiment of the gene of the invention a nucleotide substitution results in altered expression of the variant gene compared to the corresponding wild type gene.
In another embodiment the present invention relates to a vector comprising the polynucleotide of the invention or the gene of the invention.
Said vector may be, for example, a phage, plasmid, viral or retroviral vector.
Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.
The polynucleotides or genes of the invention may be joined to a vector containing selectable markers for propagation in a host. Generally, a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate, or in a complex with a charged lipid or in carbon-based clusters. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.
In a more preferred embodiment of the vector of the invention the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.
Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poIy-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDMδ, pRc/CMV, pcDNAI, pcDNA3 (Invitrogen), pSPORTI (GIBCO BRL), pFastBac (Invitrogen), pYES (Invitrogen). Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
The term "isolated fractions thereof refers to fractions of eukaryotic or prokaryotic cells or tissues which are capable of transcribing or transcribing and translating RNA from the vector of the invention. Said fractions comprise proteins which are required for transcription of RNA or transcription of RNA and translation of said RNA into a polypeptide. Said isolated fractions may be, e.g., nuclear and cytoplasmic fractions of eukaryotic cells such as of reticulocytes.
The present invention furthermore relates to a host cell genetically engineered with the polynucleotide of the invention, the gene of the invention or the vector of the invention.
Said host cell may be a prokaryotic or eukaryotic cell; see supra. The polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally. In this respect, it is also to be understood that the recombinant DNA molecule of the invention can be used for "gene targeting" and/or "gene replacement", for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, mammalian or, preferably, human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of a variant polypeptide of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. A polynucleotide coding for a mutant form of variant polypeptides of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Methods for preparing fused, operably linked genes and expressing them in bacteria or animal cells are well-known in the art (Sambrook, supra). The genetic constructs and methods described therein can be utilized for expression of variant polypeptides of the invention in, e.g., prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. The transformed prokaryotic hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The proteins of the invention can then be isolated from the grown medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the microbially or otherwise expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
Thus, in a further embodiment the invention relates to a method for producing a molecular variant CYP1A2 polypeptide or fragment thereof comprising culturing the above described host cell; and recovering said protein or fragment from the culture.
In another embodiment the present invention relates to a method for producing cells capable of expressing a molecular variant CYP1A2 polypeptide comprising genetically engineering cells with the polynucleotide of the invention, the gene of the invention or the vector of the invention.
The cells obtainable by the method of the invention can be used, for example, to test drugs according to the methods described in D. L. Spector, R. D. Goldman, L. A. Leinwand, Cells, a Lab manual, CSH Press 1998. Furthermore, the cells can be used to study known drugs and unknown derivatives thereof for their ability to complement the deficiency caused by mutations in the CYP1A2 gene. For these embodiments the host cells preferably lack a wild type allele, preferably both alleles of the CYP1A2 gene and/or have at least one mutated from thereof. Ideally, the gene comprising an allele as comprised by the polynucleotides of the invention could be introduced into the wild type locus by homologous replacement. Alternatively, strong overexpression of a mutated allele over the normal allele and comparison with a recombinant cell line overexpressing the normal allele at a similar level may be used as a screening and analysis system. The cells obtainable by the above-described method may also be used for the screening methods referred to herein below.
Furthermore, the invention relates to a polypeptide or fragment thereof encoded by the polynucleotide of the invention, the gene of the invention or obtainable by the method described above or from cells produced by the method described above. In this context it is also understood that the variant polypeptide of the invention can be further modified by conventional methods known in the art. By providing said variant proteins according to the present invention it is also possible to determine the portions relevant for their biological activity or inhibition of the same. The terms "polypeptide" and "protein" as used herein are exchangeable. Moreover, what is comprised by said terms is standard textbook knowledge.
The present invention furthermore relates to an antibody which binds specifically to the polypeptide of the invention.
Advantageously, the antibody specifically recognizes or binds an epitope containing one or more amino acid substitution(s) as defined above. Antibodies against the variant polypeptides of the invention can be prepared by well known methods using a purified protein according to the invention or a (synthetic) fragment derived therefrom as an antigen. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. In a preferred embodiment of the invention, said antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, human or humanized antibody, primatized, chimerized or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. Furthermore, antibodies or fragments thereof to the aforementioned polypeptides can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of the variant polypeptides of the invention as well as for the monitoring of the presence of said variant polypeptides, for example, in recombinant organisms, and for the identification of compounds interacting with the proteins according to the invention. For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
In a preferred embodiment the antibody of the present invention specifically recognizes an epitope containing one or more amino acid substitution(s) resulting from a nucleotide exchange as defined supra.
Antibodies which specifically recognize modified amino acids such as phospho- Tyrosine residues are well known in the art. Similarly, in accordance with the present invention antibodies which specifically recognize even a single amino acid exchange in an epitope may be generated by the well known methods described supra.
In light of the foregoing, in a more preferred embodiment the antibody of the present invention is monoclonal or polyclonal.
The invention also relates to a transgenic non-human animal comprising at least one polynucleotide of the invention, the gene of the invention or the vector of the invention as described supra. The present invention also encompasses a method for the production of a transgenic non-human animal comprising introduction of a polynucleotide or vector of the invention into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom. The non-human animal can be used in accordance with the method of the invention described below and may be a non-transgenic healthy animal, or may have a disease or disorder, preferably a disease caused by at least one mutation in the gene of the invention. Such transgenic animals are well suited for, e.g., pharmacological studies of drugs in connection with variant forms of the above described variant polypeptides since these polypeptides or at least their functional domains are conserved between species in higher eukaryotes, particularly in mammals. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryos can be analyzed using, e.g., Southern blots with an appropriate probe or based on PCR techniques. A transgenic non-human animal in accordance with the invention may be a transgenic mouse, rat, hamster, dog, monkey, rabbit, pig, frog, nematode such as Caenorhabditis elegans, fruitfly such as Drosophila melanogaster or fish such as torpedo fish or zebrafish comprising a polynucleotide or vector of the invention or obtained by the method described above, preferably wherein said polynucleotide or vector is stably integrated into the genome of said non-human animal, preferably such that the presence of said polynucleotide or vector leads to the expression of the variant polypeptide of the invention. It may comprise one or several copies of the same or different polynucleotides or genes of the invention. This animal has numerous utilities, including as a research model for cardiovascular research and therefore, presents a novel and valuable animal in the development of therapies, treatment, etc. for diseases caused by cardiovascular diseases. Accordingly, in this instance, the mammal is preferably a laboratory animal such as a mouse or rat.
Thus, in a preferred embodiment the transgenic non-human animal of the invention is a mouse, a rat or a zebrafish.
Numerous reports revealed that said animals are particularly well suited as model organisms for the investigation of the drug metabolism and its deficiencies or cancer. Advantageously, transgenic animals can be easily created using said model organisms, due to the availability of various suitable techniques well known in the art. The invention also relates to a solid support comprising one or a plurality of the polynucleotide, the gene, the vector, the polypeptide, the antibody or the host cell of the invention in immobilized form.
The term "solid support" as used herein refers to a flexible or non-flexible support that is suitable for carrying said immobilized targets. Said solid support may be homogenous or inhomogeneous. For example, said solid support may consist of different materials having the same or different properties with respect to flexibility and immobilization, for instance, or said solid support may consist of one material exhibiting a plurality of properties also comprising flexibility and immobilization properties. Said solid support may comprise glass-, polypropylene- or silicon-chips, membranes oligonucleotide-conjugated beads or bead arrays. The term "immobilized" means that the molecular species of interest is fixed to a solid support, preferably covalently linked thereto. This covalent linkage can be achieved by different means depending on the molecular nature of the molecular species. Moreover, the molecular species may be also fixed on the solid support by electrostatic forces, hydrophobic or hydrophilic interactions or Van-der-Waals forces. The above described physico-chemical interactions typically occur in interactions between molecules. For example, biotinylated polypeptides may be fixed on a avidin- coated solid support due to interactions of the above described types. Further, polypeptides such as antibodies, may be fixed on an antibody coated solid support. Moreover, the immobilization is dependent on the chemical properties of the solid support. For example, the nucleic acid molecules can be immobilized on a membrane by standard techniques such as UV-crosslinking or heat.
In a preferred embodiment of the invention said solid support is a membrane, a glass- or poylpropylene- or silicon-chip, are membranes oligonucleotide-conjugated beads or a bead array, which is assembled on an optical filter substrate.
Moreover, the present invention relates to an in vitro method for identifying a polymorphism said method comprising the steps of:
(a) isolating a polynucleotide or the gene of the invention from a plurality of subgroups of individuals, wherein one subgroup has no prevalence for a CYP1A2 associated disease and at least one or more further subgroup(s) do have prevalence for a CYP1A2 associated disease; and (b) identifying a polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having no prevalence for a CYP1 A2 associated disease with said at least one or more further subgroup(s) having a prevalence for a CYP1A2 associated disease.
The term "prevalence" as used herein means that individuals are susceptible for one or more disease(s) which are associated with CYP1A2 dysfuntion or dysregulation or could already have one or more of said disease(s). Thereby, one CYP1A2 associated disease can be used to determine the susceptibility for another CYP1A2 associated disease, e.g. impaired drug metabolism may be indicative for a prevalence for, e.g. cancer. Moreover, symptoms which are indicative for a prevalence for developing said diseases are very well known in the art and have been sufficiently described in standard textbooks such as Pschyrembel. Advantageously, polymorphisms according to the present invention which are associated with CYP1A2 dysfunction or dysregulation or one or more disease(s) based thereon should be enriched in subgroups of individuals which have a prevalence for said diseases versus subgroups which have no prevalence for said diseases. Thus, the above described method allows the rapid and reliable detection of polymorphism which are indicative for one or more CYP1A2 associated disease(s) or a susceptibility therefor. Advantageously, due to the phenotypic preselection a large number of individuals having no prevalence might be screened for polymorphisms in general. Thereby, a reference sequences comprising polymorphisms which do not correlate to one or more CYP1A2 associated disease(s) can be obtained. Based on said reference sequences it is possible to efficiently and reliably determine the relevant polymorphisms.
In a further embodiment the present invention relates to a method for identifying and obtaining a pro-drug or a drug capable of modulating the activity of a molecular variant of a CYP1 A2 polypeptide comprising the steps of:
(a) contacting the polypeptide, the solid support of the invention, a cell expressing a molecular variant gene comprising a polynucleotide of the invention, the gene or the vector of the invention in the presence of components capable of providing a detectable signal in response to drug activity with a compound to be screened for pro-drug or drug activity; and (b) detecting the presence or absence of a signal or increase or decrease of a signal generated from the pro-drug or the drug activity, wherein the absence, presence, increase or decrease of the signal is indicative for a putative pro- drug or drug.
The term "compound" in a method of the invention includes a single substance or a plurality of substances which may or may not be identical.
Said compound(s) may be chemically synthesized or produced via microbial fermentation but can also be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compounds may be known in the art but hitherto not known to be useful as an inhibitor, respectively. The plurality of compounds may be, e.g., added to the culture medium or injected into a cell or non-human animal of the invention.
If a sample containing (a) compound(s) is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound, in question or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample. It can then be determined whether said sample or compound displays the desired properties, for example, by the methods described herein or in the literature (Spector et al., Cells manual; see supra). Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s). Preferably said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical. The methods of the present invention can be easily performed and designed by the person skilled in the art, for example in accordance with other cell based assays described in the prior art or by using and modifying the methods as described herein. Furthermore, the person skilled in the art will readily recognize which further compounds may be used in order to perform the methods of the invention, for example, enzymes, if necessary, that convert a certain compound into a precursor. Such adaptation of the method of the invention is well within the skill of the person skilled in the art and can be performed without undue experimentation.
Compounds which can be used in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like. Said compounds may act as agonists or antagonists of the inveniton. Said compounds can also be functional derivatives or analogues of known drugs. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA. Furthermore, said derivatives and analogues can be tested for their effects according to methods known in the art or as described. Furthermore, peptide mimetics and/or computer aided design of appropriate drug derivatives and analogues can be used, for example, according to the methods described below. Such analogs comprise molecules may have as the basis structure of known CYP1 A2 substrates and/or inhibitors and/or modulators; see infra. Appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptides of the invention by computer assistant searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above- described computer analysis can be used in combination with the method of the invention for, e.g., optimizing known inhibitors, analogs, antagonists or agonists. Appropriate peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709- 715. Furthermore, the three-dimensional and/or crystallographic structure of said compounds and the polypeptides of the invention can be used for the design of peptidomimetic drugs (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558). It is very well known how to obtain said compounds, e.g. by chemical or biochemical standard techniques. Thus, also comprised by the method of the invention are means of making or producing said compounds. In summary, the present invention provides methods for identifying and obtaining compounds which can be used in specific doses for the treatment of specific forms of CYP1 A2 associated diseases, e.g. dysfunctions or dysreguiations of the drug metabolism such as cancer.
The above definitions apply mutatis mutandis to all of the methods described in the following.
In a further embodiment the present invention relates to a method for identifying and obtaining an inhibitor of the activity of a molecular variant of a CYP1A2 polypeptide comprising the steps of:
(a) contacting the protein, the solid support of the invention or a cell expressing a molecular variant gene comprising a polynucleotide or the gene or the vector of the invention in the presence of components capable of providing a detectable signal in response to drug activity with a compound to be screened for inhibiting activity; and
(b) detecting the presence or absence of a signal or increase or decrease of a signal generated from the inhibiting activity, wherein the absence or decrease of the signal is indicative for a putative inhibitor.
In a preferred embodiment of the method of the invention said cell is a cell, obtained by the method of the invention or can be obtained from the transgenic non-human animal as described supra.
In a still further embodiment the present invention relates to a method of identifying and obtaining a pro-drug or drug capable of modulating the activity of a molecular variant of a CYP1 A2 polypeptide comprising the steps of:
(a) contacting the host cell, the cell obtained by the method of the invention, the polypeptide or the solid support of the invention with the first molecule known to be bound by a CYP1A2 polypeptide to form a first complex of said polypeptide and said first molecule;
(b) contacting said first complex with a compound to be screened, and
(c) measuring whether said compound displaces said first molecule from said first complex.
Advantageously, in said method said measuring step comprises measuring the formation of a second complex of said protein and said inhibitor candidate. Preferably, said measuring step comprises measuring the amount of said first molecule that is not bound to said protein.
In a particularly preferred embodiment of the above-described method of said first molecule is a agonist or antagonist or a substrate and/or a inhibitor and/or a modulator of the polypeptide of the invention, e.g., with a radioactive or fluorescent label.
In a still another embodiment the present invention relates to a method of identifying and obtaining an inhibitor capable of modulating the activity of a molecular variant of a CYP1A2 polypeptide comprising the steps of:
(a) contacting the host cell or the cell obtained by the method of the invention, the protein or the solid support of the invention with the first molecule known to be bound by the CYP1A2 polypeptide to form a first complex of said protein and said first molecule;
(b) contacting said first complex with a compound to be screened, and
(c) measuring whether said compound displaces said first molecule from said first complex.
In a preferred embodiment of the method of the invention said measuring step comprises measuring the formation of a second complex of said protein and said compound.
In another preferred embodiment of the method of the invention said measuring step comprises measuring the amount of said first molecule that is not bound to said protein. In a more preferred embodiment of the method of the invention said first molecule is labeled.
The invention furthermore relates to a method for the production of a pharmaceutical composition comprising the steps of the method as described supra; and the further step of formulating the compound identified and obtained or a derivative thereof in a pharmaceutically acceptable form.
The therapeutically useful compounds identified according to the methods of the invention can be formulated and administered to a patient as discussed above. For uses and therapeutic doses determined to be appropriate by one skilled in the art and for definitions of the term "pharmaceutical composition" see infra.
Furthermore, the present invention encompasses a method for the preparation of a pharmaceutical composition comprising the steps of the above-described methods; and formulating a drug or pro-drug in the form suitable for therapeutic application and preventing or ameliorating the disorder of the subject diagnosed in the method of the invention.
Drugs or pro-drugs after their in vivo administration are metabolized in order to be eliminated either by excretion or by metabolism to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459). Thus, rather than using the actual compound or inhibitor identified and obtained in accordance with the methods of the present invention a corresponding formulation as a pro-drug can be used which is converted into its active in the patient. Precautionary measures that may be taken for the application of pro-drugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329).
In a preferred embodiment of the method of the present invention said drug or prodrug is a derivative of a medicament as defined hereinafter.
The present invention also relates to a method of diagnosing a disorder related to the presence of a molecular variant of the CYP1A2 gene or susceptibility to such a disorder comprising determining the presence of a polynucleotide or the gene of the invention in a sample from a subject.
In accordance with this embodiment of the present invention, the method of testing the status of a disorder or susceptibility to such a disorder can be effected by using a polynucleotide gene or nucleic acid of the invention, e.g., in the form of a Southern or Northern blot or in situ analysis. Said nucleic acid sequence may hybridize to a coding region of either of the genes or to a non-coding region, e.g. intron. In the case that a complementary sequence is employed in the method of the invention, said nucleic acid molecule can again be used in Northern blots. Additionally, said testing can be done in conjunction with an actual blocking, e.g., of the transcription of the gene and thus is expected to have therapeutic relevance. Furthermore, a primer or oligonucleotide can also be used for hybridizing to one of the above mentioned CYP1A2 gene or corresponding mRNAs. The nucleic acids used for hybridization can, of course, be conveniently labeled by incorporating or attaching, e.g., a radioactive or other marker. Such markers are well known in the art. The labeling of said nucleic acid molecules can be effected by conventional methods. Additionally, the presence or expression of variant CYP1A2 gene can be monitored by using a primer pair that specifically hybridizes to either of the corresponding nucleic acid sequences and by carrying out a PCR reaction according to standard procedures. Specific hybridization of the above mentioned probes or primers preferably occurs at stringent hybridization conditions. The term "stringent hybridization conditions" is well known in the art; see, for example, Sambrook et al., "Molecular Cloning, A Laboratory Manual" second ed., CSH Press, Cold Spring Harbor, 1989; "Nucleic Acid Hybridisation, A Practical Approach", Hames and Higgins eds., IRL Press, Oxford, 1985. Furthermore, the mRNA, cRNA, cDNA or genomic DNA obtained from the subject may be sequenced to identify mutations which may be characteristic fingerprints of mutations in the polynucleotide or the gene of the invention. The present invention further comprises methods wherein such a fingerprint may be generated by RFLPs of DNA or RNA obtained from the subject, optionally the DNA or RNA may be amplified prior to analysis, the methods of which are well known in the art. RNA fingerprints may be performed by, for example, digesting an RNA sample obtained from the subject with a suitable RNA-Enzyme, for example RNase Ti, RNase T2 or the like or a ribozyme and, for example, electrophoretically separating and detecting the RNA fragments as described above. Further modifications of the above-mentioned embodiment of the invention can be easily devised by the person skilled in the art, without any undue experimentation from this disclosure; see, e.g., the examples. An additional embodiment of the present invention relates to a method wherein said determination is effected by employing an antibody of the invention or fragment thereof. The antibody used in the method of the invention may be labeled with detectable tags such as a histidine flags or a biotin molecule.
The invention relates to a method of diagnosing a disorder related to the presence of a molecular variant of a CYP1A2 gene or susceptibility to such a disorder comprising determining the presence of a polypeptide or the antibody of the invention in a sample from a subject.
In a preferred embodiment of the above described method said disorder is cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia.
In a preferred embodiment of the present invention, the above described method is comprising PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques, single-strand conformation polymorphism analysis (SSCA), denaturating gradient gel electrophoresis (DGGE), direct sequencing, HPLC-based techniques, invasive cleavage assay, mass spectroscopy, microarray, a rolling circle extension assay, a primer extension assay and a molecular beacon assay, hybridization techniques or immunoassays. Said techniques are very well known in the art.
Moreover, the invention relates to a method of detection of the polynucleotide or the gene of the invention in a sample comprising the steps of
(a) contacting the solid support described supra with the sample under conditions allowing interaction of the polynucleotide or the gene of the invention with the immobilized targets on a solid support and;
(b) determining the binding of said polynucleotide or said gene to said immobilized targets on a solid support.
The invention also relates to an in vitro method for diagnosing a disease comprising the steps of the method described supra, wherein binding of said polynucleotide or gene to said immobilized targets on said solid support is indicative for the presence or the absence of said disease or a prevalence for said disease. The invention furthermore relates to a diagnostic composition comprising the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention.
In addition, the invention relates to a pharmaceutical composition comprising the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention. These pharmaceutical compositions comprising, e.g., the antibody may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation. Acceptable salts comprise acetate, methylester, HCI, sulfate, chloride and the like. The compounds may be administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. Furthermore, the use of pharmaceutical compositions which comprise antisense- oligonucleotides which specifically hybridize to RNA encoding mutated versions of the polynucleotitde or gene according to the invention or which comprise antibodies specifically recognizing a mutated polypeptide of the invention but not or not substantially the functional wild-type form is conceivable in cases in which the concentration of the mutated form in the cells should be reduced. Thanks to the present invention the particular drug selection, dosage regimen and corresponding patients to be treated can be determined in accordance with the present invention. The dosing recommendations will be indicated in product labeling by allowing the prescriber to anticipate dose adjustments depending on the considered patient group, with information that avoids prescribing the wrong drug to the wrong patients at the wrong dose.
In another embodiment the present invention relates to the use of the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention for the preparation of a diagnostic composition for diagnosing a disease. A gene encoding a functional and expressible polypeptide of the invention can be introduced into the cells which in turn produce the protein of interest. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO94/29469; WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The gene may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell.
As is evident from the above, it is preferred that in the use of the invention the nucleic acid sequence is operatively linked to regulatory elements allowing for the expression and/or targeting of the polypeptides of the invention to specific cells. Suitable gene delivery systems that can be employed in accordance with the invention may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acids to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729). Standard methods for transfecting cells with recombinant DNA are well known to those skilled in the art of molecular biology, see, e.g., WO 94/29469; see also supra. Gene therapy may be carried out by directly administering the recombinant DNA molecule or vector of the invention to a patient or by transfecting cells with the polynucleotide or vector of the invention ex vivo and infusing the transfected cells into the patient.
In light of the foregoing, in a further embodiment the present invention relates to the use of the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention for the preparation of a pharmaceutical composition for treating a disease.
In a more preferred embodiment of the use of the present invention said disease is cancer or diseases including congenital jaundice, porphyria cutanea tarda, tardive dyskinesia in schizophrenia.
The present invention also encompasses an in vitro method for identifying a single nucleotide polymorphism said method comprising the steps of
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low expression level of CYP1A2 and at least one or more further subgroup(s) do have a higher CYP1A2 expression level, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low CYP1A2 expression level with said at least one or more further subgroup(s) having a higher CYP1A2 expression level.
Furthermore, the present invention relates to an in vitro method for identifying a single nucleotide polymorphism said method comprising the steps of (a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low CYP1A2 protein level and at least one or more further subgroup(s) do have a higher CYP1 A2 protein level, and (b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low CYP1A2 protein level with said at least one or more further subgroup(s) having a higher CYP1A2 protein level.
Furthermore the present invention encompasses an in vitro method for identifying a single nucleotide polymorphism said method comprising the steps of
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low CYP1A2 activity level and at least one or more further subgroup(s) do have a higher CYP1 A2 activity level, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low CYP1A2 activity level with said at least one or more further subgroup(s) having a higher CYP1 A2 activity level.
Moreover, an in vitro method for identifying a single nucleotide polymorphism is encompassed by the present invention said method comprising the steps of
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup shows unusual effects upon administration of a drug and at least one or more further subgroup(s) do have no unusual effects, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having unusual effects upon administration of a drug with said at least one or more further subgroup(s) having no unusual effects upon administration of a drug.
Also, an in vitro method for identifying a single nucleotide polymorphism is encompassed by the present invention said method comprising the steps of (a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low plasma concentration of a drug and at least one or more further subgroup(s) do have a higher plasma concentration of a drug, and (b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low plasma concentration of a drug with said at least one or more further subgroup(s) having a higher plasma concentration of a drug.
In accordance with the foregoing, preferably said drug is a substrate of CYP1A2.
More preferably, said substrate is caffeine, theophylline, phenacetine, acetaminophen, nicotine, tacrine, imipramine, antipyrine, aminopyrine, clozapine or olanzapine.
Finally, the present invention relates to a diagnostic kit for detection of a single nucleotide polymorphism comprising the polynucleotide, the gene, the vector, the polypeptide, the antibody, the host cell, the transgenic non-human animal or the solid support of the invention.
The kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic cells and animals. The kit of the invention can be used for carrying out a method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as research tool. The parts of the kit of the invention can be packaged individually in vials or other appropriate means depending on the respective ingredient or in combination in suitable containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art. The kit may be used for methods for detecting expression of a mutant form of the polypeptides, genes or polynucleotides in accordance with any one of the above-described methods of the invention, employing, for example, immunoassay techniques such as radioimmunoassay or enzymeimmunoassay or preferably nucleic acid hybridization and/or amplification techniques such as those described herein before and in the Examples as well as pharmacokinetic studies when using non-human transgenic animals of the invention. The figures illustrate the invention:
Figure 1: Polymorphic genomic DNA sequences and the resulting changes in the protein sequence. Sequences are listed in 5' — > 3' orientation. Letters in lowercase indicate non-coding sequences, letters in uppercase indicate coding sequences. Primer regions are underlined. Variant sites are shown framed, wherein the mutated nucleotide or amino acid is depicted. Sequences are listed according to their localisation in the CYP1A2 gene (5' → 3').
Figure 2: CYP1A2 protein variants referred to in the present invention are located on the surface of the CYP protein as shown by comparative protein modelling (Guex, Electrophoresis (1997) 18: 2714-2723). A protein model of CYP1A2 was generated by using SWISS-MODEL which is a fully automated protein structure homology- modeling server, accessible via the Expasy web server (http://www.expasv.ch/swissmod/SWISS-MODEL.html) and further processing with the Deep View (Swiss-PdbViewer) software (http://www.expasy.ch/spdbv/). This alterations will change protein protein interactions and will thereby alter the activity of the CYP 1A2 enzyme.
The invention will now be described by reference to the following biological Examples which are merely illustrative and are not constructed as a limitation of the scope of the present invention.
Example 1: Isolation of genomic DNA from human blood, generation and purification of CYP1A2 gene fragments
Genomic DNA was isolated from blood samples using Qiagen blood DNA isolation kits. Oligonucleotides used in the screen were designed based on the recently determined sequence and organisation of the human CYP1A locus (Corchero, Pharmacognetics (2001), 1-6). Primer sequences and PCR fragment lengths are given in Table 2. Amplified fragments were processed through PCR purification columns (Qiagen) and sequenced on PE ABI 3700 DNA Analysers using the same primers as for PCR. The sequences were analysed for the presence of polymorphisms using the PHRED/PHRAP/POLYPHRED/CONSED software package (University of Washington, Seattle, WA, USA).
Example 2: Determination of genetic variations within the CYP1A2 locus
Sequence diversity within the CYP1A locus in African-Americans, Japanese, Chinese and Koreans was determined by PCR amplification and sequencing of 45, 50, 47 and 50 DNA samples, respectively. The PCR fragments encompass the entire protein- coding region of CYP1A2 and the non-coding exon 1 , a portion of the 3'-UTR in exon 7, as well as a portion of the 5'-flanking region.
The already known CYP1A2*1 E (cb-v-027) and CYP1A2*1B (cb-v-003) alleles were also found in the screen. The CYP1A2*1 E allele had a frequency of 5% in Japanese (i.e. similar to the 8.2% reported previously, see Tablel), 8.8% in African-Americans and Chinese samples, and 11.6% in Korean samples. The CYP1A2*1 B allele had a frequency of 77.9% in Japanese, 89.7% in African-Americans, 86.9% in Chinese and 86% in Korean samples. The allele frequency of CYP1A2*1B allele in all mentioned ethnic groups is much higher than in Caucasians (33%, see Table 1). While in Caucasians the homozygous T/T is the major variant, in Japanese, African- Americans, Chinese and Koreans the homozygous C/C is the major variant. A total of 18 new variants were detected in the screen and their allelic frequencies in the four ethnic groups ranged between 0 % and 90 % (Table 3 and Table 4). One variant is located within the 5' flanking sequence upstream the transcriptional start site of CYP1A2, one variant is located in the untranslated exon 1 , one variant is located in the 3' untranslated region of exon 7, eight variants are located in introns, whereas seven have been found in the protein-coding sequence. Among the latter ones, four variants result in amino acid substitutions of the CYP1A2 protein. The frequencies of the four protein variants which were not found in 100 analysed Caucasian samples (Chevalier, Hum Mutat 17 (2001), 355-6.) were determined in the Japanese and African-Americans samples (see Table 3) and in Chinese and Korean samples (see Table 4). The g.182C>T variant (cb-v-005, Table 3) results in a P61L amino acid exchange in exon 2 and was found only in African-Americans samples in 1 out of 35 samples. The g.1513C>A variant (cb-v-008, Table 3) leads to a S298R amino acid exchange in exon 3 and was found only in African-Americans samples in 7 out of 44 samples. The g.3482T>C variant (cb-v-014, Table 3) results in a 1401 T amino acid exchange in exon 6 and was found only in the Japanese samples in 1 out of 40 samples. The fourth g.5113C>T variant (cb-v-020, Table 4) results in a T438I aminoacid exchange in exon 7 and was found only in the Korean samples in 3 out of 43 samples.
The silent g.2045C>T variant (cb-v-011 , Table 3) has been found only in Japanese in 1 out of 50 samples; the silent g.615C>T variant (cb-v-018, Table 4) has been found only in Chinese in 1 out of 46 samples, while the silent g.1471G>A variant (cb-v-019, Table 4) has been found only in Koreans in 1 out of 46 samples.
Tablel. CYP1A2 alleles identified by others and their frequencies in the ethnical groups investigated.
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
1The first nucleotide A of the first ATG of the nucleotide sequence of CYP1 A2 protein has been taken as position +1
Table 2: Primers used to screen for polymorphisms within the CYP1A2 gene.
Figure imgf000039_0002
Ref.: Reference sequence. The positions of amplified fragments refer to GenBank sequences with AccessionAccession numbers (1) AF253322, Gl:13430063
Table 3: New polymorphisms detected in the CYP1A2 gene in Japanese and African-American ethnic groups.
o
Figure imgf000041_0001
Figure imgf000042_0001
Variants are listed according to their localisation along the gene. Polymorphism nomenclature is based on Antonarakis et al. (Antonarakis, Hum Mutat 11 (1998), 1-3) using the sequence AF253322, Gl:13430063 as genomic reference sequence wherein the A of the ATG at position 34942 is +1. Sequence context, local alignment at the polymorphic site with the major allele sequence given at the top and the variant sequence given below wherein in the variant sequence nucleotides which are identical to those at the top sequence are represented by a dot. N: number of chromosomes analysed (calculated by doubling the number of individuals). All variants were detected in the heterozygous state except for 1 homozygous individual for variant cb-v-013 and a mix of homo/heterozygous individuals for variant cb-v-012 (the major allele in Japanese and African-American ethnic groups). Variants cb-v-004 to cb-v-010 and cb-v-015 were found only in African-Americans. Variants cb-v-011 and cb-v-014 were found only in Japanese.
Table 4: New polymorphisms detected in the CYP1A2 gene in Chinese and Korean ethnic groups.
Figure imgf000043_0001
ividuals). All variants were detected in the heterozygous state except for 1 homozygous individual for variant cb-v-013 and a f homo/heterozygous individuals for variant cb-v-012 (the major allele in Chinese and Korean ethnic groups). Variants cb-v- and cb-v-021 was found only in Chinese. Variants cb-v-017, cb-v-019 and cb-v-020 were found only in Koreans.
References
1. Landi, M.T., Sinha, R., Lang, N.P. & Kadlubar, F.F. Chapter 16. Human cytochrome P4501A2. lARC Sci Pu / 148, 173-95 (1999).
2. Eaton, D.L., Gallagher, E.P., Bammler, T.K. & Kunze, K.L. Role of cytochrome P4501A2 in chemical carcinogenesis: implications for human variability in expression and enzyme activity. Pharmacogenetics 5, 259-74. (1995).
3. Catteau, A., Bechtel, Y.C., Poisson, N., Bechtel, P.R. & Bonaiti-Pellie, C. A population and family study of CYP1 A2 using caffeine urinary metabolites. Eur J Clin Pharmacol 47, 423-30 (1995).
4. Tantcheva-Poor, I., Zaigler, M., Rietbrock, S. & Fuhr, U. Estimation of cytochrome P-450 CYP1 A2 activity in 863 healthy Caucasians using a saliva- based caffeine test. Pharmacogenetics 9, 131-44. (1999).
5. Ikeya, K. et al. Human CYP1A2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1 A2 mRNA expression. Mol Endocrinol 3, 1399-408 (1989).
6. Nakajima, M. et al. Phenotyping of CYP1 A2 in Japanese population by analysis of caffeine urinary metabolites: absence of mutation prescribing the phenotype in the CYP1A2 gene. Cancer Epidemiol Biomarkers Prev 3, 413-21 (1994).
7. Yokoi, T., Sawada, M. & Kamataki, T. Polymorphic drug metabolism: studies with recombinant Chinese hamster cells and analyses in human populations. Pharmacogenetics , S65-9 (1995).
8. Kiyohara, C. Genetic polymorphism of enzymes involved in xenobiotic metabolism and the risk of colorectal cancer. J Epidemiol 10, 349-60. (2000).
9. Landi, M.T. er a/. Cytochrome P4501A2: enzyme induction and genetic control in determining 4-aminobiphenyl-hemogIobin adduct levels. Cancer Epidemiol Biomarkers Prev 5, 693-8. (1996).
10. Quattrochi, L.C. & Tukey, R.H. The human cytochrome Cyp1A2 gene contains regulatory elements responsive to 3-methylcholanthrene. Mol Pharmacol 36, 66-71 (1989).
11. Aitchison, K.J. et al. Identification of novel polymorphisms in the 5' flanking region of CYP1 A2, characterization of interethnic variability, and investigation of their functional significance. Pharmacogenetics 10, 695-704 (2000). 12. Corchero, P., Shioko Kimura, Gonzalez. Organization of the CYP1 A cluster on human chromosome 15: implications for gene regulation. Pharmacognetics 11 ,1-6(2001).
13. Devonshire, H.W. et al. The contribution of genetically determined oxidation status to inter- individual variation in phenacetin disposition. Br J Clin Pharmacol 16, 157-66. (1983).
14. Zaccaro, C. etal. Role of cytochrome P450 1A2 in bilirubin degradation Studies in Cyp1a2 (-/-) mutant mice. Biochem Pharmacol 61 , 843-9. (2001).
15. Christiansen, L. et al. Association between CYP1A2 polymorphism and susceptibility to porphyria cutanea tarda.[ln Process Citation]. Hum Genet 107, 612-4 (2000).
16. Basile, V.S. et al. A functional polymorphism of the cytochrome P450 1 A2 (CYP1A2) gene: association with tardive dyskinesia in schizophrenia. Mol Psychiatry 5, 410-7. (2000).
17. Chevalier, D. et al. Five novel natural allelic variants-951A>C, 1042G>A (D348N), 1156A>T (I386F), 1217G>A (C406Y) and 1291C>T (C431Y)-of the human CYP1A2 gene in a French Caucasian population. Hum Mutat 17, 355- 6. (2001).
18. Nakai, K. & Sakamoto, H. Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene 141 , 171-7 (1994).
19. Welfare, M.R., Aitkin, M., Bassendine, M.F. & Daly, A.K. Detailed modelling of caffeine metabolism and examination of the CYP1 A2 gene: lack of a polymorphism in CYP1A2 in Caucasians [published erratum appears in Pharmacogenetics 1999 Dec;9(6):782]. Pharmacogenetics 9, 367-75 (1999).
20. Nakajima, M. etal. Genetic polymorphism in the 5'-flanking region of human CYP1 A2 gene: effect on the CYP1 A2 inducibility in humans. J Biochem (Tokyo) 125, 803-8 (1999).
21. Chida, M. et al. Detection of three genetic polymorphisms in the 5'-flanking region and intron 1 of human CYP1 A2 in the Japanese population. Jpn J Cancer Res 90, 899-902 (1999).
22. Sachse, C, Brockmoller, J., Bauer, S. & Roots, I. Functional significance of a C->A polymorphism in intron 1 of the cytochrome P450 CYP1 A2 gene tested with caffeine. BrJ Clin Pharmacol 47, 445-9 (1999). 23. Huang, J.D., Guo, W.C, Lai, M.D., Guo, Y.L. & Lambert, G.H. Detection of a novel cytochrome P-450 1A2 polymorphism (F21 L) in Chinese. Drug Metab Dispos 27, 98-101 (1999).
24. Antonarakis, S.E. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 11 , 1-3 (1998).

Claims

1. A polynucleotide comprising a polynucleotide selected from the group consisting of:
(a) a polynucleotide having the nucleic acid sequence of any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 53, 54, 55, 57, 58, 59, 60, 61 , 63, 64, 65, 66, 67, 68, 70, 71 , 72, 74, or 75;
(b) a polynucleotide encoding a polypeptide having the amino acid sequence of any one of SEQ ID NO: 56, 62, 69, or 73;
(c) a polynucleotide capable of hybridizing to a CYP1A2 gene, wherein said polynucleotide is having at a position corresponding to position -1045, - 881 , 182, 615, 1253, 1352, 1471 , 1513, 1877, 1953, 2045, 2159, 2321 , 3482, 3606, 3614, 5113, or 5371 of the CYP1A2 gene (GenBank Accession No: AF253322, Gl:13430063 wherein position 34942 has been numbered +1), a nucleotide exchange of at least one nucleotide;
(d) a polynucleotide capable of hybridizing to a CYP1A2 gene, wherein said polynucleotide is having at a position corresponding to position -1045, 1352, 1471 , 1513, 1877 or 5371 , of the CYP1A2 gene (GenBank Accession No: AF253322, GI: 13430063 wherein position 34942 has been numbered +1) an A, at a position corresponding to position 2321 , 3482 or 3614 of the CYP1A2 gene (GenBank Accession No: AF253322, Gl:13430063 wherein position 34942 has been numbered +1) a C, at a position corresponding to position -881 , 1253, 1953, 2159 or 3606 of the CYP1A2 gene (GenBank Accession No: AF253322, Gl:13430063 wherein position 34942 has been numbered +1) a G, at a position corresponding to position 182, 615, 2045 or 5113 of the CYP1A2 gene (GenBank Accession No: AF253322, Gl:13430063 wherein position 34942 has been numbered +1) a T;
(e) a polynucleotide encoding an CYP1A2 polypeptide or a fragment thereof, wherein said polypeptide comprises an amino acid substitution at position 61 , 298, 401 or 438 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , GI: 13325061); and
(f) a polynucleotide encoding an CYP1A2 polypeptide or a fragment thereof, wherein said polypeptide comprises an amino acid substitution of. P to L at position corresponding to position 61 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061), S to R at position corresponding to position 298 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761 , Gl:13325061), I to T at position corresponding to position 401 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761, Gl:13325061) or T to I at position corresponding to position 438 of the CYP1A2 polypeptide (GenBank Accession No: NM_000761, Gl:13325061).
2. A polynucleotide of claim 1, wherein said polynucleotide is associated with cancer, congenital jaundice, porphyria cutanea tarda, or tardive dyskinesia in schizophrenia.
3. A polynucleotide of any one of claims 1 or 2 which is DNA or RNA.
4. A gene comprising the polynucleotide of any one of claims 1 or 2.
5. The gene of claim 4, wherein a nucleotide deletion, addition and/or substitution results in altered expression of the variant gene compared to the corresponding wild type gene.
6. A vector comprising a polynucleotide of any one of claims 1 to 3 or the gene of claim 4 or 5.
7. The vector of claim 6, wherein the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.
8. A host cell genetically engineered with the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5 or the vector of claim 6 or 7.
9. A method for producing a molecular variant CYP1A2 polypeptide or fragment thereof comprising
(a) culturing the host cell of claim 8; and (b) recovering said protein or fragment from the culture.
10. A method for producing cells capable of expressing a molecular variant CYP1A2 polypeptide comprising genetically engineering cells with the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5 or the vector of claim 6 or 7.
11. A polypeptide or fragment thereof encoded by the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5 or obtainable by the method of claim 9 or from cells produced by the method of claim 10.
12. An antibody which binds specifically to the polypeptide of claim 11.
13. The antibody of claim 12 which specifically recognizes an epitope containing one or more amino acid substitution(s) resulting from a nucleotide exchange as defined in claim 1 or 5.
14. The antibody of claim 12 or 13 which is monoclonal or polyclonal.
15. A transgenic non-human animal comprising at least one polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5 or the vector of claim 6 or 7.
16. The transgenic non-human animal of claim 15 which is a mouse, a rat or a zebrafish.
17. A solid support comprising one or a plurality of the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5, the vector of claim 6 or 7, the polypeptide of claim 11 , the antibody of claim 12 or 13 or the host cell of claim 8 in immobilized form.
18. The solid support of claim 17, wherein said solid support is a membrane, a glass-or polypropylene- or silicon-chip, are oligonucleotide-conjugated beads or a bead array, which is assembled on an optical filter substrate.
19. An in vitro method for identifying a single nucleotide polymorphism said method comprising the steps of:
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has no prevalence for a CYP1 A2 associated disease and at least one or more further subgroup(s) do have prevalence for a CYP1A2 associated disease; and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having no prevalence for a CYP1A2 associated disease with said at least one or more further subgroup(s) having a prevalence for a CYP1A2 associated disease.
20. A method for identifying and obtaining a pro-drug or a drug capable of modulating the activity of a molecular variant of a CYP1A2 polypeptide comprising the steps of:
(a) contacting the polypeptide of claim 11 , the solid support of claim 17 or 18, a cell expressing a molecular variant gene comprising a polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5 or the vector of claim 6 or 7 in the presence of components capable of providing a detectable signal in response to drug activity with a compound to be screened for pro-drug or drug activity; and
(b) detecting the presence or absence of a signal or increase or decrease of a signal generated from the pro-drug or the drug activity, wherein the absence, presence, increase or decrease of the signal is indicative for a putative pro-drug or drug.
21. A method for identifying and obtaining an inhibitor of the activity of a molecular variant of a CYP1 A2 polypeptide comprising the steps of:
(a) contacting the protein of claim 11 , the solid support of claim 17 or 18 or a cell expressing a molecular variant gene comprising a polynucleotide of any one of claims 1 to 3 or the gene of claim 4 or 5 or the vector of claim 6 or 7 in the presence of components capable of providing a detectable signal in response to drug activity with a compound to be screened for inhibiting activity; and (b) detecting the presence or absence of a signal or increase or decrease of a signal generated from the inhibiting activity, wherein the absence or decrease of the signal is indicative for a putative inhibitor.
22. The method of claim 20 or 21 , wherein said cell is a cell of claim 8, obtained by the method of claim 10 or can be obtained by the transgenic non-human animal of claim 15 or 16.
23. A method of identifying and obtaining a pro-drug or drug capable of modulating the activity of a molecular variant of a CYP1A2 polypeptide comprising the steps of:
(a) contacting the host cell of claim 8, the cell obtained by the method of claim 10, the polypeptide of claim 11 or the solid support of claim 17 or 18 with the first molecule known to be bound by a CYP1A2 polypeptide to form a first complex of said polypeptide and said first molecule;
(b) contacting said first complex with a compound to be screened, and
(c) measuring whether said compound displaces said first molecule from said first complex.
24. A method of identifying and obtaining an inhibitor capable of modulating the activity of a molecular variant of a CYP1A2 polypeptide or its gene product comprising the steps of:
(a) contacting the host cell of claim 8, the cell obtained by the method of claim 10, the protein of claim 11 or the solid support of claim 17 or 18 with the first molecule known to be bound by a CYP1A2 polypeptide to form a first complex of said polypeptide and said first molecule;
(b) contacting said first complex with a compound to be screened, and
(c) measuring whether said compound displaces said first molecule from said first complex.
25. The method of claim 23 or 24, wherein said measuring step comprises measuring the formation of a second complex of said polypeptide and said compound.
26. The method of any one of claims 23 to 25, wherein said measuring step comprises measuring the amount of said first molecule that is not bound to said polypeptide.
27. The method of any one of claims 23 to 26, wherein said first molecule is labeled.
28. A method for the production of a pharmaceutical composition comprising the steps of the method of any one of claims 20 to 27; and the further step of formulating the compound identified and obtained or a derivative thereof in a pharmaceutically acceptable form.
29. A method of diagnosing a disorder related to the presence of a molecular variant of a CYP1A2 gene or susceptibility to such a disorder comprising determining the presence of a polynucleotide of any one of claims 1 to 3 or the gene of claim 4 or 5 in a sample from a subject.
30. The method of claim 29 further comprising determining the presence of a polypeptide of claim 11 or the antibody of any one of claims 12 to 14.
31. A method of diagnosing a disorder related to the presence of a molecular variant of a CYP1A2 gene or susceptibility to such a disorder comprising determining the presence of a polypeptide of claim 11 or the antibody of any one of claims 12 to 14 in a sample from a subject.
32. The method of any one of claims 29 to 31 , wherein said disorder is cancer, congenital jaundice, porphyria cutanea tarda, or tardive dyskinesia in schizophrenia.
33. The method of any one of claims 29 to 32 comprising PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques, single-strand conformation polymorphism analysis (SSCA), denaturating gradient gel electrophoresis (DGGE), direct sequencing, HPLC- based techniques, invasive cleavage assay, mass spectroscopy, microarray, a rolling circle extension assay, a primer extension assay and a molecular beacon assay, hybridization techniques or immunoassays.
34. A method of detection of the polynucleotide of any one of claims 1 to 3 or the gene of claim 4 or 5 in a sample comprising the steps of
(a) contacting the solid support of claim 17 or 18 with the sample under conditions allowing interaction of the polynucleotide of claim 1 to 3 or the gene of claim 4 or 5 with the immobilized targets on a solid support and;
(b) determining the binding of said polynucleotide or said gene to said immobilized targets on a solid support.
35. An in vitro method for diagnosing a disease comprising the steps of the method of claim 34, wherein binding of said polynucleotide or gene to said immobilized targets on said solid support is indicative for the presence or the absence of said disease or a prevalence for said disease.
36. A diagnostic composition comprising the polynucleotide of any one of claims 1 to 3, the gene of claim 4 to 5, the vector of claim 6 or 7, the polypeptide of claim 11 or the antibody of any one of the claims 12 to 14.
37. A pharmaceutical composition comprising the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5, the vector of claim 6 or 7, the polypeptide of claim 11 or the antibody of any of the claims 12 to 14.
38. Use of the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5, the vector of claim 6 or 7, the polypeptide of claim 11 or the antibody of any of the claims 12 to 14 for the preparation of a diagnostic composition for
39. Use of the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5, the vector of claim 6 or 7, the polypeptide of claim 11 or the antibody of any of the claims 12 to 14 for the preparation of a pharmaceutical composition for treating a disease.
40. The use of claim 38 or 39, wherein said disease is cancer, congenital jaundice, porphyria cutanea tarda, or tardive dyskinesia in schizophrenia.
41. A method for identifying a single nucleotide polymorphism said method comprising the steps of
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low expression level of CYP1A2 and at least one or more further subgroup(s) do have a higher CYP1A2 expression level, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low CYP1A2 expression level with said at least one or more further subgroup(s) having a higher CYP1 A2 expression level.
42. A method for identifying a single nucleotide polymorphism said method comprising the steps of
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low CYP1A2 protein level and at least one or more further subgroup(s) do have a higher CYP1 A2 protein level, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low CYP1 A2 protein level with said at least one or more further subgroup(s) having a higher CYP1 A2 protein level.
43. A method for identifying a single nucleotide polymorphism said method (a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low CYP1A2 activity level and at least one or more further subgroup(s) do have a higher CYP1 A2 activity level, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low CYP1 A2 activity level with said at least one or more further subgroup(s) having a higher CYP1 A2 activity level.
44. A method for identifying a single nucleotide polymorphism said method comprising the steps of
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup shows unusual effects upon administration of a drug and at least one or more further subgroup(s) do have no unusual effects, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having unusual effects upon administration of a drug with said at least one or more further subgroup(s) having no unusual effects upon administration of a drug.
45. A method for identifying a single nucleotide polymorphism said method comprising the steps of
(a) isolating a polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5 from a plurality of subgroups of individuals, wherein one subgroup has a low plasma concentration of a drug and at least one or more further subgroup(s) do have a higher plasma concentration of a drug, and
(b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having a low plasma concentration of a drug with said at least one or more further subgroup(s) having a higher plasma concentration of a drug.
46. A method according to claim 41 to 45 wherein said drug is a substrate of CYP1A2.
47. A method according to claim 41 to 45 wherein said drug is caffeine, theophylline, phenacetine, acetaminophen, nicotine, tacrine, imipramine, antipyrine, aminopyrine, clozapine or olanzapine.
48. A diagnostic kit for detection of a single nucleotide polymorphism comprising the polynucleotide of any one of claims 1 to 3, the gene of claim 4 or 5, the vector of claim 6 or 7, the polypeptide of claim 11 , the antibody of any of the claims 12 to 14, the host cell of claim 8, the transgenic non-human animal of claim 15 or 16 or the solid support of claim 17 or 18.
PCT/EP2002/008893 2001-08-08 2002-08-08 Polymorphisms in the human gene for cyp1a2 and their use in diagnostic and therapeutic applications WO2003014387A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP01118770.5 2001-08-08
EP01118770 2001-08-08

Publications (2)

Publication Number Publication Date
WO2003014387A2 true WO2003014387A2 (en) 2003-02-20
WO2003014387A3 WO2003014387A3 (en) 2003-09-25

Family

ID=8178236

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/008893 WO2003014387A2 (en) 2001-08-08 2002-08-08 Polymorphisms in the human gene for cyp1a2 and their use in diagnostic and therapeutic applications

Country Status (1)

Country Link
WO (1) WO2003014387A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013113252A1 (en) * 2012-02-02 2013-08-08 北京大学 Method for regulating expression level of cyp gene and use thereof
CN106290671A (en) * 2016-08-25 2017-01-04 广东电网有限责任公司电力科学研究院 The detection method of aminophenazone in a kind of transformer oil

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BASILE V S ET AL: "A functional polymorphism of the cytochrome P450 1A2 (CYP1A2) gene: Association with tardive dyskinesia in schizophrenia." MOLECULAR PSYCHIATRY, vol. 5, no. 4, July 2000 (2000-07), pages 410-417, XP002228674 ISSN: 1359-4184 cited in the application *
CHRISTIANSEN L ET AL: "Association between CYP1A2 polymorphism and susceptibility to porphyria cutanea tarda." HUMAN GENETICS, vol. 107, no. 6, December 2000 (2000-12), pages 612-614, XP002228675 ISSN: 0340-6717 cited in the application *
DATABASE GENBANK [Online] 11 May 1998 (1998-05-11) Database accession no. AA959044 XP002228676 *
JOSEPHY P DAVID ET AL: "Activation of MelQ (2-amino-3,4-dimethylimidazo-(4,5-f)quinol ine) by sequence variants of recombinant human cytochrome P450 1A2." ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, vol. 35, no. 4, 2000, pages 328-335, XP009004549 ISSN: 0893-6692 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013113252A1 (en) * 2012-02-02 2013-08-08 北京大学 Method for regulating expression level of cyp gene and use thereof
CN106290671A (en) * 2016-08-25 2017-01-04 广东电网有限责任公司电力科学研究院 The detection method of aminophenazone in a kind of transformer oil

Also Published As

Publication number Publication date
WO2003014387A3 (en) 2003-09-25

Similar Documents

Publication Publication Date Title
NO20100798L (en) Polymorphisms in the human MDR-1 gene and their diagnostic and therapeutic use
US20110131671A1 (en) Polymorphisms in the human gene for cytochrome p450 polypeptide 2c8 and their use in diagnostic and therapeutic applications
ES2288173T3 (en) IDENTIFICATION OF THE GENETIC DETERMINANTS OF THE EXPRESSION OF CYP3A5 POLYMORPH.
US20150018229A1 (en) Polymorphisms in the human gene for the multidrug resistance-associated protein 1 (mrp-1) and their use in diagnostic and therapeutic applications
EP1088900A1 (en) Polymorphisms in the human CYP3A4, CYP3A7 and hPXR genes and their use in diagnostic and therapeutic applications
US20100009366A1 (en) Polymorphisms in the human genes for oct1 and their use in diagnostic and therapeutic applications
WO2003014387A2 (en) Polymorphisms in the human gene for cyp1a2 and their use in diagnostic and therapeutic applications
US20060078879A1 (en) Polymorphisms in the human gene for tpmt and their use in diagnostic and therapeutic applications
WO2003097873A2 (en) Polymorphisms in the human gene for htr3b and their use in diagnostic and therapeutic applications
WO2002024747A2 (en) Polymorphisms in human genes of cardiovascular regulators and their use in diagnostic and therapeutic applications
Brinkmann Penger et al.
WO2003104381A2 (en) Methods

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VC VN YU ZA ZM

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP