WO2001029176A2 - Drug target isogenes: polymorphisms in the cholinergic receptor, muscarinic 3 gene - Google Patents
Drug target isogenes: polymorphisms in the cholinergic receptor, muscarinic 3 gene Download PDFInfo
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- WO2001029176A2 WO2001029176A2 PCT/US2000/028247 US0028247W WO0129176A2 WO 2001029176 A2 WO2001029176 A2 WO 2001029176A2 US 0028247 W US0028247 W US 0028247W WO 0129176 A2 WO0129176 A2 WO 0129176A2
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- 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
- This invention relates to variation in genes that encode pharmaceutically important proteins.
- this invention provides genetic variants of the human cholinergic receptor, muscarinic 3 (CHRM3) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
- CHRM3 human cholinergic receptor, muscarinic 3
- a target protein currently used to screen drugs typically is expressed by a gene cloned from an individual who was arbitrarily selected.
- the nucleotide sequence of a particular gene may vary tremendously among individuals.
- Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a target protein may be manifested as significant variation in expression of or in the structure and/or function of the protein. Such alterations may explain the relatively high degree of uncertainty inherent in treatment of individuals with drugs whose design is based upon a single representative example of the target.
- haplotype The organization of single nucleotide variations (polymorphisms) in the primary sequence of a gene into one of the limited number of combinations that exist as units of inheritance is termed a haplotype. Each haplotype therefore contains significantly more information than individual unorganized polymorphisms. Haplotypes provide an accurate measurement of the genomic variation in the two chromosomes of an individual and can be used to identify and distinguish between isoforms of genes coding for drug targets. It is well-established that many diseases are associated with specific variations in gene sequences.
- CHRM3 muscarinic 3
- HM3 muscarinic cholinergic receptor 3
- mAChR muscarinic acetylcholine receptors
- CHRM3 plays a key role in salivary secretion, pupillary constriction, and bladder detrusor contractions (Matsui et al. Proc. Natl. Acad. Sci. 15; 97(17):9579-84, 2000).
- CHRM3 antagonists have also been implicated in the treatment of disorders associated with smooth muscle contractility or tone. These disorders include irritable bowel syndrome, chronic obstructive airway disease, and urinary incontinence (Walhs. Life Sci. 1995;56(1 1-12):861-8) Specifically, the treatment of obstructive airway disease may be improved by antimusca ⁇ nic agents, which selectively block CHRM3, but do not inhibit prejunctional cholinergic autoreceptors that limit release of acetylcholme (Alabaster. Life Sci 1997,60(13-14)- 1053-60). Furthermore, CHRM3 may also be involved m central respiratory d ⁇ ve and control (Richardson et al.
- the cholinergic receptor, musca ⁇ nic 3 gene is located on chromosome Iq41-q44 and contains 1 exon that encodes a 596 a ino acid protein Reference sequences for the CHRM3 gene (GenBank Accession No U29589 1, SEQ ID NO 1), coding sequence, and protein are shown m Figures 1, 2 and 3, respectively
- polymorphic sites correspond to the following nucleotide positions in the indicated GenBank Accession Number: 53 (PS1), 394 (PS2), 1387 (PS3) and 1493 (PS4) in U29589 1
- PS1 GenBank Accession Number
- PS2 GenBank Accession Number
- PS3 1387
- PS4 1493
- the polymorphisms at these sites are thymine or cytosme at PS 1 , guanine or ade ne at PS2, cytosine or adenine at PS3 and thymine or cytosine at PS4.
- the inventors have determined the identity of the alternative nucleotides present at these sites in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups Af ⁇ can descent, Asian, Caucasian and Hispanic/Latino. It is believed that CHRM3-encod ⁇ ng polynucleotides containing one or more of the novel polymorphic sites reported herein will be useful in studying the expression and biological function of CHRM3, as well as in developing drugs targetmg this protein. In addition, information on the combinations of polymorphisms in the CHRM3 gene may have diagnostic and forensic applications.
- the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic va ⁇ ant of a reference sequence for the CHRM3 gene or a fragment thereof.
- the reference sequence comprises SEQ ID NO: 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of cytosine at PSl, adenine at PS2, adenine at PS3 and cytosine at PS4.
- a particularly preferred polymorphic variant is a naturally- occurring isoform (also referred to herein as an "isogene") of the CHRM3 gene.
- a CHRM3 isogene of the invention comprises thymine or cytosine at PSl, guanine or adenine at PS2, cytosine or adenine at PS3 and thymine or cytosine at PS4.
- the invention also provides a collection of CHRM3 isogenes, referred to herein as a CHRM3 genome anthology.
- a CHRM3 isogene may be defined by the combination and order of these polymorphisms in the isogene, which is referred to herein as a CHRM3 haplotype.
- the invention also provides data on the number of different CHRM3 haplotypes found in the above four population groups. This haplotype data is useful in methods for deriving a CHRM3 haplotype from an individual's genotype for the CHRM3 gene and for determining an association between a CHRM3 haplotype and a particular trait.
- the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a CHRM3 cDNA or a fragment thereof.
- the reference sequence comprises SEQ ID NO:2 (Fig. 2) and the polymorphic cDNA comprises at least one polymorphism selected from the group consisting of adenine at a position corresponding to nucleotide 193, adenine at a position corresponding to nucleotide 1 186 and cytosine at a position corresponding to nucleotide 1292.
- Polynucleotides complementary to these CHRM3 genomic and cDNA variants are also provided by the invention.
- the invention provides a recombinant expression vector comprising one of the polymorphic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector.
- the recombinant vector and host cell may be used to express CHRM3 for protein structure analysis and drug binding studies.
- the invention provides a polypeptide comprising a polymorphic variant of a reference amino acid sequence for the CHRM3 protein.
- the reference amino acid sequence comprises SEQ ID NO:3 (Fig.
- the polymorphic variant comprises at least one variant amino acid selected from the group consisting of isoleucine at a position corresponding to amino acid position 65, methionine at a position corresponding to amino acid position 396, and proline at a position corresponding to amino acid position 431.
- a polymorphic variant of CHRM3 is useful in studying the effect of the variation on the biological activity of CHRM3 as well as on the binding affinity of candidate drugs targeting CHRM3 for the treatment of Alzheimer's disease, Sjogren's syndrome, disorders associated with smooth muscle contractility and sudden infant death syndrome.
- the present invention also provides antibodies that recognize and bind to the above polymorphic CHRM3 protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.
- the invention provides methods, compositions, and kits for haplotyping and/or genotyping the CHRM3 gene in an individual.
- the methods involve identifying the nucleotide or nucleotide pair present at one or more polymorphic sites selected from PSl, PS2, PS3, and PS4 in one or both copies of the CHRM3 gene from the individual.
- the compositions contain oligonucleotide probes and primers designed to specifically hybridize to one or more target regions containing, or that are adjacent to, a polymorphic site.
- the methods and compositions for establishing the genotype or haplotype of an individual at the novel polymorphic sites described herein are useful for studying population diversity, anthropological lineage, the significance of diversity and lineage at the phenotypic level, paternity testing, forensic applications, and for identifying associations between the CHRM3 genetic variation and a trait such as level of drug response or susceptibility to disease.
- the invention provides a method for identifying an association between a genotype or haplotype and a trait.
- the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
- Such methods have applicability in developing diagnostic tests and therapeutic treatments for Alzheimer's disease, Sjogren's syndrome, disorders associated with smooth muscle contractility and sudden infant death syndrome.
- the present invention also provides nonhuman transgenic animals comprising one of the CHRM3 genomic polymorphic variants described herein and methods for producing such animals.
- the transgenic animals are useful for studying expression of the CHRM3 isogenes in vivo, for in vivo screening and testing of drugs targeted against CHRM3 protein, and for testing the efficacy of therapeutic agents and compounds for Alzheimer's disease, Sjogren's syndrome, disorders associated with smooth muscle contractility and sudden infant death syndrome in a biological system.
- the present invention also provides a computer system for storing and displaying polymo ⁇ hism data determined for the CHRM3 gene.
- the computer system comprises a computer processing unit; a display; and a database containing the polymo ⁇ hism data.
- the polymo ⁇ hism data includes the polymo ⁇ hisms, the genotypes and the haplotypes identified for the CHRM3 gene in a reference population.
- the computer system is capable of producing a display showing CHRM3 haplotypes organized according to their evolutionary relationships.
- Figure 1 illustrates a reference sequence for the CHRM3 gene (Genbank Version Number U29589.1; contiguous lines; SEQ ID NO: l), with the start and stop positions of each region of coding sequence indicated below the sequence by the numbers within the brackets and the polymo ⁇ hic sites and polymo ⁇ hisms identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
- Figure 2 illustrates a reference sequence for the CHRM3 coding sequence (contiguous lines; SEQ ID NO:2), with the polymo ⁇ hic sites and polymo ⁇ hisms identified by Applicants in a reference population indicated by the va ⁇ ant nucleotide positioned below the polymo ⁇ hic site in the sequence
- Figure 3 illustrates a reference sequence for the CHRM3 protem (contiguous lines; SEQ ID NO:3), with the va ⁇ ant ammo acids caused by the polymo ⁇ hisms of Fig. 2 positioned below the polymo ⁇ hic site in the sequence.
- the present invention is based on the discovery of novel va ⁇ ants of the CHRM3 gene.
- the inventors herein discovered 4 novel polymo ⁇ hic sites by characte ⁇ zing the CHRM3 gene found in genomic DNAs isolated from an Index Repository that contains immortalized cell lines from one chimpanzee and 93 human individuals.
- the human individuals included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups- Caucasian (22 individuals), Af ⁇ can descent (20 individuals) Asian (20 individuals) Hispanic/Latino (17 individuals) To the extent possible, the members of this reference population were organized into population subgroups by the self-identified ethnogeographic origin of their four grandparents as shown in Table 1 below.
- the Index Repository contains three unrelated indigenous Ame ⁇ can Indians (one from each of North, Central and South Ame ⁇ ca), one three-generation Caucasian family (from the CEPH Utah cohort) and one two-generation African-American family.
- the CHRM3 genotypes identified in the Index Repository and the methodology described in the Examples below also determined the haplotypes found on each chromosome for most human members of this repository.
- the CHRM3 genotypes and haplotypes found in the repository include those shown in Tables 4 and 5, respectively.
- the polymo ⁇ hism and haplotype data disclosed herein are useful for studying population diversity, anthropological lineage, the significance of diversity and lineage at the phenotypic level, paternity testing, forensic applications, and for identifying associations between the CHRM3 genetic variation and a trait such as level of drug response or susceptibility to disease.
- the following terms shall be defined as follows unless otherwise indicated:
- Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.
- Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.
- Genotype An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at one or more polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in an individual.
- genotype includes a full-genotype and/or a sub-genotype as described below.
- Sub-genotype The unphased 5 ' to 3 ' sequence of nucleotides seen at a subset of the known polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in a single individual.
- Genotyping A process for determining a genotype of an individual.
- Haplotype - A 5' to 3' sequence of nucleotides found at one or more polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
- haplotype includes a full- haplotype and/or a sub-haplotype as described below.
- Full-haplotype The 5 ' to 3 ' sequence of nucleotides found at all known polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
- Sub-haplotype The 5 ' to 3 ' sequence of nucleotides seen at a subset of the known polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
- Haplotype pair The two haplotypes found for a locus in a single individual.
- Haplotyping A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
- Haplotype data Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.
- Isoform - A particular form of a gene, mRNA, cDNA or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
- Isogene - One of the isoforms of a gene found in a population.
- An isogene contains all of the polymo ⁇ hisms present in the particular isoform of the gene.
- Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.
- Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
- Naturally-occurring A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.
- Nucleotide pair The nucleotides found at a polymo ⁇ hic site on the two copies of a chromosome from an individual.
- phased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, phased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is known.
- Polymorphic site (PS) - A position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.
- Polymorphism The sequence variation observed in an individual at a polymo ⁇ hic site.
- Polymo ⁇ hisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
- Polymorphism data Information concerning one or more of the following for a specific gene: location of polymo ⁇ hic sites; sequence variation at those sites; frequency of polymo ⁇ hisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.
- Polymorphism Database A collection of polymo ⁇ hism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
- Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.
- Reference Population A group of individuals sharing a common ethnogeographic origin.
- Reference Population A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population.
- the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
- SNP Single Nucleotide Polymorphism
- Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
- Treatment A stimulus administered internally or externally to a subject.
- Unphased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is not known.
- the inventors herein have discovered 4 novel polymo ⁇ hic sites in the CHRM3 gene.
- the polymo ⁇ hic sites identified by the inventors are referred to as PS 1-4 to designate the order in which they are located in the gene (see Table 3 below).
- the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the CHRM3 gene or a fragment of the gene which contains at least one of the novel polymo ⁇ hic sites described herein.
- nucleotide sequence of a variant CHRM3 gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymo ⁇ hic sites PSl, PS2, PS3, and PS4.
- nucleotide sequence of a variant fragment of the CHRM3 gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymo ⁇ hic sites described herein.
- the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence (or other reported CHRM3 sequences) or to portions of the reference sequence (or other reported CHRM3 sequences), except for genotyping oligonucleotides as described below.
- the location of a polymo ⁇ hism in a variant gene or fragment is identified by aligning its sequence against SEQ ID NO: l.
- the polymo ⁇ hism is selected from the group consisting of cytosine at PSl, adenine at PS2, adenine at PS3 and cytosine at PS4.
- the polymo ⁇ hic variant comprises a naturally-occurring isogene of the CHRM3 gene which is defined by any one of haplotypes 1-3 shown in Table 5 below.
- Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the CHRM3 gene from a human genomic library.
- the clone may be sequenced to determine the identity of the nucleotides at the polymo ⁇ hic sites desc ⁇ bed herein.
- Any particular va ⁇ ant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art CHRM3 isogenes may be isolated using any method that allows separation of the two "copies" of the CHRM3 gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles.
- Separation methods include targeted in vivo cloning (TIVC) in yeast as desc ⁇ bed m WO 98/01573, U.S. Patent No 5,866,404, and U.S. Patent No. 5,972,614.
- Another method which is desc ⁇ bed in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with p ⁇ mer extension and exonuclease degradation to generate hemizygous DNA targets.
- Yet other methods are single molecule dilution (SMD) as desc ⁇ bed in Ruano et al., Proc. Natl. Acad. Sci.
- CHRM3 genome anthologies are collections of CHRM3 isogenes found in a given population
- the population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same sex population
- a CHRM3 genome anthology may compnse individual CHRM3 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like.
- two or more groups of the CHRM3 isogenes m the anthology may be stored in separate containers.
- a preferred CHRM3 genome anthology of the invention comp ⁇ ses a set of isogenes defined by the haplotypes shown in Table 5 below
- An isolated polynucleotide containing a polymo ⁇ hic va ⁇ ant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded CHRM3 protein in a prokaryotic or a eukaryotic host cell.
- expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters de ⁇ ved from vaccinia virus, adenovirus, retroviruses, or SV40
- Other regulatory elements include, but are not limited to, approp ⁇ ate leader sequences, termination codons, polyadenylation signals, and other sequences required for the approp ⁇ ate transc ⁇ ption and subsequent translation of the nucleic acid sequence in a given host cell.
- the correct combinations of expression regulatory elements will depend on the host system used.
- the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell.
- Such elements include, but are not limited to, o ⁇ gins of replication and selectable markers.
- Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons. New York, New York).
- Host cells which may be used to express the variant CHRM3 sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
- the recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York).
- eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used.
- Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, he ⁇ es virus vectors, and baculovirus transfer vectors.
- Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282: 1145-1 147). Particularly preferred host cells are mammalian cells.
- polymo ⁇ hic variants of the CHRM3 gene will produce CHRM3 mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules.
- These mRNAs can be used for the preparation of a CHRM3 cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the CHRM3 reference coding sequence shown in Figure 2.
- the invention also provides CHRM3 mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
- polymo ⁇ hisms selected from the group consisting of adenine at a position corresponding to nucleotide 193, adenine at a position corresponding to nucleotide 1 186 and cytosine at a position corresponding to nucleotide 1292. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain the novel polymo ⁇ hisms described herein.
- the invention specifically excludes polynucleotides identical to previously identified and characterized CHRM3 cDNAs and fragments thereof. Polynucleotides comprising a variant RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
- Polymo ⁇ hic variants of fragments according to the invention comprise at least one novel polymo ⁇ hism identified herein and have a length of at least 10 nucleotides and may range up to the full length of the gene.
- such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
- nucleic acid molecules containing the CHRM3 gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the co ⁇ esponding site on the complementary antisense strand.
- reference may be made to the same polymo ⁇ hic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymo ⁇ hic site.
- the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the CHRM3 genomic variants described herein.
- Polynucleotides comprising a polymo ⁇ hic gene variant or fragment may be useful for therapeutic pu ⁇ oses.
- an expression vector encoding the isoform may be administered to the patient.
- the patient may be one who lacks the CHRM3 isogene encoding that isoform or may already have at least one copy of that isogene.
- CHRM3 isogene In other situations, it may be desirable to decrease or block expression of a particular CHRM3 isogene.
- Expression of a CHRM3 isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA for the isogene.
- oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3 ' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred.
- inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
- Antisense oligonucleotides may also be designed to block translation of CHRM3 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of CHRM3 mRNA transcribed from a particular isogene.
- the oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo.
- the oligonucleotides may be formulated as a pharmaceutical composition for administration to the patient.
- Oligoribonucleotides and or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life.
- Possible modifications include, but are not limited to phosphorothioate or 2 ' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
- the invention also provides an isolated polypeptide comprising a polymo ⁇ hic variant of the reference CHRM3 amino acid sequence shown in Figure 3.
- the location of a variant amino acid in a CHRM3 polypeptide or fragment of the invention is identified by aligning its sequence against SEQ ID NO:3 (Fig. 3).
- a CHRM3 protein variant of the invention comprises an amino acid sequence identical to SEQ ID NO: 3 except for having one or more variant amino acids selected from the group consisting of isoleucine at a position corresponding to amino acid position 65, methionine at a position corresponding to amino acid position 396, and proline at a position corresponding to amino acid position 431.
- CHRM3 protein variants included within the invention comprise all amino acid sequences based on SEQ ID NO: 3 and having the combination of amino acid variations described in Table 2 below.
- a CHRM3 protein variant of the invention is encoded by an isogene defined by one of the observed haplotypes shown in Table 5.
- the invention also includes CHRM3 peptide variants, which are any fragments of a CHRM3 protein variant that contains one or more of the amino acid variations shown in Table 2.
- a CHRM3 peptide variant is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long.
- Such CHRM3 peptide variants may be useful as antigens to generate antibodies specific for one of the above CHRM3 isoforms.
- the CHRM3 peptide variants may be useful in drug screening assays.
- a CHRM3 variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing one of the variant CHRM3 genomic and cDNA sequences as described above.
- the CHRM3 protein variant may be isolated from a biological sample of an individual having a CHRM3 isogene which encodes the variant protein. Where the sample contains two different CHRM3 isoforms (i.e., the individual has different CHRM3 isogenes), a particular CHRM3 isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular CHRM3 isoform but does not bind to the other CHRM3 isoform.
- CHRM3 protein may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isoform of the CHRM3 protein as discussed further below.
- CHRM3 variant proteins can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Current Protocols in Molecular Biology John Wiley and Sons, New York, New York). In the case of immunoaffinity chromatography, antibodies specific for a particular polymo ⁇ hic variant may be used.
- a polymo ⁇ hic variant CHRM3 gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric CHRM3 protein.
- the non-CHRM3 portion of the chimeric protein may be recognized by a commercially available antibody.
- the chimeric protein may also be engineered to contain a cleavage site located between the CHRM3 and non- CHRM3 portions so that the CHRM3 protein may be cleaved and pu ⁇ fied away from the non-CHRM3 portion.
- An additional embodiment of the invention relates to using a novel CHRM3 protein isoform in any of a va ⁇ ety of drug screening assays.
- Such screening assays may be performed to identify agents that bind specifically to all known CHRM3 protein isoforms or to only a subset of one or more of these isoforms.
- the agents may be from chemical compound hbra ⁇ es, peptide hbra ⁇ es and the like.
- the CHRM3 protein or peptide va ⁇ ant may be free in solution or affixed to a solid support
- high throughput screening of compounds for binding to a CHRM3 va ⁇ ant may be accomplished using the method desc ⁇ bed in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pms or some other surface, contacted with the CHRM3 prote ⁇ n(s) of interest and then washed Bound CHRM3 protem(s) are then detected using methods well-known in the art.
- a novel CHRM3 protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the CHRM3 protein
- the invention provides antibodies specific for and immunoreactive with one or more of the novel CHRM3 va ⁇ ant proteins desc ⁇ bed herein
- the antibodies may be either monoclonal or polyclonal in o ⁇ gin
- the CHRM3 protein or peptide va ⁇ ant used to generate the antibodies may be from natural or recombinant sources or produced by chemical synthesis using synthesis techniques known in the art If the CHRM3 protein va ⁇ ant is of insufficient size to be antigenic, it may be conjugated, complexed, or otherwise covalently linked to a earner molecule to enhance the antigemcity of the peptide Examples of earner molecules, include, but are not limited to, albumins (e g , human, bovine, fish, ovine), and keyhole limpet hemocyamn (Basic and Clinical Immunology, 1991 , Eds D P Stites, and A
- Antibodies specific for and immunoreactive with one of the novel CHRM3 protein isoform desc ⁇ bed herein may be used to immunoprecipitate the CHRM3 protein va ⁇ ant from solution as well as react with CHRM3 protein isoforms on Western or lmmunoblots of polyacrylamide gels on membrane supports or substrates.
- the antibodies will detect CHRM3 protein isoforms in paraffin or frozen tissue sections, or m cells which have been fixed or unfixed and prepared on slides, covershps, or the like, for use m immunocytochemical, immunohistochemical, and immunofluorescence techniques.
- an antibody specifically immunoreactive with one of the novel CHRM3 protem va ⁇ ants descnbed herein is used in immunoassays to detect this va ⁇ ant m biological samples.
- an antibody of the present invention is contacted with a biological sample and the formation of a complex between the CHRM3 protein variant and the antibody is detected.
- suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J.
- Neoman Stockton Press, New York, New York; Current Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York).
- Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and Campbell et al., 1984, Methods in Immunology, W.A. Benjamin, Inc.).
- Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g., Principles and Practice of Immunoassay, 1991 , Eds. Christopher P. Price and David J.
- Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Current Protocols in Molecular Biology, supra.
- Exemplary antibody molecules for use in the detection and therapy methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site.
- Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam).
- the antibodies or antigen binding fragments thereof may also be produced by genetic engineering.
- the technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication number WO 901443, WO 901443 and WO 9014424 and in Huse et al., 1989, Science, 246: 1275-1281.
- the antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci. 86; 10029).
- Effect(s) of the polymo ⁇ hisms identified herein on expression of CHRM3 may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the CHRM3 gene.
- expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into CHRM3 protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
- the desired CHRM3 isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
- the CHRM3 isogene is introduced into a cell in such a way that it recombmes with the endogenous CHRM3 gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired CHRM3 gene polymo ⁇ hism.
- Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known m the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
- Examples of cells into which the CHRM3 isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and p ⁇ mary or culture cells of the relevant tissue type, i.e., they express the CHRM3 isogene. Such recombinant cells can be used to compare the biological activities of the different protein va ⁇ ants.
- Recombinant nonhuman organisms, 1 e., transgenic animals, expressing a va ⁇ ant CHRM3 gene are prepared using standard procedures known in the art
- a construct compnsing the va ⁇ ant gene is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage.
- Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art.
- One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harbo ⁇ ng the insulated gene(s) as a transgene, see e.g., U.S. Patent No.
- Another method involves directly injecting a transgene into the embryo.
- a third method involves the use of embryonic stem cells Examples of animals into which the CHRM3 isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman p ⁇ mates (see "The Introduction of Foreign Genes into Mice” and the cited references therein, In. Recombinant DNA, Eds. J D. Watson, M. Gilman, J. Witkowski, and M. Zoller, W.H. Freeman and Company, New York, pages 254-272).
- Transgenic animals stably expressing a human CHRM3 isogene and producing human CHRM3 protein can be used as biological models for studying diseases related to abnormal CHRM3 expression and/or activity, and for screening and assaying va ⁇ ous candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
- An additional embodiment of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel CHRM3 isogene descnbed herein.
- the pharmaceutical composition may comp ⁇ se any of the following active ingredients: a polynucleotide compnsing one of these novel CHRM3 isogenes; an antisense oligonucleotide directed against one of the novel CHRM3 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel CHRM3 isogene desc ⁇ bed herein.
- the composition contains the active ingredient in a therapeutically effective amount.
- therapeutically effective amount is meant that one or more of the symptoms relating to disorders affected by expression or function of a novel CHRM3 isogene is reduced and/or eliminated.
- composition also comp ⁇ ses a pharmaceutically acceptable earner, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
- a pharmaceutically acceptable earner examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
- Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
- the pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound.
- Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, mtra-arte ⁇ al, intramedullary, intrathecal, intraventncular, mtradermal, transdermal, subcutaneous, intrape ⁇ toneal, intranasal, enteral, topical, subhngual, or rectal. Further details on techniques for formulation and administration may be found m the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
- the dose can be estimated initially either in cell culture assays or in animal models
- the animal model may also be used to determine the approp ⁇ ate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- the exact dosage will be determined by the practitioner, in light of factors relating to the patient requi ⁇ ng treatment, including but not limited to seventy of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
- the invention also provides compositions and methods for detecting the novel CHRM3 polymo ⁇ hisms identified herein
- compositions compnse at least one CHRM3 genotyping oligonucleotide.
- a CHRM3 genotyping oligonucleotide is a probe or p ⁇ mer capable of hyb ⁇ dizing to a target region that is located close to, or that contains, one of the novel polymo ⁇ hic sites descnbed herein.
- the term "oligonucleotide” refers to a polynucleotide molecule having less than about 100 nucleotides.
- a preferred oligonucleotide of the invention is 10 to 35 nucleotides long.
- the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides m length.
- the oligonucleotide may be comp ⁇ sed of any phosphorylation state of nbonucleotides, deoxy ⁇ bonucleotides, and acyclic nucleotide denvatives, and other functionally equivalent denvatives.
- oligonucleotides may have a phosphate-free backbone, which may be comp ⁇ sed of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R.
- Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
- the oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
- Genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a CHRM3 polynucleotide, i.e., a CHRM3 isogene.
- specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with a non-target region or a non-CHRM3 polynucleotide under the same hybridizing conditions.
- the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
- the skilled artisan can readily design and test oligonucleotide probes and primers suitable for detecting polymo ⁇ hisms in the CHRM3 gene using the polymo ⁇ hism information provided herein in conjunction with the known sequence information for the CHRM3 gene and routine techniques.
- a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect” or “complete” complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the conesponding position of the other molecule.
- a nucleic acid molecule is "substantially complementary” to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D.
- an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary to the target region.
- non-complementary nucleotides may be interspersed into the oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
- Preferred genotyping oligonucleotides of the invention are allele-specific oligonucleotides.
- ASO allele-specific oligonucleotide
- allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps.
- Allele-specific oligonucleotide probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 lh or 8 th position in a 15 mer, the 8 th or 9 th position in a 16mer, the 10 lh or 1 1 th position in a 20 mer).
- a preferred ASO probe for detecting CHRM3 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:
- ATGAACTTGATGTTT (SEQ ID NO: :4) and its complement
- ATGAACTCGATGTTT (SEQ ID NO: :5) and its complement
- ATCCAGCTAGAGTCA (SEQ ID NO: ;10) and its complement
- ATCCAGCCAGAGTCA (SEQ ID NO: :11) and its complement .
- An allele-specific oligonucleotide primer of the invention has a 3 ' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present. Allele-specific oligonucleotide primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
- a prefe ⁇ ed ASO primer for detecting CHRM3 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:
- genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymo ⁇ hic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymo ⁇ hisms described herein and therefore such genotyping oligonucleotides are referred to herein as "primer-extension oligonucleotides".
- the 3 '-terminus of a primer-extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymo ⁇ hic site.
- a particularly preferred oligonucleotide primer for detecting CHRM3 gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
- a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymo ⁇ hic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymo ⁇ hic site.
- CHRM3 genotyping oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized genotyping oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized CHRM3 genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymo ⁇ hisms in multiple genes at the same time.
- the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers.
- the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
- the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
- CHRM3 genotype and “CHRM3 haplotype” mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymo ⁇ hic sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymo ⁇ hic sites in the CHRM3 gene.
- the additional polymo ⁇ hic sites may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
- One embodiment of the genotyping method involves isolating from the individual a nucleic acid mixture compnsing the two copies of the CHRM3 gene, or a fragment thereof, that are present m the individual, and determining the identity of the nucleotide pair at one or more of the polymo ⁇ hic sites selected from PSl, PS2, PS3, and PS4 m the two copies to assign a CHRM3 genotype to the individual.
- the two "copies" of a gene in an individual may be the same allele or may be different alleles.
- the genotyping method comp ⁇ ses determining the identity of the nucleotide pair at each of PS 1-4
- the nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample.
- tissue samples include whole blood, semen, saliva, tears, unne, fecal mate ⁇ al, sweat, buccal, skin and hair.
- the nucleic acid mixture may be compnsed of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from an organ m which the CHRM3 gene is expressed Furthermore it will be understood by the skilled artisan that mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in mtrons or in 5 and 3' nontransc ⁇ bed regions If a CHRM3 gene fragment is isolated, it must contain the polymo ⁇ hic s ⁇ te(s) to be genotyped
- One embodiment of the haplotyping method compnses isolating from the individual a nucleic acid molecule containing only one of the two copies of the CHRM3 gene, or a fragment thereof, that is present m the individual and determining in that copy the identity of the nucleotide at one or more of the polymo ⁇ hic sites PSl, PS2, PS3, and PS4 in that copy to assign a CHRM3 haplotype to the individual.
- the nucleic acid may be isolated using any method capable of separating the two copies of the CHRM3 gene or fragment such as one of the methods desc ⁇ bed above for prepa ⁇ ng CHRM3 isogenes, with targeted in vivo cloning being the prefened approach.
- any individual clone will only provide haplotype information on one of the two CHRM3 gene copies present in an individual If haplotype information is desired for the individual's other copy, additional CHRM3 clones will need to be examined Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the CHRM3 gene in an individual
- the nucleotide at each of PS 1-4 is identified
- a CHRM3 haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more of the polymo ⁇ hic sites selected from PSl, PS2, PS3, and PS4 in each copy of the CHRM3 gene that is present m the individual.
- the haplotyping method comp ⁇ ses identifying the phased sequence of nucleotides at each of PS 1-4 in each copy of the CHRM3 gene
- the identifying step is preferably performed with each copy of the gene being placed in separate containers.
- the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be possible in some cases to perform the method in the same container.
- first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymo ⁇ hic s ⁇ te(s), then detecting a combination of the first and third dyes would identify the polymo ⁇ hism m the first gene copy while detecting a combination of the second and third dyes would identify the polymo ⁇ hism in the second gene copy.
- the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic s ⁇ te(s) may be determined by amplifying a target reg ⁇ on(s) containing the polymo ⁇ hic s ⁇ te(s) directly from one or both copies of the CHRM3 gene, or fragment thereof, and the sequence of the amplified reg ⁇ on(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymo ⁇ hic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
- the polymo ⁇ hism may be identified directly, known as positive-type identification, or by inference, refe ⁇ ed to as negative-type identification.
- a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site Alternatively, the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guamne).
- the identity of the allele(s) present at any of the novel polymo ⁇ hic sites descnbed herein may be indirectly determined by genotyping a polymo ⁇ hic site not disclosed herein that is in linkage disequihb ⁇ um with the polymo ⁇ hic site that is of interest. Two sites are said to be m linkage disequilibrium if the presence of a particular va ⁇ ant at one site enhances the predictability ot another vanant at the second site (Stevens, JC 1999, Mol Diag.
- Polymo ⁇ hic sites in linkage disequihb ⁇ um with the presently disclosed polymo ⁇ hic sites may be located in regions of the gene or in other genomic regions not examined herein Genotyping of a polymo ⁇ hic site in linkage disequihbnum with the novel polymo ⁇ hic sites descnbed herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymo ⁇ hic
- the target reg ⁇ on(s) may be amplified using any ohgonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U S Patent No 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- Oligonucleotides useful as pnmers or probes in such methods should specifically hyb ⁇ dize to a region of the nucleic acid that contains or is adjacent to the polymo ⁇ hic site.
- the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length Most preferably, the oligonucleotides are 20 to 25 nucleotides long. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
- nucleic acid amplification procedures may be used to amplify the target region including transcnption-based amplification systems (U.S Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5, 169,766, WO89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992).
- a polymo ⁇ hism in the target region may also be assayed before or after amplification using one of several hybndization-based methods known in the art.
- allele-specific oligonucleotides are utilized in performing such methods.
- the allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one vanant of a target sequence and the other member showing a perfect match to a different vanant.
- more than one polymo ⁇ hic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs.
- the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hyb ⁇ dizing to each of the polymo ⁇ hic sites being detected.
- Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
- Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads.
- the solid support may be treated, coated or denvatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
- the genotype or haplotype for the CHRM3 gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene to nucleic acid anays and subanays such as desc ⁇ bed in WO 95/1 1995.
- the arrays would contain a battery of allele-specific oligonucleotides representing each of the polymo ⁇ hic sites to be included in the genotype or haplotype.
- polymo ⁇ hisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using nboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coh mutS protein (Modnch, P. Ann. Rev. Genet. 25:229-253, 1991).
- a mismatch detection technique including but not limited to the RNase protection method using nboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coh mutS protein (Modnch, P. Ann. Rev. Genet. 25:229-253, 1991).
- variant alleles can be identified by single strand conformation polymo ⁇ hism (SSCP) analysis (Onta et al., Genomics 5:874-879, 1989; Humphnes et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denatunng gradient gel electrophoresis (DGGE) (Wartell et al, Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).
- SSCP single strand conformation polymo ⁇ hism
- DGGE denatunng gradient gel electrophoresis
- a polymerase-mediated p ⁇ mer extension method may also be used to identify the polymo ⁇ h ⁇ sm(s).
- Several such methods have been desc ⁇ bed in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the hgase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524.
- Related methods are disclosed in W091/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283.
- Extended pnmers containing a polymo ⁇ hism may be detected by mass spectrometry as desc ⁇ bed in U.S. Patent No. 5,605,798.
- Another p ⁇ mer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res. 17 8392, 1989; Ruano et al , Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95: 1635-1641, 1995).
- multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific pnmers as desc ⁇ bed in Wallace et al. (WO89/10414).
- an individual's CHRM3 haplotype pair is predicted from its CHRM3 genotype using information on haplotype pairs known to exist in a reference population.
- the haplotyping prediction method compnses identifying a CHRM3 genotype for the individual at two or more polymo ⁇ hic sites selected from PS l, PS2, PS3, and PS4, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing CHRM3 haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data.
- the reference haplotype pairs include the CHRM3 haplotype pairs shown in Table 4
- the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world.
- a prefe ⁇ ed reference population for use in the methods of the present invention comp ⁇ ses an approximately equal number of individuals from Caucasian, Af ⁇ can Ame ⁇ can, Asian and Hispanic-Latino population groups with the minimum number of each group being chosen based on how rare a haplotype one wants to be guaranteed to see.
- a prefe ⁇ ed reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comp ⁇ ses about 20 unrelated individuals from each of the four population groups named above
- a particularly preferred reference population includes a 3-generat ⁇ on family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures
- the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equi b ⁇ um.
- Hardy- Weinberg equihb ⁇ um (D.L. Haiti et al., P ⁇ nciples of Population Genomics, Smauer Associates (Sunderland, MA), 3 rd Ed., 1997) postulates that the frequency of finding the haplotype pair H, / H 2 is equal to p H .
- a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long- range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
- the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
- the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System' technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al.,
- the invention also provides a method for determining the frequency of a CHRM3 genotype or CHRM3 haplotype in a population.
- the method comprises determining the genotype or the haplotype pair for the CHRM3 gene that is present in each member of the population, wherein the genotype or haplotype comprises the nucleotide pair or nucleotide detected at one or more of the polymo ⁇ hic sites PSl, PS2, PS3, and PS4 in the CHRM3 gene; and calculating the frequency any particular genotype or haplotype is found in the population.
- the population may be a reference population, a family population, a same sex population, a population group, a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).
- frequency data for CHRM3 genotypes and/or haplotypes found in a reference population are used in a method for identifying an association between a trait and a CHRM3 genotype or a CHRM3 haplotype.
- the trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment.
- the method involves obtaining data on the frequency of the genotype(s) or haplotype(s) of interest in a reference population as well as in a population exhibiting the trait.
- Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods descnbed above
- the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach desc ⁇ bed above.
- the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in w ⁇ tten or electronic form.
- the frequency data may be present in a database that is accessible by a computer Once the frequency data is obtained, the frequencies of the genotype(s) or haplotype(s) of interest in the reference and trait populations are compared.
- the frequencies of all genotypes and/or haplotypes observed in the populations are compared. If a particular genotype or haplotype for the CHRM3 gene is more frequent in the trait population than in the reference population at a statistically significant amount, then the trait is predicted to be associated with that CHRM3 genotype or haplotype
- the CHRM3 genotype or haplotype being compared in the trait and reference populations is selected from the full-genotypes and full-haplotypes shown in Tables 4 and 5, respectively, or from sub-genotypes and sub-haplotypes de ⁇ ved from these genotypes and haplotypes
- the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting CHRM3 or response to a therapeutic treatment for a medical condition
- medical condition includes but is not limited to any condition or disease manifested as one or more physical and/or
- clinical data may be obtained by analyzing the results of a clinical tnal that has already been run and/or the clinical data may be obtained by designing and carrying out one or more new clinical t ⁇ als
- the term "clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase III clinical t ⁇ als Standard methods are used to define the patient population and to enroll subjects It is prefe ⁇ ed that the individuals included in the clinical population have been graded for the existence of the medical condition of interest This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same.
- the therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses.
- the CHRM3 gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
- Conelations may be produced in several ways. In one method, individuals are grouped by their CHRM3 genotype or haplotype (or haplotype pair) (also referred to as a polymo ⁇ hism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated. These results are then analyzed to determine if any observed variation in clinical response between polymo ⁇ hism groups is statistically significant. Statistical analysis methods which may be used are described in L.D. Fisher and G.
- a second method for finding co ⁇ elations between CHRM3 haplotype content and clinical responses uses predictive models based on e ⁇ or-minimizing optimization algorithms.
- One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Their Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997).
- Simulated annealing Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K.
- Co ⁇ elations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymo ⁇ hic sites in the CHRM3 gene.
- ANOVA analysis of variation
- a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of CHRM3 genotype or haplotype content.
- the model is validated in one or more follow-up clinical trials designed to test the model.
- the identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the CHRM3 gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug.
- the diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the CHRM3 gene), a serological test, or a physical exam measurement.
- this diagnostic method uses the predictive haplotyping method described above.
- any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer.
- the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the CHRM3 gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymo ⁇ hism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
- the CHRM3 polymo ⁇ hism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files).
- polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD ROM or on one or more other storage devices accessible by the computer.
- the data may be stored on one or more databases in communication with the computer via a network.
- EXAMPLE 1 This example illustrates examination of various regions of the CHRM3 gene for polymo ⁇ hic sites.
- the following target regions of the CHRM3 gene were amplified using the PCR primer pairs listed below, with the sequences presented in the 5 ' to 3 ' direction and nucleotide positions shown for each region co ⁇ esponding to the indicated GenBank Accession No.
- Amplification profile 94°C - 2 min. 1 cycle
- PCR products were purified by Solid Phase Reversible Immobilization using the protocol developed by the Whitehead Genome Center. A detailed protocol can be found at http://www.genome.wi.mit.edu/sequencing/protocols/pure/SPRI_pcr.html.
- the purified PCR products were sequenced in both directions using the primer sets described previously.
- This example illustrates analysis of the CHRM3 polymo ⁇ hisms identified in the Index Repository for human genotypes and haplotypes.
- the different genotypes containing these polymo ⁇ hisms that were observed in the reference population are shown in Table 4 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
- Table 4 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 4 were infe ⁇ ed based on linkage disequilibrium and/or Mendelian inheritance.
- haplotype pairs shown in Table 4 were estimated from the unphased genotypes using an extension of Clark's algorithm (Clark, A.G. (1990) Mol Bio Evol 1, 1 1 1-122), as described in U.S. Provisional Application Serial No. 60/198,340 entitled "A Method and System for Determining Haplotypes from a Collection of Polymo ⁇ hisms".
- haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites.
- the list of haplotypes is augmented with haplotypes obtained from a three- generation Caucasian family and a two-generation African- American family. This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
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WO2002046421A2 (en) * | 2000-10-30 | 2002-06-13 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods and compositions for analysis of m3 muscarinic acetylchloine receptors |
CN1306037C (en) * | 2004-10-19 | 2007-03-21 | 哈尔滨医科大学 | Novel use of cardiocyte M3 receptor and IkM3 in screening medicament for treating arrhythmia |
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Non-Patent Citations (3)
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BONNER ET AL.: 'Identification of a family or muscarinic acetylcholine receptor genes' SCIENCE vol. 237, 31 July 1987, pages 527 - 532, XP002939145 * |
DATABASE CAPLUS [Online] DOC. NO. 132:147377 MATSUI ET AL.: 'Mapping of five subtype genes for muscarinic acetylcholine receptor to mouse chromosomes', XP002939147 Retrieved from STN Database accession no. 1999:599375 & GENES GENET. SYST. vol. 74, no. 1, 1999, pages 15 - 21, ISSN: 1341-75681 * |
DATABASE CAPLUS [Online] DOC. NO. 134:158204 ARMSTRONG ET AL.: 'Suspension arrays for high throughput, moltiplexed single nucleotide polymorphism genotyping', XP002939146 Retrieved from STN Database accession no. 2000:406810 & CYTOMETRY vol. 40, no. 2, 2000, pages 102 - 108 * |
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WO2002046421A2 (en) * | 2000-10-30 | 2002-06-13 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods and compositions for analysis of m3 muscarinic acetylchloine receptors |
WO2002046421A3 (en) * | 2000-10-30 | 2003-07-31 | Us Health | Methods and compositions for analysis of m3 muscarinic acetylchloine receptors |
CN1306037C (en) * | 2004-10-19 | 2007-03-21 | 哈尔滨医科大学 | Novel use of cardiocyte M3 receptor and IkM3 in screening medicament for treating arrhythmia |
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