WO2002090512A2 - Haplotypes du gene nnmt - Google Patents

Haplotypes du gene nnmt Download PDF

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Publication number
WO2002090512A2
WO2002090512A2 PCT/US2002/014538 US0214538W WO02090512A2 WO 2002090512 A2 WO2002090512 A2 WO 2002090512A2 US 0214538 W US0214538 W US 0214538W WO 02090512 A2 WO02090512 A2 WO 02090512A2
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nnmt
haplotype
ofthe
seq
gene
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PCT/US2002/014538
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WO2002090512A3 (fr
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Anne Chew
Christopher Raleigh Gilson
Amir Kazemi
Beena Koshy
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2002257254A priority Critical patent/AU2002257254A1/en
Publication of WO2002090512A2 publication Critical patent/WO2002090512A2/fr
Publication of WO2002090512A3 publication Critical patent/WO2002090512A3/fr

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    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates to variation in genes that encode pharmaceutically-important proteins.
  • this invention provides genetic variants ofthe human nicotinamide N-methyltransferase (NNMT) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • NNMT nicotinamide N-methyltransferase
  • haplotype The standard for measuring genetic variation among individuals is the haplotype, which ⁇ s the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymorphisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses AD supra; Ulbrecht M et al. 2000 Am J Respir Crit Care Med 161: 469-74) and drug response (Wolfe CR et al.
  • NNMT nicotinamide N-methyltransferase
  • a person with a higher level of NNMT activity may be predisposed to the neurodegenerative disorder Parkinson's disease (PD) (Parsons et al., J Neuropathol. Exp Neurol. 2002; 61:111-124; Aoyama et al., Neurosci Lett. 2001; 298:78-80).
  • PD patients showed an increase in NNMT protein levels in both their cerebrospinal fluid and in those brain regions relevant to PD pathology.
  • NNMT activity measured in relevant brain regions was increased in PD patients as compared to non-PD controls.
  • NNMT is also thought to be associated with the neurons that degenerate in PD, as NNMT expression decreases with disease duration. Taken together, these data suggest that NNMT is a candidate gene for PD.
  • NNMT may also be a marker for cancer cachexia, a common cause of death in advanced cancer patients (Okamura et al., Jpn. J Cancer Res. 1998; 89:649-656; Barber et al., Surg. Oncol. 1999; 8: 133-141). While cachexia is a general state of physical ill health associated with chronic disese, cachexia associated with cancer is specifically characterized by metabolic abnormalities that can result in severe weight loss. Mice in which cachexia was induced showed a marked progessive increase in liver NNMT activity that paralleled weight loss, and continued until death. Agents that inhibited NNMT activity in these mice also prevented weight loss. Therefore, therapeutics that target NNMT may be useful in treating cancer cachexia.
  • the nicotinamide N-methyltransferase gene is located on chromosome 1 lq23.1 and contains 3 exons that encode a 264 amino acid protein.
  • a reference sequence for the NNMT gene comprises the non-contigous sequences shown in the contiguous lines of Figure 1, which is a composite genomic sequence based on Genaissance Reference No. 447335 (SEQ ED NO: 1).
  • Reference sequences for the coding sequence (GenBank Accession No. NM_006169.1) and protein are shown in Figures 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3), respectively.
  • polymorphic sites correspond to the following nucleotide positions in Figure 1 : 394 (PSI), 928 (PS2) and 2696 (PS3).
  • PSI polymorphic sites
  • PS2 polymorphic sites
  • PS3 polymorphic sites
  • the polymorphisms at these sites are adenine or thymine at PSI, thymine or cytosine at PS2 and thymine or cytosine at PS3.
  • the inventors have determined the identity ofthe alleles at these sites in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic Latino.
  • each of these NNMT haplotypes constitutes a code, or genetic marker, that defines the variant nucleotides that exist in the human population at this set of polymorphic sites in the NNMT gene.
  • each NNMT haplotype also represents a naturally-occurring isoform (also referred to herein as an "isogene") ofthe NNMT gene.
  • the frequency of each haplotype and haplotype pair within the total reference population and within each ofthe four major population groups included in the reference population was also determined.
  • the invention provides a method, composition and kit for genotyping the NNMT gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PSI, PS2 and PS3 in both copies ofthe NNMT gene from the individual.
  • a genotyping composition ofthe invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these NNMT polymorphic sites.
  • a genotyping kit ofthe invention comprises a set of oligonucleotides designed to genotype each of these novel NNMT polymorphic sites. The genotyping method, composition, and kit are useful in determining whether an individual has one ofthe haplotypes in Table 4 below or has one ofthe haplotype pairs in Table 3 below.
  • the invention also provides a method for haplotyping the NNMT gene in an individual.
  • the haplotyping method comprises determining, for one copy ofthe NNMT gene, the identity ofthe nucleotide at one or more polymorphic sites selected from the group consisting of PSI, PS2 and PS3.
  • the haplotyping method comprises determining whether one copy ofthe individual's NNMT gene is defined by one ofthe NNMT haplotypes shown in Table 4, below, or a sub-haplotype thereof.
  • the haplotyping method comprises determining whether both copies ofthe individual's NNMT gene are defined by one ofthe NNMT haplotype pairs shown in Table 3 below, or a sub-haplotype pair thereof.
  • Establishing the NNMT haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs metabolized by NNMT or drugs for treating diseases associated with NNMT activity, e.g., Parkinson's disease and cancer cachexia.
  • the haplotyping method can be used by the pharmaceutical research scientist to validate NNMT as a candidate target for treating a specific condition or disease predicted to be associated with NNMT activity. Determining for a particular population the frequency of one or more ofthe individual NNMT haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue NNMT as a target for treating the specific disease of interest. In particular, if variable NNMT activity is associated with the disease, then one or more NNMT haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
  • variable NNMT activity has little, if any, involvement with that disease.
  • the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any NNMT haplotype or haplotype pair, apply the information derived from detecting NNMT haplotypes in an individual to decide whether modulating NNMT activity would be useful in treating the disease.
  • the claimed invention is also useful in screening for compounds targeting NNMT to treat a specific condition or disease predicted to be associated with NNMT activity. For example, detecting which ofthe NNMT haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each ofthe NNMT isoforms present in the disease population, or for only the most frequent NNMT isoforms present in the disease population.
  • the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.
  • Haplotyping the NNMT gene in an individual is also useful to control for genetically-based bias in the design of candidate drugs that target or are metabolized by NNMT.
  • the pharmaceutical scientist of ordinary skill would be concerned that a favorable efficacy and/or side effect profile shown in a Phase II or IH trial may not be replicated in the general population if a higher (or lower) percentage of patients in the treatment group, compared to the general population, have a form ofthe NNMT gene that makes them genetically predisposed to metabolize the drug more efficiently than patients with other forms ofthe NNMT gene.
  • this pharmaceutical scientist would recognize the potential for bias in the results of a Phase II or Phase HI clinical trial of a drug targeting NNMT that could be introduced if individuals whose NNMT gene structure makes them genetically predisposed to respond well to the drug are present in a higher (or lower) frequency in the treatment group than in the control group (Bacanu et al., 2000, AM. J. Hum. Gen. 66:1933-44; Pritchard et al., 2000, Am. J. Hum. Gen. 67: 170-81).
  • the pharmaceutical scientist can immediately reduce this potential for genetically-based bias in the results of clinical trials of drugs metabolized by or targeting NMT by practicing the claimed invention.
  • the pharmaceutical scientist can then assign that individual to the treatment or control group as appropriate to ensure that approximately equal frequencies of different NNMT haplotypes (or haplotype pairs) are represented in the two groups and/or the frequencies of different NNMT haplotypes or haplotype pairs are similar to the frequencies in the general population.
  • the pharmaceutical scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any NNMT haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and a NNMT genotype, haplotype, or haplotype pair for one or more ofthe novel polymorphic sites described herein.
  • the method comprises comparing the frequency ofthe NNMT genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency ofthe NNMT genotype or haplotype in a reference population.
  • a different frequency ofthe NNMT genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the NNMT genotype, haplotype, or haplotype pair.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • the NNMT haplotype is selected from the haplotypes shown in Table 4, or a sub-haplotype thereof.
  • Such methods have applicability in developing diagnostic tests for assessing potential drug metabolism by NNMT and for developing diagnostic tests and therapeutic treatments for Parkinson's disease or cancer cachexia.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the NNMT gene or a fragment thereof.
  • the reference sequence comprises the contiguous sequences shown in Figure 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of thymine at PSI, cytosine at PS2 and cytosine at PS3.
  • a particularly preferred polymorphic variant is an isogene ofthe NNMT gene.
  • a NNMT isogene ofthe invention comprises adenine or thymine at PSI, thymine or cytosine at PS2 and thymine or cytosine at PS3.
  • the invention also provides a collection of NNMT isogenes, referred to herein as a NNMT genome anthology.
  • the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a NNMT cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymorphic cDNA comprises cytosine at a position corresponding to nucleotide 426.
  • a particularly preferred polymorphic cDNA variant is A represented in Table 7.
  • Polynucleotides complementary to these NNMT genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants ofthe NNMT gene will be useful in studying the expression and function of NNMT, and in expressing NNMT protein for use in screening for candidate drugs that may be metabolized by NNMT or to treat diseases related to NNMT activity.
  • the invention provides a recombinant expression vector comprising one ofthe polymorphic genomic and cDNA 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 NNMT for protein structure analysis and drug binding studies.
  • the present invention also provides nonhuman transgenic animals comprising one or more of the NNMT polymorphic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression ofthe NNMT isogenes in vivo, for in vivo screening and testing of drugs targeted against NNMT protein, and for testing the efficacy of therapeutic agents and compounds for Parkinson's disease and cancer cachexia in a biological system.
  • the present invention also provides a computer system for storing and displaying polymorphism data determined for the NNMT gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymorphism data.
  • the polymorphism data includes one or more ofthe following: the polymorphisms, the genotypes, the haplotypes, and the haplotype pairs identified for the NNMT gene in a reference population.
  • the computer system is capable of producing a display showing NNMT haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the NNMT gene (Genaissance Reference No. 447335; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • SEQ ID NO: 1 is equivalent to
  • SEQ ID NO:21 is a modified version of SEQ ID NO:l that shows the context sequence of each polymorphic site, PS 1-PS3, in a uniform format to facilitate electromc searching.
  • SEQ ID NO:21 contains a block of 60 bases ofthe nucleotide sequence encompassing the centrally-located polymorphic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein.
  • Figure 2 illustrates a reference sequence for the NNMT coding sequence (contiguous lines; SEQ ID NO:2), with the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • Figure 3 illustrates a reference sequence for the NNMT protein (contiguous lines; SEQ ID NO:3). DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention is based on the discovery of novel variants ofthe NNMT gene.
  • the inventors herein discovered 5 isogenes ofthe NNMT gene by characterizing the NNMT 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 (21 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic/Latino (18 individuals). To the extent possible, the members of this reference population were organized into population subgroups by their self-identified ethnogeographic origin as shown in Table 1 below.
  • the Index Repository contains three 'unrelated indigenous American Indians (one from each of North, Central and South America), one three-generation Caucasian family (from the CEPH Utah cohort) and one two-generation African- American family.
  • the NNMT isogenes present in the human reference population are defined by haplotypes for 3 polymorphic sites in the NNMT gene, all of which are believed to be novel.
  • the novel NNMT polymorphic sites identified by the inventors are referred to as PS1-PS3 to designate the order in which they are located in the gene (see Table 2 below).
  • PS1-PS3 novel NNMT polymorphic sites identified by the inventors
  • the inventors herein also determined the pair of haplotypes for the NNMT gene present in individual human members of this repository.
  • the human genotypes and haplotypes found in the repository for the NNMT gene include those shown in Tables 3 and 4, respectively.
  • the polymorphism and haplotype data disclosed herein are useful for validating whether NNMT is a suitable target for drugs to treat Parkinson's disease and cancer cachexia, screening for such drugs and reducing bias in clinical trials of such drugs. These data are also useful to control for genetically-based bias in the design of drugs metabolized by NNMT.
  • 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 polymorphic 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 ofthe polymorphic sites examined herein 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 polymorphic 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 polymorphic sites examined herein in a locus on a single chromosome from a single individual.
  • Sub-haplotype The 5' to 3' sequence of nucleotides seen at a subset ofthe polymorphic sites examined herein 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 ofthe following for a specific gene: a listing ofthe haplotype pairs in each individual in a population; a listing ofthe 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, coding sequence or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
  • Isogene - One ofthe isoforms e.g., alleles
  • An isogene (or allele) contains all ofthe polymorphisms present in the particular isoform ofthe 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 ofthe present invention.
  • Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature, where physical features include polymorphic sites.
  • 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 polymorphic site on the two copies of a chromosome from an individual.
  • Phased As applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, phased means the combination of nucleotides present at those polymorphic sites on a single copy ofthe locus is known.
  • Polymorphic site (PS) A position on a chromosome or DNA molecule at which at least two alternative sequences are found in a population.
  • Polymorphic variant (variant)- A gene, mRNA, cDNA, polypeptide, protein or peptide whose nucleotide or amino acid sequence varies from a reference sequence due to the presence of a polymorphism in the gene.
  • Polymorphism The sequence variation observed in an individual at a polymorphic site.
  • Polymorphisms 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 ofthe following for a specific gene: location of polymorphic sites; sequence variation at those sites; frequency of polymorphisms 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 polymorphism 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.
  • Population Group A group of individuals sharing a common ethnogeographic origin.
  • Reference Population A group of subjects or individuals who are predicted to be representative ofthe genetic variation found in the general population. Typically, 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%.
  • Single Nucleotide Polymorphism SNP - Typically, the specific pair of nucleotides observed at a single polymorphic site. In rare cases, three or four nucleotides may be found.
  • 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 polymorphic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy ofthe locus is not known.
  • the invention also provides compositions and methods for detecting the novel NNMT polymo ⁇ hisms, haplotypes and haplotype pairs identified herein.
  • compositions comprise at least one oligonucleotide for detecting the variant nucleotide or nucleotide pair located at a NNMT polymo ⁇ hic site in one copy or two copies ofthe NNMT gene.
  • oligonucleotides are referred to herein as NNMT haplotyping oligonucleotides or genotyping oligonucleotides, respectively, and collectively as NNMT oligonucleotides.
  • a NNMT haplotyping or genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, one ofthe novel polymo ⁇ hic sites described herein.
  • oligonucleotide refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a preferred oligonucleotide ofthe invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length. The exact length ofthe oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
  • oligonucleotide may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives.
  • oligonucleotides may have a phosphate-free backbone, which may be comprised 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. Meyers, VCH Publishers, Inc. (1995), pages 617-620).
  • Oligonucleotides ofthe 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.
  • Haplotyping or genotyping oligonucleotides ofthe invention must be capable of specifically hybridizing to a target region of a NNMT polynucleotide.
  • the target region is located in a NNMT 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 another region in the NNMT polynucleotide or with a non-NNMT polynucleotide under the same hybridizing conditions.
  • the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • oligonucleotide probes and primers suitable for detecting polymo ⁇ hisms in the NNMT gene using the polymo ⁇ hism information provided herein in conjunction with the known sequence information for the NNMT 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 ofthe molecules is complementary to the nucleotide at the corresponding position ofthe 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 ofthe primer being complementary to the target region.
  • non-complementary nucleotides may be interspersed into the probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • 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. Examples of hybridization and washing conditions typically used for ASO probes are found in Kogan et al.,
  • an ASO will be perfectly complementary to one allele while containing a single mismatch for another allele.
  • Allele-specific oligonucleotides ofthe invention include ASO probes and ASO primers.
  • ASO probes which usually provide good discrimination between different alleles are those in which a central position ofthe oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 th or 8 th position in a 15mer, the 8 th or 9 th position in a 16mer, and the 10 th or 11 th position in a 20mer).
  • An ASO primer ofthe 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.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a preferred ASO probe for detecting NNMT gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • CGAGCTCWAGTGCTC (SEQ ID NO: 4) and its complement
  • AGTCATAYAGATGGA (SEQ ID NO: 5) and its complement
  • AGTGTGAYGTGACTC (SEQ ID NO: 6) and its complement.
  • a preferred ASO primer for detecting NNMT gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • TCCTGACGAGCTCWA (SEQ ID NO: 7); CAGAGGGAGCACT G (SEQ ID NO: 8); AATGTGAGTCATAYA (SEQ ' ID NO: 9); TGAGACTCCATCTRT (SEQ ID NO: 10); TGCTGAAGTGTGAYG (SEQ ID NO: 11) and GGCTCTGAGTCACRT (SEQ ID NO: 12).
  • oligonucleotides ofthe invention hybridize to a target region located one to several nucleotides downstream of one ofthe novel polymo ⁇ hic sites identified herein.
  • Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one ofthe novel polymo ⁇ hisms described herein and therefore such 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 NNMT gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • TGACGAGCTC SEQ ID NO: 13
  • AGGGAGCACT SEQ ID NO: 14
  • GTGAGTCATA SEQ ID NO: 15
  • GACTCCATCT SEQ ID NO:16
  • TGAAGTGTGA SEQ ID NO:17
  • TCTGAGTCAC SEQ ID NO: 18
  • a composition contains two or more differently labeled NNMT oligonucleotides for simultaneously probing the identity of nucleotides or nucleotide pairs 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.
  • NNMT oligonucleotides ofthe 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 oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized NNMT oligonucleotides ofthe 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 NNMT 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.
  • the above described oligonucleotide compositions and kits are useful in methods for genotyping and/or haplotyping the NNMT gene in an individual.
  • the terms "NNMT genotype” and “NNMT haplotype” mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more ofthe 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 NNMT gene.
  • the additional polymo ⁇ hic sites may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
  • a genotyping method ofthe invention involves examining both copies of the individual's NNMT gene, or a fragment thereof, to identify the nucleotide pair at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2 and PS3 in the two copies to assign a NNMT genotype to the individual.
  • "examining a gene” may include examining one or more of: DNA containing the gene, mRNA transcripts thereof, or cDNA copies thereof.
  • the two "copies" of a gene, mRNA or cDNA (or fragment of such NNMT molecules) in an individual may be the same allele or may be different alleles.
  • a genotyping method ofthe invention comprises determining the identity of the nucleotide pair at each of PS 1 -PS3.
  • nucleic acid sample comprising the two copies ofthe NNMT gene, mRNA transcripts thereof or cDNA copies thereof, or a fragment of any ofthe foregoing, that are present in the individual.
  • the nucleic acid sample is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample. Suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the nucleic acid sample may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from a tissue in which the NNMT gene is expressed.
  • a haplotyping method ofthe invention comprises examining one copy of the individual's NNMT gene, or a fragment thereof, to identify the nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2 and PS3 in that copy to assign a NNMT haplotype to the individual.
  • the nucleotide at each of PS1-PS3 is identified.
  • the NNMT haplotype assigned to the individual is selected from the group consisting ofthe NNMT haplotypes shown in Table 4.
  • "examining a gene” may include examining one or more of: DNA containing the gene, mRNA transcripts thereof, or cDNA copies thereof.
  • One method of examining one copy ofthe individual's NNMT gene is by isolating from the individual a nucleic acid sample containing only one ofthe two copies ofthe NNMT gene, mRNA or cDNA, or a fragment of such NNMT molecules, that is present in the individual and determining in that copy the identity ofthe nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2 and PS3 in that copy to assign a NNMT haplotype to the individual.
  • the nucleotide at each of PS1-PS3 is identified.
  • the nucleic acid used in the above haplotyping methods ofthe invention may be isolated using any method capable of separating the two copies of the NNMT gene or fragment such as one of the methods described above for preparing NNMT isogenes, with targeted in vivo cloning being the preferred approach.
  • any individual clone will typically only provide haplotype information on one ofthe two NNMT gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional NNMT clones will usually need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies ofthe NNMT gene in an individual.
  • the haplotype for the other allele may be inferred if the individual has a known genotype for the polymo ⁇ hic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known.
  • the haplotyping method comprises determining whether an individual has one or more ofthe NNMT haplotypes shown in Table 4. This can be accomplished by identifying the phased sequence of nucleotides present at PS1-PS3 for at least one copy ofthe individual's NNMT gene and assigning to that copy a NNMT haplotype that is consistent with the phased sequence, wherein the NNMT haplotype is selected from the group consisting ofthe NNMT haplotypes shown in Table 4 and wherein each ofthe NNMT haplotypes in Table 4 comprises a sequence of polymo ⁇ hisms whose positions and identities are set forth in the table. This identifying step does not necessarily require that each of PS1-PS3 be directly examined.
  • PS1-PS3 typically only a subset of PS1-PS3 will need to be directly exammed to assign to an individual one or more ofthe haplotypes shown in Table 4- This is because at least one polymo ⁇ hic site in a gene is frequently in strong linkage disequilibrium with one or more other polymo ⁇ hic sites in that gene (Drysdale, CM et al. 2000 PNAS 97: 10483-10488; Rieder MJ et al. 1999 Nature Genetics 22:59-62).
  • Two nucleotide alleles are said to be in linkage disequilibrium if the presence of a particular allele at one polymo ⁇ hic site predicts the presence ofthe other allele at a second polymo ⁇ hic site (Stephens, JC 1999, Mol. Diag. 4:309-317).
  • Techniques for determining whether any two polymo ⁇ hic sites are in linkage disequilibrium are well- known in the art (Weir B.S. 1996 Genetic Data Analysis II, Sinauer Associates, Inc. Publishers, Sunderland, MA).
  • Johnson et al. (2001 Nature Genetics 29: 233-237) presented one possible method for selection of subsets of polymo ⁇ hic sites suitable for identifying known haplotypes.
  • a NNMT haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2 and PS3 in each copy ofthe NNMT gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS3 in each copy ofthe NNMT gene.
  • the haplotyping method comprises determining whether an individual has one ofthe NNMT haplotype pairs shown in Table 3.
  • One way to accomplish this is to identify the phased sequence of nucleotides at PS1-PS3 for each copy ofthe individual's NNMT gene and assigning to the individual a NNMT haplotype pair that is consistent with each ofthe phased sequences, wherein the NNMT haplotype pair is selected from the group consisting ofthe NNMT haplotype pairs shown in Table 3.
  • the identifying step does not necessarily require that each of PS1-PS3 be directly examined. As a result of linkage disequilibrium, typically only a subset of PS1-PS3 will need to be directly examined to assign to an individual a haplotype pair shown in Table 3.
  • the identifying step is preferably performed with each copy ofthe 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 ofthe gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oHgonucleotide labeled with yet a third different fluorescent dye is used to assay the polymo ⁇ hic site(s), then detecting a combination ofthe first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination ofthe 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 site(s) may be determined by amplifying a target region(s) containing the polymo ⁇ hic site(s) directly from one or both copies ofthe NNMT gene, or a fragment thereof, and the sequence ofthe amplified region(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, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for an mdividual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanme/guanine).
  • the target region(s) may be amplified using any oligonucleotide-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. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241:1077-1080, 1988).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No.
  • a polymo ⁇ hism in the target region may also be assayed before or after amplification using one of several hybridization-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 ofthe pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymo ⁇ hic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs.
  • the members ofthe set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing 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 avid n-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 ofthe 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 derivatized to facilitate the immobilization ofthe allele-specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the NNMT gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies ofthe gene, mRNA, cDNA or fragment(s) thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995.
  • the arrays would contain a battery of allele-specific oligonucleotides representing each ofthe 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 riboprobes (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. coli mutS protein (Modricb, P. Ann. Rev. Genet. 25:229-253,
  • variant alleles can be identified by single strand conformation polymo ⁇ hism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymo ⁇ hism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524.
  • Related methods are disclosed in W091/02087, WO90/09455,
  • Extended primers containing a polym ⁇ hism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions ofthe nucleic acid using sets of allele-specific primers as described in
  • the identity ofthe allele(s) present at any ofthe novel polymo ⁇ hic sites described herein may be indirectly determined by haplotyping or genotyping another polymo ⁇ hic site that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest.
  • Polymo ⁇ hic sites in linkage disequilibrium with the presently disclosed polymo ⁇ hic sites may be located in regions ofthe gene or in other genomic regions not examined herein.
  • Detection ofthe allele(s) present at a polymo ⁇ hic site in linkage disequilibrium with the novel polymo ⁇ hic sites described herein may be performed by, but is not limited to, any ofthe above-mentioned methods for detecting the identity ofthe allele at a polymo ⁇ hic site.
  • an individual's NNMT haplotype pair is predicted from its NNMT genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying a NNMT genotype for the individual at two or more NNMT polymo ⁇ hic sites described herein, accessing data containing NNMT haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the individual's NNMT genotype.
  • the reference haplotype pairs include the NNMT haplotype pairs shown in Table 3.
  • the NNMT haplotype pair can be assigned by comparing the individual's genotype with the genotypes corresponding to the haplotype pairs known to exist in the general population or in a specific population group, and determining which haplotype pair is consistent with the genotype ofthe individual.
  • the comparing step may be performed by visual inspection (for example, by consulting Table 3).
  • frequency data (such as that presented in Table 6) may be used to determine which of these haplotype pairs is most likely to be present in the individual. This determination may also be performed in some embodiments by visual inspection, for example by consulting Table 6.
  • the comparison may be made by a computer-implemented algorithm with the genotype ofthe individual and the reference haplotype data stored in computer-readable formats.
  • one computer-implemented algorithm to perform this comparison entails enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing NNMT haplotype pairs frequency data determined in a reference population to determine a probability that the individual has a possible haplotype pair, and analyzing the determined probabilities to assign a haplotype pair to the mdividual.
  • the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups ofthe world.
  • a preferred reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each ofthe four population groups named above.
  • a particularly preferred reference population includes a 3-generation family representing one or more ofthe 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 equilibrium. Hardy-Weinberg equilibrium (D.L.
  • haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), single molecule dilution, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
  • CLASPER System TM technology U.S. Patent No. 5,866,404
  • single molecule dilution single molecule dilution
  • 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 ofthe possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one ofthe 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 haplotype pair in an individual may be predicted from the individual's genotype for that gene using reported methods (e.g., Clark et al. 1990 Mol Bio Evol 7:111-22 or WO 01/80156) or through a commercial haplotyping service such as offered by Genaissance Pharmaceuticals, Inc. (New Haven, CT).
  • a commercial haplotyping service such as offered by Genaissance Pharmaceuticals, Inc. (New Haven, CT).
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No.
  • the invention also provides a method for determining the frequency of a NNMT genotype, haplotype, or haplotype pair in a population.
  • the method comprises, for each member ofthe population, determining the genotype, haplotype or the haplotype pair for the novel NNMT polymo ⁇ hic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population.
  • the population may be e.g., a reference population, a family population, a same gender population, a population group, or 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).
  • 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.
  • NNMT haplotype frequencies in a trait population having a medical condition and a control population lacking the medical condition are used in a method of validating the NNMT protein as a candidate target for treating a medical condition predicted to be associated with NNMT activity.
  • the method comprises comparing the frequency of each NNMT haplotype shown in Table 4 in the trait population and in a control population and making a decision whether to pursue NNMT as a target.
  • the composition ofthe control population will be dependent upon the specific study and may be a reference population or it may be an appropriately matched population with regards to age, gender, and clinical symptoms for example.
  • each ofthe trait and controls populations may be comprised of different ethnogeographic origins, including but not limited to
  • the trait and reference population may be comprised of just one ethnogeographic origin.
  • frequency data for NNMT haplotypes are determined in a population having a condition or disease predicted to be associated with NNMT activity and used in a method for screening for compounds targeting the NNMT protein to treat such condition or disease.
  • frequency data are determined in the population of interest for the NNMT haplotypes shown in Table 4.
  • the frequency data for this population may be obtained by genotyping or haplotyping each individual in the population using one or more ofthe methods described above.
  • the haplotypes for this population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as described above.
  • the frequency data for this population are obtained by accessing previously determined frequency data, which may be in written or electronic form.
  • the frequency data may be present in a database that is accessible by a computer.
  • the NNMT isoforms corresponding to NNMT haplotypes occurring at a frequency greater than or equal to a desired frequency in this population are then used in screening for a compound, or compounds, that displays a desired agonist (enhancer) or antagonist (inhibitor) activity for each NNMT isoform.
  • the desired frequency for the haplotypes might be chosen to be the frequency ofthe most frequent haplotype, greater than some cut-off value, such as 10% in the population, or the desired frequency might be determined by ranking the haplotypes by frequency and then choosing the frquency ofthe third most frequent haplotype as the cut-off value.
  • a desired frequency is chosen to be greater than or equal to a cut-off value, such as activity levels in the top 10% of values determined.
  • Embodiments may employ cell-free or cell- based screening assays known in the art.
  • the compounds used in the screening assays may be from chemical compound libraries, peptide libraries and the like.
  • the NNMT isoforms used in the screening assays may be free in solution, affixed to a solid support, or expressed in an appropriate cell line.
  • the condition or disease associated with NNMT activity is Parkinson's disease or cancer cachexia.
  • frequency data for NNMT genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and a NNMT genotype, haplotype, or haplotype pair.
  • 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 ofthe genotype(s), • haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait.
  • Frequency data for one or both ofthe reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one or more ofthe methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as described above.
  • the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written 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 ofthe genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared.
  • the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared. If a particular NNMT genotype, haplotype, or haplotype pair is different in the trait population than in the reference population to a statistically significant degree, then the trait is predicted to be associated with that NNMT genotype, haplotype or haplotype pair.
  • the NNMT genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the full- genotypes and full-haplotypes shown in Tables 3 and 4, or from sub-genotypes and sub-haplotypes derived 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 or metabolized by NNMT 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 psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders.
  • clinical response means any or all ofthe following: a quantitative measure ofthe response, no response, and/or adverse response (i.e., side effects).
  • clinical population In order to deduce a correlation between clinical response to a treatment and a NNMT genotype, haplotype, or haplotype pair, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population".
  • This clinical data may be obtained by analyzing the results of a clinical trial that has already been run and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials.
  • 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 in clinical trials. Standard methods are used to define the patient population and to enroll subjects.
  • the individuals included in the clinical population have been graded for the existence ofthe 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 ofthe underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome.
  • This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.
  • 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 NNMT gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after a ⁇ ninistering the treatment. After both the clinical and polymo ⁇ hism data have been obtained, correlations between individual response and NNMT genotype or haplotype content are created. Correlations may be produced in several ways.
  • individuals are grouped by their NNMT 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.
  • a second method for finding correlations between NNMT haplotype content and clinical responses uses predictive models based on error-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.
  • Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much ofthe variation in the clinical data is explained by different subsets ofthe polymo ⁇ hic sites in the NNMT gene.
  • ANOVA analysis of variation
  • ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10).
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of NNMT 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 NNMT 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 will detect the presence in an individual ofthe genotype, haplotype or haplotype pair that is associated with the clinical response and may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more ofthe polymo ⁇ hic sites in the NNMT gene), a serological test, or a physical exam measurement.
  • a direct DNA test i.e., genotyping or haplotyping one or more ofthe polymo ⁇ hic sites in the NNMT gene
  • serological test i.e., a serological test
  • a physical exam measurement i.e., a physical exam measurement.
  • this diagnostic method uses the predictive haplotyping method described above.
  • Another embodiment ofthe invention comprises a method for reducing the potential for bias in a clinical trial of a candidate drug that targets or is metabolized by NNMT.
  • Haplotyping one or both copies ofthe NNMT gene in those individuals participating in the trial will allow the pharmaceutical scientist conducting the clinical trial to assign each individual from the trial one ofthe haplotypes or haplotype pairs shown in Tables 4 and 3, respectively, in the NNMT gene.
  • the haplotypes may be determined directly, or alternatively, by a predictive genotype to haplotype approach as decribed above.
  • this can be accomplished by haplotyping individuals participating in a clinical trial by identifying, for example, in one or both copies ofthe individual's NNMT gene, the phased sequence of nucleotides present at each of PSI - PS3. Determining the NNMT haplotype or haplotype pair present in individuals participating in the clinical trial enables the pharmaceutical scientist to assign individuals possessing a specific haplotype or haplotype pair evenly to treatment and control groups.
  • Typical clinical trials conducted may include, but are not limited to, Phase I, ⁇ , and III clinical trials. Each individual in the trial may produce a specific response to the candidate drug based upon the individual's haplotype or haplotype pair.
  • each treatment and control group are assigned an even distribution (or equal numbers) of individuals having a particular NNMT haplotype or haplotype pair.
  • the pharmaceutical scientist requires no a priori knowledge of any effect a NNMT haplotype or haplotype pair may have on the results ofthe trial.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant ofthe NNMT gene or a fragment ofthe gene which contains at least one ofthe novel polymo ⁇ hic sites described herein.
  • the nucleotide sequence of a variant NNMT gene is identical to the reference genomic sequence for those portions ofthe gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more ofthe novel polymo ⁇ hic sites PSI, PS2 and PS3.
  • the nucleotide sequence of a variant fragment ofthe NNMT gene is identical to the corresponding portion ofthe reference sequence except for having a different nucleotide at one or more ofthe novel polymo ⁇ hic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence ofthe NNMT gene, which is defined by haplotype 3, (or other reported NNMT sequences) or to portions ofthe reference sequence (or other reported NNMT sequences), except for the haplotyping and genotyping oligonucleotides described above.
  • the location of a polymo ⁇ hism in a variant NNMT gene or fragment is preferably identified by aligning its sequence against SEQ ID NO:l.
  • the polymo ⁇ hism is selected from the group consisting of thymine at PSI, cytosine at PS2 and cytosine at PS3.
  • the polymo ⁇ hic variant comprises a naturally-occurring isogene ofthe NNMT gene which is defined by any one of haplotypes 1- 2 and 4 - 5 shown in Table 4 below.
  • Polymo ⁇ hic variants ofthe invention may be prepared by isolating a clone containing the NNMT gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel polymo ⁇ hic sites described herein.
  • Any particular variant or fragment thereof, that is claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
  • Any particular NNMT variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.
  • NNMT isogenes, or fragments thereof may be isolated using any method that allows separation ofthe two "copies" ofthe NNMT 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 described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • TIVC targeted in vivo cloning
  • Another method which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • NNMT genome anthologies which are collections of at least two NNMT 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 gender population.
  • a NNMT genome anthology may comprise individual NNMT isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups ofthe NNMT isogenes in the anthology may be stored in separate containers. Individual isogenes or groups of such isogenes in a genome anthology may be stored in any convenient and stable form, including but not limited to in buffered solutions, as DNA precipitates, freeze-dried preparations and the like.
  • a preferred NNMT genome anthology ofthe invention comprises a set of isogenes defined by the haplotypes shown in Table 4 below.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence ofthe 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 NNMT 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 derived from vaccinia virus, adenovirus, retroviruses, or SV40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation ofthe nucleic acid sequence in a given host cell.
  • the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins 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 NNMT sequences ofthe 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: 1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • NNMT gene will produce NNMT 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 NNMT cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of he NNMT reference coding sequence shown in Figure 2.
  • the invention also provides NNMT mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
  • a particularly preferred polymo ⁇ hic cDNA variant is A represented in Table 7. Fragments of these variant mRNAs and cDNAs are included in the scope ofthe invention, provided they contain the novel polymorphism described herein.
  • the invention specifically excludes polynucleotides identical to previously identified NNMT mRNAs or cDNAs, and previously described fragments thereof.
  • Polynucleotides comprising a variant NNMT RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
  • a polymo ⁇ hic variant of a NNMT gene, mRNA or cDNA fragment comprises at least one novel polymo ⁇ hism identified herein and has a length of at least 10 nucleotides and may range up to the full length ofthe 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 200 and 750 nucleotides in length.
  • nucleic acid molecules containing the NNMT gene or cDNA may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding 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 ofthe NNMT genomic, mRNA and cDNA variants described herein.
  • Polynucleotides comprising a polymo ⁇ hic gene variant or fragment ofthe invention 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 NNMT isogene encoding that isoform or may already have at least one copy of that isogene.
  • NNMT neuropeptide
  • Expression of a NNMT 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 or antisense RNA for the isogene or fragment thereof.
  • oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3' untranslated region) of he 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) ofthe 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 NNMT mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of NNMT mRNA transcribed from a particular isogene.
  • the untranslated mRNA, antisense RNA or antisense 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. Alternatively, such molecules 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' 0-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.
  • Effect(s) ofthe polymo ⁇ hisms identified herein on expression of NNMT may be investigated by various means known in the art, such as by in vitro translation of mRNA transcripts ofthe NNMT gene, cDNA or fragment thereof, or by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant ofthe NNMT gene.
  • expression includes but is not limited to one or more ofthe following: transcription of the gene into precursor mRNA; splicing and other processing ofthe precursor mRNA to produce mature mRNA; mRNA stability; translation ofthe mature mRNA(s) into NNMT protein(s) (including effects of polymo ⁇ hisms on codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the desired NNMT isogene, cDNA or coding sequence may be introduced into the cell in a vector such that the isogene, cDNA or coding sequence remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the NNMT isogene, cDNA or coding sequence is introduced into a cell in such a way that it recombines with the endogenous NNMT gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired NNMT gene polymo ⁇ hism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in 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 ofthe skilled practitioner.
  • Examples of cells into which the NNMT isogene, cDNA or coding sequence may be introduced include, but are not limited to, continuous culture cells, such as COS, CHO, NIH/3T3, and primary or culture cells ofthe relevant tissue type, i.e., they express the NNMT isogene, cDNA or coding sequence. Such recombinant cells can be used to compare the biological activities ofthe different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant NNMT gene, cDNA or coding sequence are prepared using standard procedures known in the art.
  • a construct comprising the variant gene, cDNA or coding sequence is introduced into a nonhuman animal or an ancestor ofthe 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 ofthe 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 (or cDNA or coding sequence) of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent
  • a third method involves directly injecting a transgene into the embryo.
  • a third method involves the use of embryonic stem cells.
  • animals into which the NNMT isogene, cDNA or coding sequences may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (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 NNMT isogene, cDNA or coding sequence and producing the encoded human NNMT protein can be used as biological models for studying diseases related to abnormal NNMT expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • An additional embodiment ofthe invention relates to pharmaceutical compositions affected by expression or function of a novel NNMT isogene described herein.
  • the pharmaceutical composition may comprise any ofthe following active ingredients: a polynucleotide comprising one of these novel NNMT isogenes (or cDNAs or coding sequences); an antisense oligonucleotide directed against one of the novel NNMT isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel NNMT isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • a pharmaceutically acceptable carrier 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 ofthe pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in 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 appropriate 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 requiring treatment, including but not limited to severity ofthe disease state, general health, age, weight and gender ofthe patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • any or all analytical and mathematical operations involved in practicing the methods ofthe 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 NNMT 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 NNMT 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.
  • This example illustrates examination of various regions ofthe NNMT gene for polymo ⁇ hic sites.
  • the universal 'tail' sequence for the forward PCR primers comprises the sequence 5 '-TGTAAAACGACGGCCAGT-3 '
  • Amplification profile 97°C - 2 min. 1 cycle
  • the PCR products were purified using a Whatman/Polyfiltronics 100 ⁇ l 384 well unifilter plate essentially according to the manufacturers protocol.
  • the purified DNA was eluted in 50 ⁇ l of distilled water.
  • Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol.
  • the purified PCR products were sequenced in both directions using either the primer sets represented below with the positions of their first and last nucleotide corresponding to positions in Figure 1, or the appropriate universal 'tail' sequence as a primer. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
  • Fragment 1 150-171 complement of 671-652 Fragment 2 442-461 complement of 951-932 Fragment 3 495-514 complement of 912-893 Fragment 4 Tailed Seq. Fragment 5 Tailed Seq. Fragment 6 Tailed Seq. Fragment 7 2754-2773 complement of 3244-3224
  • Polyld is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc. (R) Reported previously.
  • This example illustrates analysis ofthe NNMT polymo ⁇ hisms identified in the Index Repository for human genotypes and haplotypes.
  • the different genotypes containing these polymo ⁇ hisms that were observed in unrelated members ofthe reference population are shown in Table 3 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
  • Table 3 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 3 were inferred based on linkage disequilibrium and/or Mendelian inheritance. Table 3 . Genotypes Observed for the NNMT Gene
  • haplotype pairs shown in Table 3 were estimated from the unphased genotypes using a computer-implemented algorithm for assigning haplotypes to unrelated individuals in a population sample, as described in WO 01/80156.
  • haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one ofthe variable sites.
  • This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
  • the list of haplotypes was augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African- American family).
  • each ofthe NNMT haplotypes comprises a 5 ' - 3 ' ordered sequence of 3 polymo ⁇ hisms whose positions in SEQ ID NO: 1 and identities are set forth in Table 4.
  • the column labeled "Region Examined” provides the nucleotide positions in SEQ ID NO:l corresponding to sequenced regions ofthe gene.
  • PS No. and PS Position provide the polymo ⁇ hic site number designation (see Table 2) and the corresponding nucleotide position of this polymo ⁇ hic site within SEQ ID NO: 1 or SEQ ID NO:21.
  • the columns beneath the "Haplotype Number” heading are labeled to provide a unique number designation for each NNMT haplotype.
  • Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO : 1 of the regions sequenced;
  • SEQ ID NO:l refers to Figure 1, with the two alternative allelic variants of each polymo ⁇ hic site indicated by the appropriate nucleotide symbol.
  • SEQ ID NO:21 is a modified version of SEQ ID NO: 1 that shows the context sequence of each of PS1-PS3 in a uniform format to facilitate electronic searching ofthe NNMT haplotypes.
  • SEQ ID NO:21 contains a block of 60 bases ofthe nucleotide sequence encompassing the centrally-located polymo ⁇ hic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each polymo ⁇ hic site is separated by genomic sequence whose composition is defined elsewhere herein.
  • the size and composition ofthe Index Repository were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population.
  • this repository contains approximately equal sample sizes of African-descent, Asian- American, European- American, and Hispanic-Latino population groups. Almost all individuals representing each group had all four grandparents with the same ethnogeographic background.
  • the number of unrelated individuals in the Index Repository provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population.
  • a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group.
  • the size and composition ofthe Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pairs ofthe NNMT gene are likely to be similar to the relative frequencies of these NNMT haplotypes and haplotype pairs in the general U.S. population and in the four population groups represented in the Index Repository.
  • the genetic diversity observed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.
  • Each NNMT haplotype shown in Table 4 defines a NNMT isogene.
  • the NNMT isogene defined by a given NNMT haplotype comprises the examined regions of SEQ ID NO: 1 indicated in
  • Each NNMT isogene defined by one ofthe haplotypes shown in Table 4 will further correspond to a particular NNMT coding sequence variant.
  • Each of these NNMT coding sequence variants comprises the regions of SEQ ID NO:2 examined and is defined by the 5 ' - 3 ' ordered sequence of nucleotides occurring at each polymo ⁇ hic site within the coding sequence ofthe NNMT gene, as shown in Table 7.
  • the column labeled 'Region Examined' provides the nucleotide positions in SEQ ID NO:2 corresponding to sequenced regions ofthe gene; the columns labeled 'PS No.' and 'PS Position' provide the polymo ⁇ hic site number designation (see Table 2) and the corresponding nucleotide position of this polymo ⁇ hic site within SEQ ID NOJ2.
  • the columns beneath the 'Coding Sequence Number' heading are numbered to correspond to the haplotype number defining the NNMT isogene from which the coding sequence variant is derived.
  • NNMT coding sequence variants that differ from the reference NNMT coding sequence are denoted in Table 7 by a letter (A, B, etc) identifying each unique novel coding sequence.
  • the same letter at the top of more than one column denotes that a given novel coding sequence is present in multiple novel NNMT isogenes.
  • Region examined represents the nucleotide positions in SEQ ID NO:2 defining the start and stop positions ofthe regions sequenced;

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Abstract

La présente invention concerne des variantes génétiques du gène de la Nicotinamide N-Méthyltransférase (NNMT). L'invention concerne plus particulièrement divers génotypes, haplotypes, et paires de haplotypes présents généralement dans la population des Etats Unis d'Amérique pour le gène NNMT. L'invention concerne également des compositions et procédés de haplotypage et/ou de génotypage du gène NNMT chez un individu. L'invention concerne enfin des polynucléotides définis par les haplotypes de l'invention.
PCT/US2002/014538 2001-05-07 2002-05-07 Haplotypes du gene nnmt WO2002090512A2 (fr)

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WO2004057336A2 (fr) * 2002-12-20 2004-07-08 Roche Diagnostics Gmbh Utilisation de la nicotinamide n-methyltransferase comme marqueur du cancer colorectal
EP1541695A1 (fr) * 2003-12-09 2005-06-15 Nanogen Recognomics GmbH Méthode à déterminer le caractère maligne de melanoma utilisant une mutation dans le BRAF gène
JP2008514188A (ja) * 2004-09-24 2008-05-08 アンジオブラスト システムズ，インコーポレーテッド 間葉系前駆細胞（ｍｐｃ）の増殖および／または生存を増強させる方法

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Title
AKSOY ET AL.: 'Human nicotinamide N-methyltransferase gene: molecular cloning, structural characterization and chromosomal localization' GENOMICS vol. 29, 1995, pages 555 - 561, XP002959533 *
DATABASE GENBANK [Online] 09 September 2000 BIRREN ET AL.: 'Homo sapiens clone RP11-649M10, working draft sequence, 15 unordered pieces', XP002959532 Database accession no. (AC019290) *
SAITO ET AL.: 'Identification of 197 genetic variations in six human methyltransferase genes in the Japanese population' J. HUM. GENET. vol. 46, 2001, pages 529 - 537, XP002959535 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004057336A2 (fr) * 2002-12-20 2004-07-08 Roche Diagnostics Gmbh Utilisation de la nicotinamide n-methyltransferase comme marqueur du cancer colorectal
WO2004057336A3 (fr) * 2002-12-20 2005-01-20 Roche Diagnostics Gmbh Utilisation de la nicotinamide n-methyltransferase comme marqueur du cancer colorectal
US7205118B2 (en) 2002-12-20 2007-04-17 Roche Diagnostics Operations, Inc. Nicotinamide N-methyltransferase as a marker for colorectal cancer
CN100437115C (zh) * 2002-12-20 2008-11-26 霍夫曼-拉罗奇有限公司 烟酰胺n-甲基转移酶作为结肠直肠癌标志的用途
EP1541695A1 (fr) * 2003-12-09 2005-06-15 Nanogen Recognomics GmbH Méthode à déterminer le caractère maligne de melanoma utilisant une mutation dans le BRAF gène
WO2005059171A1 (fr) * 2003-12-09 2005-06-30 Nanogen Recognomics Gmbh Utilisation d'une mutation du gene braf pour determiner la malignite de cellules de melanome
JP2008514188A (ja) * 2004-09-24 2008-05-08 アンジオブラスト システムズ，インコーポレーテッド 間葉系前駆細胞（ｍｐｃ）の増殖および／または生存を増強させる方法
JP2013099347A (ja) * 2004-09-24 2013-05-23 Mesoblast Inc 間葉系前駆細胞（ｍｐｃ）の増殖および／または生存を増強させる方法

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