WO2001079551A1 - Haplotypes of the nat1 gene - Google Patents

Abstract

Novel single nucleotide polymorphisms in the human N-acetyltransferase 1(arylamine N-acetyltransferase) (NAT1) gene are described. In addition, various genotypes, haplotypes and haplotype pairs for the NAT1 gene that exist in the population are described. Compositions and methods for haplotyping and/or genotyping the NAT1 gene in an individual are also disclosed. Polynucleotides containing one or more of the NAT1 polymorphisms disclosed herein are also described.

Description

HAPLOTNPES OF THE ΝAT1 GENE

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 60/196,773 filed April 12, 2000.

FIELD OF THE INVENTION

This invention relates to variation in genes that encode pharmaceutically-important proteins. In particular, this invention provides genetic variants of the human N-acetyltransferase l(arylamine N-acetyltransferase) (NATl) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.

BACKGROUND OF THE INVENTION

Current methods for identifying pharmaceuticals to treat disease often start by identifying, cloning, and expressing an important target protein related to the disease. A determination of whether an agonist or antagonist is needed to produce an effect that may benefit a patient with the disease is then made. Then, vast numbers of compounds are screened against the target protein to find new potential drugs. The desired outcome of this process is a lead compound that is specific for the target, thereby reducing the incidence of the undesired side effects usually caused by activity at non-intended targets. The lead compound identified in this screening process then undergoes further in vitro and in vivo testing to determine its absorption, disposition, metabolism and toxicological profiles. Typically, this testing involves use of cell lines and animal models with limited, if any, genetic diversity.

What this approach fails to consider, however, is that natural genetic variability exists between individuals in any and every population with respect to pharmaceutically-important proteins, including the protein targets of candidate drugs, the enzymes that metabolize these drugs and the proteins whose activity is modulated by such drug targets. Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a pharmaceutically-important protein may be manifested as significant variation in expression, structure and/or function of the protein. Such alterations may explain the relatively high degree of uncertainty inherent in the treatment of individuals with a drug whose design is based upon a single representative example of the target or enzyme(s) involved in metabolizing the drug. For example, it is well-established that some drugs frequently have lower efficacy in some individuals than others, which means such individuals and their physicians must weigh the possible benefit of a larger dosage against a greater risk of side effects. Also, there is significant variation in how well people metabolize drugs and other exogenous chemicals, resulting in substantial interindividual variation in the toxicity and/or efficacy of such exogenous substances (Evans et al., 1999, Science 286:487-491). This variability in efficacy or toxicity of a drug in genetically-diverse patients makes many drugs ineffective or even dangerous in certain groups of the population, leading to the failure of such drugs in clinical trials or their early withdrawal from the market even though they could be highly beneficial for other groups in the population. This problem significantly increases the time and cost of drug discovery and development, which is a matter of great public concern.

It is well-recognized by pharmaceutical scientists that considering the impact of the genetic variability of pharmaceutically-important proteins in the early phases of drug discovery and development is likely to reduce the failure rate of candidate and approved drugs (Marshall A 1997 Nature Biotech 15:1249-52; Kleyn PW et al. 1998 Science 281: 1820-21; Kola 1 1999 Curr Opin Biotech 10:589-92; Hill AVS et al. 1999 in Evolution in Health and Disease Stearns SS (Ed.) Oxford University Press, New York, pp 62-76; Meyer U.A. 1999 in Evolution in Health and Disease Stearns SS (Ed.) Oxford University Press, New York, pp 41-49; Kalow W et al. 1999 Clin. Pharm. Therap. 66:445-7; Marshall, E 1999 Science 284:406-7; Judson R et al. 2000 Pharmacogenomics 1:1-12; Roses AD 2000 Nature 405:857-65). However, in practice this has been difficult to do, in large part because of the time and cost required for discovering the amount of genetic variation that exists in the population (Chakravarti A 1998 Nature Genet 19:216-7; Wang DG et al 1998 Science 280:1077-82; Chakravarti A 1999 Nat Genet 21:56-60 (suppl); Stephens JC 1999 Mol. Diagnosis 4:309-317; Kwok PY and Gu S 1999 Mol. Med. Today 5:538-43; Davidson S 2000 Nature Biotech 18:1134-5).

The standard for measuring genetic variation among individuals is the haplotype, which is 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 JRespir Crit Care Med 161: 469-74) and drug response (Wolfe CR et al. 2000 BMJ 320:987-90; Dahl BS 1997 Acta Psychiatr Scand 96 (Suppl 391): 14-21), in many other cases an individual polymorphism may be found in a variety of genomic backgrounds, i.e., different haplotypes, and therefore shows no definitive coupling between the polymorphism and the causative site for the phenotype (Clark AG et al. 1998 Am JHum Genet 63:595-612; Ulbrecht M et al. 2000 supra; Drysdale et al. 2000 PNAS 97: 10483-10488). Thus, there is an unmet need in the pharmaceutical industry for information on what haplotypes exist in the population for pharmaceutically-important genes. Such haplotype information would be useful in improving the efficiency and output of several steps in the drug discovery and development process, including target validation, identifying lead compounds, and early phase clinical trials (Marshall et al., supra).

One pharmaceutically-important gene for the treatment of lung cancer is the Ν- acetyltransferase 1 (arylamine Ν-acetyltransferase) (ΝAT1) gene or its encoded product. ΝAT1 catalyzes the acetylation of arylamines and hydrazines, and is involved in the activation and detoxification of carcinogens. NATl is mainly expressed in erythrocytes, bladder, lymphocytes and neural tissue, as well as in liver and intestine. NATl also plays an endogenous role in the acetylation of the folate catabolite, p-aminobenzoyl-L-glutamate (pABGlu) to produce the major urinary product, N-acetyl-pABGlu (Smelt et al., Hum. Mol. Genet. 2000; 9: 1101-1107).

The N-acetyltransferase -l(arylamine N-acetyltransferase) gene is located on chromosome 8p23.1-p21.3 and contains 1 exon that encodes a 290 amino acid protein. Reference sequences for the NATl gene (Genaissance Reference No. 2517962; SEQ ID NO: 1), coding sequence (GenBank Accession No:NM_000662.1), and protein are shown in Figures 1, 2 and 3, respectively.

Several polymorphismsm in the NATl gene have been reported. The polymorphic variants of the NATl gene affect the bioactivation of carcinogens and therefore have implications in cancer risk (Butcher et al., Pharmacogenetics 1998; 8:67-72). These include the following polymorphisms in Figure 1 : a cytosine or thymine at a position corresponding to nucleotide 97, an adenine or thymiηe at a position corresponding to nucleotide position 401, a guanine or adenine at a position corresponding to nucleotide position 885, a guanine or adenine at a position corresponding to nucleotide position 899 and a thymine or guanine at a position corresponding to nucleotide position 1080. The polymorphisms at nucleotide positions 885 and 1080 result in amino acid variations of valine or isoleucine at amino acid position 149 (NATl* 11), and a serine or alanine at amino acid position 214 (NAT1*25) in Figure 3, respectively (Doll et al., Biochem. Biophys. Res. Commun. 1997; 233:584-591, SWISS-PROT.P18440).

Bucher et al., (supra) have also reported that a polymorphism of guanine or adenine at a position corresponding to nucleotide position 1000 in Figure 1 which results in the amino acid variation of an arginine or glutamine at amino acid position 187 (NATl* 14) in Figure 3. This variant has reduced acetylation capacity.

NATl polymorphisms play an important role in susceptibility to lung cancer by influencing the acetylation of tobacco derived carcinogens. Bouchardy et al. (Pharmacogenetics 1998; 8:291- 298) evaluated 150 lung cancer patients for genetic variants of NATl, all of whom were French Caucasian smokers. They found that subjects carrying the slow acetylator variant, NAT1*14, had the highest risk for lung cancer suggesting that this polymorphism modifies an individual's susceptibility to smoking-induced lung cancer. Because of the potential for variation in the NATl gene to affect the expression and function of the encoded protein, it would be useful to know whether additional polymorphisms exist in the NATl gene, as well as how such polymorphisms are combined in different copies of the gene. Such information could be applied for studying the biological function of NATl as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function. SUMMARY OF THE INVENTION

Accordingly, the inventors herein have discovered 5 novel polymorphic sites in the NATl gene. These polymorphic sites (PS) correspond to the following nucleotide positions in Figure 1: 97 (PS1), 163 (PS2), 288 (PS3), 405 (PS5), and 461 (PS6), The polymo hisms at these sites are cytosine or thymine at PS1, thymine or adenine at PS2, guanine or adenine at PS3, adenine or thymine at PS5, thymine or guanine at PS6,. In addition, the inventors have determined the identity of the alleles at these sites, as well as at the previously identified sites at nucleotide positions 401 (PS4),885 (PS7), 899 (PS8), 1000 (PS9) and 1080 (PS10), 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. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS 1-10 in the NATl gene, which are shown below in Tables 5 and 4, respectively. Each of these NATl haplotypes defines a naturally-occurring isoform (also referred to herein as an "isogene") of the NATl gene that exists in the human population.

Thus, in one embodiment, the invention provides a method, composition and kit for genotyping the NATl 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 PS1, PS2, PS3, PS5 and PS6 in both copies of the NATl gene from the individual. A genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel NATl polymorphic sites. A genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel NATl polymorphic sites. In a preferred embodiment, the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS 1-10. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 4 below or has one of the haplotype pairs in Table 3 below.

The invention also provides a method for haplotyping the NATl gene in an individual. In one embodiment, the haplotyping method comprises determining, for one copy of the NATl gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS1, PS2, PS3, PS5 and PS6. In another embodiment, the haplotyping method comprises determining whether one copy of the individual's NATl gene is defined by one of the NATl haplotypes shown in Table 4, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises deteπnining whether both copies of the individual's NATl gene are defined by one of the NATl haplotype pairs shown in Table 3 below, or a sub-haplotype pair thereof. The method for establishing the NATl 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 for treating diseases associated with NATl activity, e.g., lung cancer.

For example, the haplotyping method can be used by the pharmaceutical research scientist to validate NATl as a candidate target for treating a specific condition or disease predicted to be associated with NATl activity. Determining for a particular population the frequency of one or more of the individual NATl haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue NATl as a target for treating the specific disease of interest. In particular, if variable NATl activity is associated with the disease, then one or more NATl haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls. Conversely, if each of the observed NATl haplotypes are of similar frequencies in the disease and control groups, then it may be inferred that variable NATl activity has little, if any, involvement with that disease. In either case, the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any NATl haplotype or haplotype pair, apply the information derived from detecting NATl haplotypes in an individual to decide whether modulating NATl activity would be useful in treating the disease.

The claimed invention is also useful in screening for compounds targeting NATl to treat a specific condition or disease predicted to be associated with NATl activity. For example, detecting which of the NATl 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 of the most frequent NATl isoforms present in the disease population. Thus, without requiring any a priori knowledge of the phenotypic effect of any particular NATl haplotype or haplotype pair, the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.

^• The method of haplotyping the NATl 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 NATl. For example, for a lead compound that is metabolized by NATl, the pharmaceutical scientist of ordinary skill would be concerned that a favorable efficacy and/or side effect profile shown in a Phase II or Phase III 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 of the NATl gene that makes them genetically predisposed to metabolize the drug more efficiently than patients with other forms of the NATl gene. Similarly, this pharmaceutical scientist would recognize the potential for bias in the results of a Phase II or Phase III clinical trial of a drug targeting NATl that could be introduced if individuals whose NATl 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 NATl by practicing the claimed invention. In particular, by determining which of the NATl haplotypes disclosed herein are present in individuals recruited to participate in a clinical trial of a drug metabolized by or targeting NATl, the pharmaceutical scientist can then assign that individual to the treatment or control group as appropriate to ensure that approximately equal frequencies of the different NATl haplotypes (or haplotype pairs) are represented in the two groups and/or the frequencies of different NATl haplotypes or haplotype pairs are similar to the frequencies in the general population. Thus, by practicing the claimed invention, the pharmaceutical scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any NATl haplotype or haplotype pair.

In another embodiment, the invention provides a method for identifying -an association between a trait and a NATl genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein. The method comprises comparing the frequency of the NATl genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the NATl genotype or haplotype in a reference population. A higher frequency of the NATl genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the NATl genotype, haplotype, or haplotype pair. In preferred embodiments, the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug. In a particularly preferred embodiment, the NATl haplotype is selected from the haplotypes shown in Table 4, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic treatments for lung cancer.

In yet another embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the NATl gene or a fragment thereof. The reference sequence comprises SEQ ID NO:l and the polymorphic variant comprises at least one polymorphism selected from the group consisting of thymine at PS1, adenine at PS2, adenine at PS3, thymine at PS5, and guanine at PS6. In a preferred embodiment, the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of thymine at PS4, adenine at PS7, adenine at PS8, adenine at PS9 and guanine atPSlO.

A particularly preferred polymorphic variant is an isogene of the NATl gene. A NATl isogene of the invention comprises cytosine or thymine at PS1, thymine or adenine at PS2, guanine or adenine at PS3, adenine or thymine at PS4, adenine or thymine at PS5, thymine or guanine at PS6, guanine or adenine at PS7, guanine or adenine at PS8, guanine or adenine at PS9 and thymine or guanine at PS 10. The invention also provides a collection of NATl isogenes, referred to herein as a NATl genome anthology.

In another embodiment, the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a NATl cDNA or a fragment thereof. The reference sequence comprises SEQ LD NO:2 (Fig.2) and the polymorphic cDNA comprises guanine at a position corresponding to nucleotide 21. In a preferred embodiment, the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of adenine at a position corresponding to nucleotide 445, adenine at a position corresponding to nucleotide 459, adenine at a position corresponding to nucleotide 560 and guanine at a position corresponding to nucleotide 640. A particularly preferred polymorphic cDNA variant comprises the coding sequence of a NATl isogene defined by haplotypes 2-7.

Polynucleotides complementary to these NATl genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants of the NATl-gene will be useful in studying the expression and function of NATl, and in expressing NATl protein for use in screening for candidate drugs to treat diseases related to NATl activity.

In other embodiments, the invention provides a recombinant expression vector comprising one of the polymorphic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression.vector. The recombinant vector and host cell may be used to express NATl for protein structure analysis and drug binding studies.

The present invention also provides nonhuman transgenic animals comprising one of the NATl polymorphic genomic variants described herein and methods for producing such animals. The transgenic animals are useful for studying expression of the NATl isogenes in vivo, for in vivo screening and testing of drugs targeted against NATl protein, and for testing the efficacy of therapeutic agents and compounds for lung cancer in a biological system.

The present invention also provides a computer system for storing and displaying polymorphism data determined for the NATl gene. The computer system comprises a computer processing unit; a display; and a database containing the polymorphism data. The polymorphism data includes the polymorphisms, the genotypes and the haplotypes identified for the NATl gene in a reference population. In a preferred embodiment, the computer system is capable of producing a display showing NATl haplotypes organized according to their evolutionary relationships.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates a reference sequence for the NATl gene (Genaissance Reference No. 2517962; contiguous lines; SEQ LO NO:l), 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:29 is equivalent to Figure 1, with the two alternative allelic variants of each polymorphic site indicated by the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WLPO standard ST.25).

Figure 2 illustrates a reference sequence for the NATl coding sequence (contiguous lines; SEQ LD 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 NATl protein (contiguous lines; SEQ LD NO:3), with the variant amino acid(s) caused by the polymorphism(s) of Figure 2 positioned.below the polymorphic site in the sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the discovery of novel variants of the NATl gene. As described in more detail below, the inventors herein discovered 8 isogenes of the NATl gene by characterizing the NATl 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 (CA) (22 individuals), African descent (AF) (20 individuals), Asian (AS) (20 individuals), or Hispanic/Latino (HL) (17 individuals). To the extent possible, the members of this reference population were organized into population subgroups by the self-identified ethnogeographic origin of their four grandparents as shown in Table 1 below.

In addition, 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 NATl isogenes present in the human reference population are defined by haplotypes for 10 polymorphic sites in the NATl gene, 5 of which are believed to be novel. The NATl polymorphic sites identified by the inventors are referred to as PS1-10 to designate the order in which they are located in the gene (see Table 2 below), with the novel polymorphic sites referred to as PS1, PS2, PS3, PS5 and PS6. Using the genotypes identified in the Index Repository for PS1-10 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the NATl gene present in individual human members of this, repository. The human genotypes and haplotypes found in the repository for the NATl gene include those shown in Tables 3 and 4, respectively. The polymorphism and haplotype data disclosed herein are useful for validating whether NATl is a suitable target for drugs to treat lung cancer, screening for such drugs and reducing bias in clinical trials of such drugs.

In the context of this disclosure, the following terms shall be defined as follows unless otherwise indicated:

Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.

Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.

Gene - A segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

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. As used herein, genotype includes a full-genotype and or a sub-genotype as described below.

Full-genotype - The unphased 5 "to 3 ' sequence of nucleotide pairs found at all known polymorphic sites in a locus on a pair of homologous chromosomes in a single individual.

Sub-genotype - The unphased 5' to 3' sequence of nucleotides seen at a subset of the known polymorphic sites in a locus on a pair of homologous chromosomes in a single individual.

Genotyping - A process for determining a genotype of an individual.

Haplotype - A 5' to 3' sequence of nucleotides found at one or more polymorphic sites in a locus on a single chromosome from a single individual. As used herein, haplotype includes a full- haplotype and/or a sub-haplotype as described below.

Full-haplotype - The 5 ' to 3 ' sequence of nucleotides found at all known polymorphic sites in a locus on a single chromosome from a single individual.

Sub-haplotype - The 5' to 3' sequence of nucleotides seen at a subset of the known polymorphic sites in a locus on a single chromosome from a single individual.

Haplotype pair - The two haplotypes found for a locus in a single individual.

Haplotyping - A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.

Haplotype data - Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.

Isoform - A particular form of a gene, mRNA, cDNA or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.

Isogene — One of the isoforms of a gene found in a population. An isogene contains all of the polymorphisms present in the particular isoform of the gene.

Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term "isolated" is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.

Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.

Naturally-occurring - A term used to designate that the object it is applied to, e.g.,. naturally-occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.

Nucleotide pair - The nucleotides found at a 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 of the locus is known.

Polymorphic site (PS) - A position within a locus at which at least' wo alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.

Polymorphic variant - A gene, mRNA, cDNA, polypeptide 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 of the 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 associations) 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 singleτstranded 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 of the 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 polymorphic sites on a single copy of the locus is not known.

As discussed above, information on the identity of genotypes and haplotypes for the NATl gene of any particular individual as well as the frequency of such genotypes and haplotypes in any particular population of individuals is expected to be useful for a variety of drug discovery and development applications. Thus, the invention also provides compositions and methods for detecting the novel NATl polymorphisms and haplotypes identified herein.

The compositions comprise at least one NATl genotyping oligonucleotide. In one embodiment, a NATl genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one of the novel polymorphic sites described herein. As used herein, the term "oligonucleotide" refers to a polynucleotide molecule having less than about 100 nucleotides. A preferred oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan. The oligonucleotide may be ^■ comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives. Alternatively, 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 (Var a, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc. (1995), pages 617-620). Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion. The oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.

Genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a NATl polynucleotide, i.e., a NATl isogene. As used herein, specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with a non-target region or a non-NATl polynucleotide under the same hybridizing conditions. Preferably, the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions. The skilled artisan can readily design and test oligonucleotide probes and primers suitable for detecting polymorphisms in the NATl gene using the polymorphism ihformation provided herein in conjunction with the known sequence information for the NATl gene and routine techniques.

A nucleic acid molecule such as an oligonucleotide or polynucleotide is^" said to be a "perfect" or "complete" complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule. A nucleic acid molecule is "substantially complementary" to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D. et al. in Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). While perfectly complementary oligonucleotides are preferred for detecting polymorphisms, departures from complete complementarity are contemplated where such departures do not prevent the molecule from specifically hybridizing to the target region. For example, an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary to the target region. Alternatively, non-complementary nucleotides may be interspersed into the oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.

Preferred genotyping oligonucleotides of the invention are allele-specific oligonucleotides. As used herein, the term allele-specific oKgonucleotide (ASO) means an oligonucleotide that is able, under sufficiently stringent conditions, to hybridize specifically to one allele of a gene, or other locus, at a target region containing a polymorphic site while not hybridizing to the corresponding region in another allele(s). As understood by the skilled artisan, 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., "Genetic Prediction of Hemophilia A" in PCR Protocols, A Guide to Methods and Applications, Academic Press, 1990 and Ruafio et al., 87 Proc. Natl. Acad. Sci. USA 6296-6300, 1990. Typically, an ASO will be perfectly complementary to one allele while containing a single mismatch for another allele.

Allele-specific oligonucleotides of the invention include ASO probes and ASO primers. ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymorphic 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 of the invention has a 3 ' teπninal 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.

ASO probes and primers listed below use the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WLPO standard ST.25) at the position of the polymorphic site to represent the two alternative allelic variants observed at that polymorphic site.

A preferred ASO probe for detecting NATl gene polymorphisms comprises a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:

CAGAAGAYGTTTATA (SEQ ID NO: 4) and its complement,

AGAACCAWGAACAAG (SEQ ID NO: 5) and its complement,

TGTAAAΑRGAATTCA (SEQ ID NO: 6) and its complement,

TAAA&TGWTTTGCTT (SEQ ID NO: 7) and its complement, and

CATATCTKGAAAGAA (SEQ ID NO: 8) and its complement.

A preferred ASO primer for detecting NATl gene polymorphisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:

CTACATCAGAAGAYG (SEQ ID NO: 9); TAGGCTTATAAACRT (SEQ ID NO:10);

ACCAAGAGAACCAWG (SEQ ID NO: 11); 7AAACAGCTTGTTCWT (SEQ ID NO: 12);

TATGTGTGTAAAARG (SEQ ID -NO: 13); ATTG A GAATTCYT (SEQ ID NO: 14);

TAA^GTAAAATGWT (SEQ ID NO: 15); AAΑCGAA&GCAAAWC (SEQ ID NO: 16);

TTGAAGCATATCTKG (SEQ ID NO: 17); and AGCCAATTCTTTCMA (SEQ ID NO: 18).

Other genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymorphic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymorphisms described herein and therefore such genotyping oligonucleotides are referred to herein as "primer-extension oligonucleotides". In a preferred embodiment, the 3 '- terminus of a primer-extension oligonucleotide is a deoxynuclebtide complementary to the nucleotide located immediately adjacent to the polymorphic site.

A particularly preferred oligonucleotide primer for detecting NATl gene polymorphisms by primer extension terminates in a nucleotide sequence, listed 5' to 3', selected from the group consisting of:

CATCAGAAGA (SEQ ID NO: 19) ; GCTTATAAAC (SEQ ID NO:20);

AAGAGAACCA (SEQ ID NO: 21) ; CAGCTTGTTC (SEQ ID NO:22);

GTGTGTAAAA (SEQ ID NO: 23) ; GTATGAATTC (SEQ ID NO:24);

AAGTAAAATG (SEQ ID NO: 25) ; CGAAAGCAAA (SEQ ID NO:26);

AAGCATATCT (SEQ ID NO:27); and CAAITCTTTC (SEQ ID NO:28).

In some embodiments, a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymorphic 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 polymorphic site.

NATl genotyping oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such* s a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized genotyping oligonucleotides may be used in a variety of polymorphism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized NATl genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymoφhisms in multiple genes at the same time.

In another embodiment, the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers. The kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container. Alternatively, where the oligonucleotides are to be used to amplify a target region, 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 NATl gene in an individual. As used herein, the terms "NATl genotype" and "NATl haplotype" mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymoφhic sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymoφhic sites in the NATl gene.^' The additional polymoφhic sites may be currently known polymoφhic sites or sites that are subsequently discovered.

One embodiment of the genotyping method involves isolating from the individual a nucleic acid sample comprising the two copies of the NATl gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymoφhic sites selected from the group consisting of PS1, PS2, PS3, -PS5 and PS6 in the two copies to assign a NATl genotype to the individual. As will be readily understood by the skilled artisan, the two "copies" of a gene in an individual may be the same allele or may be different alleles. In a preferred embodiment of the genotyping method, the identity of the nucleotide pair at one or more of the polymoφhic^' sites selected from the group consisting of PS4 and PS7-PS10 is also determined. In a particularly preferred embodiment, the genotyping method comprises determining the identity of the nucleotide pair at each of PS1-10.

Typically, 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 NATl gene is expressed. Furthermore it will be understood by the skilled artisan that mRNA or cDNA preparations would not be used to detect polymoφhisms located in introns or in 5 ' and 3 ' untranslated regions. If a NATl gene fragment is isolated, it must contain the polymoφhic site(s) to be genotyped.

One embodiment of the haplotyping method comprises isolating from the individual a nucleic acid sample containing only one of the two copies of the NATl gene, or a fragment thereof, that is present in the individual and determining in that copy the identity of the nucleotide at one or more polymoφhic sites selected from the group consisting of PS1, PS2,, PS3, PS5 and PS6 in that copy to assign a NATl haplotype to the individual. The nucleic acid may be isolated using any method capable of separating the two copies of the NATl gene or fragment such as one of the methods described above for preparing NATl isogenes, with targeted in vivo cloning being the preferred approach. As will be readily appreciated by those skilled in the art, any individual clone will only provide haplotype mformation on one of the two NATl gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional NATl clones will need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the NATl gene in an individual. In some embodiments, the haplotyping method also comprises identifying the nucleotide at one or more polymoφhic sites selected from the group consisting of PS4 and PS7-PS10. In a particularly preferred embodiment, the nucleotide at each of PS1-10 is identified.

In another embodiment, the haplotyping method comprises determining whether ah individual has one or more of the NATl haplotypes shown in Table 4. This can be accomplished by identifying, for one or both copies of the individual's NATl gene, the phased sequence of nucleotides present at each of PS 1- 10. The present invention also contemplates that typically only a subset of PS1-10 will need to be directly examined to assign to an individual one or more of the haplotypes shown in Table 4. This is because at least one polymoφhic site in a gene is frequently in strong linkage disequilibrium with one or more other polymoφhic sites in that gene (Drysdale, CM et al. 2000 PNAS 97:10483-10488; Rieder MJ et al. 1999 Nature Genetics 22:59-62). Two sites are said to be in linkage disequilibrium if the presence of a particular variant at one site enhances the predictability of another variant at the second site (Stephens, JC 1999, Mol. Diag. 4:309-317). Techniques for determining whether any two polymoφhic 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).

In a preferred embodiment, a NATl haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymoφhic sites selected from the group consisting of PS1, PS2, PS3, PS5 and PS6 in each copy of the NATl gene that is present in the individual. In a particularly preferred embodiment, the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS 1-10 in each copy of the NATl gene. When haplotyping both copies of the gene, the identifying step is preferably performed with each copy of the gene being placed in separate containers. However, it is also envisioned that if 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. For example, if first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymoφhic site(s), then detecting a combination of the first and third dyes would identify the polymoφhism in the first gene copy while detecting a combination of the second and third dyes would identify the polymoφhism in the second gene copy.

In both the genotyping and haplotyping methods, the identity of a nucleotide (or nucleotide pair) at a polymoφhic site(s) may be determined by amplifying a target region(s) containing the polymoφhic site(s) directly from one or both copies of the NATl gene, or a fragment thereof, and the sequence of the 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 polymoφhic 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 polymoφhism may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification. For example, where a SNP is known to be guanine and cytosine in a reference population, a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guariine and cytosine, if the individual is heterozygous at that site. Alternatively, the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/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).

Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992).

A polymoφhism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art. Typically, allele-specific oligonucleotides are utilized in performing such methods. The allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant. In some embodiments, more than one polymoφhic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs. Preferably, the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymoφhic sites being detected.

Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross- linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis. Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads. The solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.

The genotype or haplotype for the NATl gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene, or fragments) 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 of the polymoφhic sites to be included in the genotype or haplotype.

The identity of polymoφhisms 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 (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variant alleles can be identified by single strand conformation polymoφhism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).

A polymerase-mediated primer extension method may also be used to identify the polymoφhism(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, W095/17676; U.S. Patent Nos. 5,302,509, and 5,945,283. Extended primers containing a polymoφhism may be detected by mass spectrometry as described in U.S. Parent No. 5,605,798. Another primer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res. 17:8392, 1989; Ruano et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995). In addition, multiple polymoφhic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).

In addition, the identity of the allele(s) present at any of the novel polymoφhic sites described herein may be indirectly determined by genotyping another pplymoφhic site that is in linkage disequilibrium with the polymoφhic site that is of interest. Polymoφhic sites in linkage disequilibrium with the presently disclosed polymoφhic sites may be located in regions of the gene or in other genomic regions not examined herein. Genotyping of a polymoφhic site in linkage disequilibrium with the novel polymoφhic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymoφhic site.

In another aspect of the invention, an individual's NATl haplotype pair is predicted from its NATl genotype using information on haplotype pairs known to exist in a reference population. In its broadest embodiment, the haplotyping prediction method comprises identifying a NATl genotype for the individual at two or more NATl polymoφhic sites described herein, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing NATl haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data. In one embodiment, the reference haplotype pairs include the NATl haplotype pairs shown in Table 4.

Generally, the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world. A preferred reference population for use in the methods of the present invention comprises an approximately equal number of individuals from Caucasian, African American, Asian and Hispanic-Latino population groups with the minimum number of each group being chosen based on how rare a haplotype one wants to be guaranteed to see. For example, if one wants to have a q% chance of not missing a haplotype that exists in the population at a p% frequency of occurring in the reference population, the number of individuals (n) who must be sampled is given by 2n=log(l-q)/log(l-p) where p and q are expressed as fractions. 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 of the four population groups named above. A particularly preferred reference population includes a 3- generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures.

In a preferred embodiment, the haplotype frequency data for each ethnogeόgraphic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L. Hartl et al., Principles of Population. Genomics, Sinauer Associates (Sunderland, MA), 3^rd Ed., 1997) postulates that the frequency of finding the haplotype pair H_l I H₂ is equal to p_H__w (H IH_) = 2p(H_x )p(H₂ ) if H_x ≠ H₂ and p_H__w (H IH₂) = p(H_x )p{H₂ ) if H = H₂ .

A statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System™ 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).

In one embodiment of this method for predicting a NATl haplotype pair for an individual, the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individudl is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair. Alternatively, 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 through a commercial haplotyping service such as offered by Genaissance Pharmaceuticals, Inc. (New Haven, CT). In rare cases, either no haplotypes in the reference population are consistent with the possible haplotype pairs, or alternatively, multiple reference haplotype pairs are consistent with the possible haplotype pairs. In such cases, the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System^™ technology (U.S. Patent No. 5,866,404), SMD, or allele- specific long-range PCR (Michalotos-Beloin et al., supra). The invention also provides a method for determining the frequency of a NATl genotype, haplotype, or haplotype pair in a population. The method comprises, for each member of the population, determining the genotype or the haplotype pair for the novel NATl polymoφhic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population. The population may be a reference population, a family population, a same sex 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).

In another aspect of the invention, frequency data for NATl 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 NATl 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 of the 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 of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods described above. The haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above. In another embodiment, 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. For example, the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes, haplotypes,' and/or haplotype pairs observed in the populations are compared. If a particular NATl genotype, haplotype, or haplotype pair is more frequent in the trait population than in the reference population at a statistically significant amount, then the trait is predicted to be associated with that NATl genotype, haplotype or haplotype pair. Preferably, the NATl 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 4 and 5, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.

In a preferred embodiment of the method, the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting NATl or response to a therapeutic treatment for a medical condition. As used herein, "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. As used herein the term "clinical response" means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects). In order to deduce a correlation between clinical response to a treatment and a NATl 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. As used herein, 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 IH clinical trials. Standard methods are used to define the patient population and to enroll subjects.,

It is preferred that the individuals included in the clinical population have been graded for the existence of the medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. 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. In addition, the NATl gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.

After both the clinical and polymoφhism data have been obtained, correlations between individual response and NATl genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their NATl genotype or haplotype (or haplotype pair) (also referred to as a polymoφhism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymoφhism group are calculated.

These results are then analyzed to determine if any observed variation in clinical response between polymoφhism groups is statistically significant. Statistical analysis methods which may be used are described in L.D. Fisher and G. vanBelle, "Biostatistics: A Methodology for the Health Sciences", Wiley-Interscience (New York) 1993. This analysis may also include a regression calculation of which polymoφhic sites in the NATl gene give the most significant contribution to the differences in phenotype. One regression model useful in the invention is described in PCT Application Serial No. PCT/US00/17540, entitled "Methods for Obtaining and Using Haplotype Data".

A second method for finding correlations between NATl 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. Knight, "Artificial Intelligence", 2^nd Edition (McGraw-Hill, New York, 1991, Ch. 18), standard gradient descent methods (Press et al., supra, Ch. 10), or other global or local optimization approaches (see discussion in Judson, supra) could also be used. Preferably, the correlation is found using a genetic algorithm approach as described in PCT Application Serial No. PCT/US00/17540.

Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymoφhic sites in the NATl gene. As described in PCT Application Serial No. . PCT/US00/17540, 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, siψra, Ch. 10).

From the analyses described above, a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of NATl genotype or haplotype content. Preferably, 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 NATl gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug. The diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymoφhic sites in the NATl gene), a serological test, or a physical exam measurement. The only requirement is that there be a good correlation between the diagnostic test results and the underlying NATl genotype or haplotype that is in turn correlated with the clinical response. In a preferred embodiment, this diagnostic method uses the predictive haplotyping method described above.

In another embodiment, the invention provides an isolated polynucleotide comprising a polymoφhic variant of the NATl gene or a fragment of the gene which contains at least one of the novel polymoφhic sites described herein. The nucleotide sequence of a variant NATl gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymoφhic sites PSl, PS2, PS3, PS5 and PS6, and may also comprise one or more additional polymoφhisms selected from the group consisting of thymine at PS4, adenine at PS7, adenine at PS8, adenine at PS9 and guanine at PS10. Similarly, the nucleotide sequence of a variant fragment of the NATl gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymoφhic sites described herein. Thus, the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the NATl gene, which is defined by haplotype 1, (or other reported NATl sequences) or to portions of the reference sequence (or other reported NATl sequences), except for genotyping oligonucleotides as described below.

The location of a polymoφhism in a variant gene or fragment is identified by aligning its sequence against SEQ ID NO:l. The polymoφhism is selected from the group consisting of thymine at PSl, adenine at PS2, adenine at PS3, thymine at PS5, and guanine at PS6. In a preferred embodiment, the polymoφhic variant comprises a natarally-occurring isogene of the NATl gene which is defined by any one of haplotypes 2-7 shown in Table 4 below.

Polymoφhic variants of the invention may be prepared by isolating a clone containing the NATl gene from a human genomic library. The clone may be sequenced to determine the identity of the nucleotides at the novel polymoφhic sites described herein. Any particular variant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well- known in the art.

NATl isogenes may be isolated using any method that allows separation of the two "copies" of the NATl 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. Yet other methods are single molecule dilution (SMD) as described in- Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruano et al, 1989, supra; Ruano et al., 1991, supra; Michalatos-Beloin et al., supra).

The invention also provides NATl genome anthologies, which are collections of NATl isogenes found in a given population. The population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same sex population. A NATl genome anthology may comprise individual NATl isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the NATl isogenes in the anthology may be stored in separate containers. Individual isogenes or groups of 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 NATl genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 4 below.

An isolated polynucleotide containing a polymoφhic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded NATl protein in a prokaryotic or a eukaryotic host cell. Examples of 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. Other 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 of the nucleic acid sequence in a given host cell. Of course, the correct combinations of expression regulatory elements will depend on the host system used. In addition, it is understood that 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 NATl sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art. The recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). In a preferred aspect, 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, heφes virus vectors, and baculovirus transfer vectors. Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryomc stem cells (Thomson, J. A. et al., 1998 Science 282: 1145-1147). Particularly preferred host cells are mammalian cells.

As will be readily recognized by the skilled artisan, expression of polymoφhic variants of the NATl gene will produce NATl mRNAs varying from each other at any polymoφhic site retained in the spliced and processed mRNA molecules. These mRNAs can be used for the preparation of a NATl cDNA comprising a nucleotide sequence which is a polymoφhic variant of the NATl reference coding sequence shown in Figure 2. Thus, the invention also provides NATl mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ LD NO:2 (Fig. 2), or its corresponding RNA sequence, except for having guanine at a position corresponding to nucleotide 21, and may also comprise one or more additional polymoφhisms selected from the group consisting of adenine at a position corresponding to nucleotide 445, adenine at a position corresponding to nucleotide.459, adenine at a position corresponding to nucleotide 560 and guanine at a position corresponding to nucleotide 640. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of a NATl isogene defined by haplotypes 2-7. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain the novel polymoφhism described herein. The invention specifically excludes polynucleotides identical to previously identified and characterized NATl cDNAs and fragments thereof. Polynucleotides comprising a variant RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.

As used herein, a polymoφhic variant of a NATl gene fragment comprises at least one novel polymoφhism identified herein and has a length of at least 10 nucleotides and* may range up to the full length of the gene. Preferably, such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.

In describing the NATl polymoφhic sites identified herein, reference is made to the sense strand of the gene for convenience. However, as recognized by the skilled artisan, nucleic acid molecules containing the NATl gene 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. Thus, reference may be made to the same polymoφhic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymoφhic site. Thus, the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the NATl genomic variants described herein.

Polynucleotides comprising a polymoφhic gene variant or fragment may be useful for therapeutic puφoses. For example, where a patient could benefit from expression, or increased expression, of a particular NATl protein isoform, an expression vector encoding the isoform may be administered to the patient. The patient may be one who lacks the NATl isogene encoding that isoform or may already have at least one copy of that isogene.

In other situations, it may be desirable to decrease or block expression of a particular NATl isogene. Expression of a NATl isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA for the isogene. Alternatively, oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred. Similarly, inhibition of transcription can be achieved using oligonucleotides that base- pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). Antisense oligonucleotides may also be designed to block translation of NATl mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of NATl mRNA transcribed from a particular isogene.

The oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo. Alternatively, the oligonucleotides may be formulated as a pharmaceutical composition for administration to the patient: Oligoribonucleptides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may^' be modified to increase stability and half-life. Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.

Effect(s) of the polymoφhisms identified herein on expression of NATl may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymoφhic variant of the NATl gene. As used herein, "expression" includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into NATl protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.

To prepare a recombinant cell of the invention, the desired NATl isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by.the cell from the extrachromosomal location. In a preferred embodiment, the NATl isogene is introduced into a cell in such a way that it recombines with the endogenous NATl gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired NATl gene polymoφhism. 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 of the skilled practitioner. Examples of cells into which the NATl isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NTH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the NATl isogene. Such recombinant cells can be used to compare the biological activities of the different protein variants.

Recombinant nonhuman organisms, i.e., transgenic animals, expressing a variant NATl gene are prepared using standard procedures known in the art. Preferably, a construct comprising the variant gene is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage. Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art. One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053. Another method involves directly injecting a transgene into the embryo. A third method involves the use of embryonic stem cells. Examples of animals into which the NATl isogenes 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 NATl isogene and producing human NATl protein can be used as biological models for studying diseases related to abnormal NATl 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 of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel NATl isogene described herein. The pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel NATl isogenes; an antisense oligonucleotide directed against one of the novel NATl isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel NATl isogene described herein. Preferably, the composition contains the active ingredient in a therapeutically effective amount. By therapeutically effective amount is meant that one or more of the symptoms relating to disorders affected by expression or function of a novel NATl isogene is reduced and/or eliminated. The composition also comprises 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 of the 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).

For any composition, determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within the capability of those skilled in the art. For example, 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 of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.

Any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer. In addition, 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 NATl gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymoφhism 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 NATl polymoφhism 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). These polymoφhism 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. For example, the data may be stored on one or more databases in communication with the computer via a network.

Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

EXAMPLES The Examples herein are meant to exemplify the various aspects of carrying out the invention and are not intended to limit the scope of the invention in any way. The Examples do not include detailed descriptions for conventional methods employed, such as in the performance of genomic DNA isolation, PCR and sequencing procedures. Such methods are well-known to those skilled in the art and are described in numerous publications, for example, Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual", 2^nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989).

EXAMPLE 1 This example illustrates examination of various regions of the NATl gene for polymoφhic sites.

Amplification of Target Regions

The following target regions of the NATl gene were amplified using PCR primer pairs. The primers used for each region represented below by providing the nucleotide positions of their and final nucleotides, which correspond to positions in Figure 1.

PCR Primer Pairs

Fragment No. Forward Primer Reverse Primer PCR Product

Fragment 1 22-43 complement of 579-557 558 nt

Fragment 2 148-171 complement of 840-820 693 nt

Fragment 3 432-454 complement of 1168-1147 , 737 nt

Fragment 4 ^' 741-762 complement of 1373-1351 633 nt

These primer pairs were used in PCR reactions containing genomic DNA isolated from immortalized cell lines for each member of the Index Repository. The PCR reactions were carried out under the following conditions:

Reaction volume ^{' ■} = 10 μl

10 x Advantage 2 Polymerase reaction buffer (Clontech) = 1 μl

100 ng of human genomic DNA = 1 μl lO M dNTP = 0.4 μl

Advantage 2 Polymerase enzyme mix (Clontech) = 0.2 μl

Forward Primer (10 μM) = 0.4 μl

Reverse Primer (10 μM) = 0.4 μl

Water = 6.6μl

Amplification profile: 97°C - 2 min. 1 cycle

10 cycles

97°C - 15 sec. ~ι

64°C - 45 sec. L 35 cycles

72°C - 45 sec. J

Sequencing of PCR Products

The PCR products were purified using a Whatman Polyfiltrόnics 100 μl 384 well unifilter plate essentially according to the manufacturers protocol. The purified DNA was eluted in 50 μl 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 the primer sets described previously or those represented below by the nucleotide positions of their initial and final nucleotides, which correspond to positions in Figure 1.. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.

Sequencing Primer Pairs

Fragment No. Forward Primer Reverse Primer Fragment 1 68-88 complement of 543-523 Fragment 2 243-264 complement of 76.5-746 Fragment 3 535-554 complement of 1084-1063 Fragment 4 765-784 complement of 1284-1265

Analysis of Sequences for Polymorphic Sites

Sequences were analyzed for the presence of polymoφhisms using the Polyphred program (Nickerson et al., Nucleic Acids Res. 14:2745-2751, 1997). The presence of a polymoφhism was confirmed on both strands. The polymoφhisms and their locations in the NATl gene are listed in Table 2 below.

Table 2. Polymoφhic Sites Identified in the NATl Gene

Polymoφhic Nucleotide Reference Variant CDS Variant AA

Site Number PolyId^a Position Allele Allele Position Variant

PSl 2517973 . 97 * C T

PS2 2517975 163 T A

PS3 2517977 . 288 G A

PS4^R 2517981 401 A T

PS5 2517983 405 A T '

PS6 2517987 461 T G 21 L7L

PS7^R 2517999 885 G A 445 V149I

PS8^R 2518001 899 G A 459 T153T

PS9^R 2518007 1000 G A 560 R187Q

PS10^R 2518011 1080 T G 640 S214A ^aPolyId is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc. ^R Previously reported in literature

EXAMPLE 2

This example illustrates analysis of the NATl polymoφhisms identified in the Index Repository for human genotypes and haplotypes.

The different genotypes containing these polymoφhisms that were observed in the 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. In 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 and Haplotype Pairs Observed for NATl Gene Genotype , Polymorphic Sites

Number , PSl PS2 ^• PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 HAP Pair

1 c T G A A T G G G T 1 1

2 c T G A A/T T G G G - T 1 7

3 c T G A A ^' T/G G G G T 1 5

4 C T G/A A A T G G G T 1 6

5 C T G A A T G G^' G/A T 1 ^' 4

6 C T/A G A A T G G- G T 1 3

:/τ T G A/T A T . G/A G/A G T/G 1 2

The haplotype pairs shown in Table 3 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G.1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to umelated individuals in a population sample. In this method, haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites. This list of haplotypes is augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African- American family) and then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.

By following this protocol, it was determined that the Index Repository examined herein and, by extension, the general population contains the 7 human NATl haplotypes shown in Table 4 below.

Table 4. Haplotypes Identified in the NATl Gene Haplotype Polymorphic Sites

PS PS PS PS PS PS PS PS PS PS Number 1 2 3 4 5 6 7 8 9 10

^' 1 ^' C T G A A T G G G T

2 T T G T A ^' A A G^' G

3 C A G A A T G G G T

4 C T G A A T G G A T

5 C T G A A G G G G T

6 C T A A A T G G G T

7 C T G A T T G G. G T

In Table 5 below, the number of chromosomes characterized by a given haplotype is shown, arranged by the ethnic background of the subjects in the index repository. In Table 6 below, the number of subjects characterized by a given haplotype pair is shown, arranged by the ethnic background of the subjects in the index repository. In Tables 5 and 6, the following abbreviations are used: AF, African or African- American; AS, Asian; CA, Caucasian; HL, Hispanic-Latino; and AM, Native Americans.

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above ^• description and shown in the accompanying drawings shall be inteφreted as illustrative and not in a limiting sense.

All references cited in this specification, including patents and patent applications, are hereby incoφorated in their entirety by reference. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.

Claims

What is Claimed is:

1. A method for haplotyping the N-acetyltransferase 1 (arylamine N-acetyltransferase) (NAT 1 ) gene of an individual which comprises determining whether the individual has one of the NATl haplotypes shown in Table 4 or one of the haplotype pairs shown in Table 3.

2. The method of claim 1, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PS 1-10 on at least one copy of the individual's NATl gene.

3. The method of claim 1, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PS1-10 on both copies of the individual's NATl gene.

4. A method for genotyping the N-acetyltransferase 1 (arylamine N-acetyltransferase) (NATl) gene of an individual, comprising determining for the two copies of the NATl gene present in the individual the identity of the nucleotide pair at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS5 and PS6.

5.¹ The method of claim 4, wherein the determining step comprises:

(a) isolating from the individual a nucleic acid mixture comprising both copies of the NATl . gene, or a fragment thereof, that are present in the individual;

(b) amplifying from the nucleic acid mixture a target region containing the selected polymoφhic site;

(c) hybridizing a primer extension oligonucleotide to one allele of the amplified target region;

(d) performing a nucleic acid template-dependent, primer extension reaction on the hybridized genotyping oligonucleotide in the presence of at least two different terminators of the reaction, wherein said terminators are complementary to the alternative nucleotides present at the selected polymoφhic site; and

(e) detecting the presence and identity of the terminator in the extended genotyping oligonucleotide.

6. The method of claim 4, which comprises determining for the two copies of the NATl gene present in the individual the identity of the nucleotide pair at each of PS1-10.

7. A method for haplotyping the N-acetyltransferase l(arylamine N-acetyltransferase) (NATl) gene of an individual which comprises determining, for one copy of the NATl gene present in the individual, the identity of the nucleotide at two or more polymoφhic sites selected from the group consisting of PS 1, PS2, PS3, PS5 and PS6.

8. The method of claim 7, further comprising determining the identity of the nucleotide at one or more polymoφhic sites selected from the group consisting of PS4, PS7, PS8, PS9 and PS10.

9. The method of claim 7, wherein the determining step comprises:

(a) isolating from the individual a nucleic acid sample containing only one of the two copies of the NATl gene, or a fragment thereof, that is present in the individual;

(b) amplifying from the nucleic acid molecule a target region containing the selected polymoφhic site;

(c) hybridizing a primer extension oligonucleotide to one allele of the amplified target region;

(d) performing a nucleic acid template-dependent, primer extension reaction on the hybridized genotyping oligonucleotide in the presence of at least two different terminators of the reaction, wherein said terminators are complementary to the alternative nucleotides present at the selected polymoφhic site; and

(e) detecting the presence and identity of the terminator in the extended genotyping oligonucleotide.

10. A method for predicting a haplotype pair for the N-acetyltransferase 1 (arylamine N- acetyltransferase) (NATl) gene of an individual comprising:

(a) identifying a NATl genotype for the individual, wherein the genotype comprises the nucleotide pair at two or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS5 and PS6;

(b) enumerating all possible haplotype pairs which are consistent with the genotype;

(c) comparing the possible haplotype pairs to the data in Table 4; and

(d) assigning a haplotype pair to the individual that is consistent with the data.

11. The method of claim 10, wherein the identified genotype of the individual comprises the nucleotide pair at each of PS1-10.

12. A method for identifying an association between a trait and at least one haplotype or haplotype pair of the N-acetyltransferase 1 (arylamine N-acetyltransferase) (NATl) gene which comprises comparing the frequency of the haplotype or haplotype pair in a population exhibiting the trait with the frequency of the haplotype or haplotype pair in a reference . population, wherein the haplotype is selected from haplotypes 1-7 shown in Table 4 and the haplotype pair is selected from the haplotype pairs shown in Table 4, wherein a higher frequency of the haplotype or haplotype pair in the trait population than in the reference population indicates the trait is associated with the haplotype or haplotype pair.

13. The method of claim 12, wherein the trait is a clinical response to a drug targeting or metabolized by NATl .

14. A composition comprising at least one genotyping oligonucleotide for detecting a polymoφhism in the N-acetyltransferase 1 (arylamine N-acetyltransferase) (NATl) gene at a polymoφhic site selected from the group consisting of PSl, PS2, PS3, PS5 and PS6.

15. The composition of claim 14, wherein the genotyping oligonucleotide is an allele-specific oligonucleotide that specifically hybridizes to an allele of the NATl gene at a region containing the polymoφhic site.

16. The composition of claim 15, wherein the allele-specific oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ LD NOS:4 -8, the complements of SEQ ID NOS:4-8, and SEQ ID NOS:9-18.

17. The composition of claim 14, wherein the genotyping oligonucleotide is a primer-extension oligonucleotide.

18. The composition of claim.17, wherein the primer extension oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 19-28.

19. A kit for genotyping the NATl gene of an individual, which comprises a set of oligonucleotides designed to genotype each of PSl, PS2, PS3, PS5 and PS6.

20. The kit of claim 19, which further comprises oligonucleotides designed to genotype each of PS4 and PS7, PS8, PS9 and PS10.

21. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) a first nucleotide sequence which is a polymoφhic variant of a reference .sequence for the N-acetyltransferase 1 (arylamine N-acetyltransferase) (NATl) gene or a fragment thereof, wherein the reference sequence comprises SEQ LD NO:l and the polymoφhic variant comprises a NATl isogene defined by a haplotype selected from the group consisting of haplotypes 1-7 in Table 4; and

(b) a second nucleotide sequence which is complementary to the first nucleotide sequence.

22. The isolated polynucleotide of claim 21 , which is a DNA molecule and comprises both the first and second nucleotide sequences and further comprises expression regulatory elements operably linked to the first nucleotide sequence.

23. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 21 , wherein the organism expresses a NATl protein encoded by the first nucleotide sequence.

24. The recombinant organism of claim 23, which is a nonhuman transgenic animal.

25. The isolated polynucleotide of claim 21 , wherein the first nucleotide sequence is a polymoφhic variant of a fragment of the NATl gene, the fragment comprising one or more polymoφhisms selected from the group consisting of thymine at PSl, adenine at PS2, adenine at PS3, thymine at PS5, and guanine at PS6.

26. An isolated polynucleotide comprising a nucleotide sequence which is a polymoφhic variant of a reference sequence for the NATl cDNA or a fragment thereof, wherein the reference sequence comprises SEQ LD NO:2 and the polymoφhic variant comprises the coding sequence of a NATl isogene defined by one of the haplotypes shown in Table 4.

27. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 26, wherein the organism expresses a N-acetyltransferase 1 (arylamine N-acetyltransferase) (NATl) protein encoded by the polymoφhic variant sequence.

28. The recombinant organism of claim 27, which is a nonhuman transgenic animal.

29. A computer system for storing and analyzing polymoφhism data for the N-acetyltransferase 1 (arylamine N-acetyltransferase) gene, comprising:

(a) a central processing unit (CPU);

(b) a communication interface;

(c) a display device;

(d) an input device; and

(e) a database containing the polymoφhism data; wherein the polymoφhism data comprises the genotypes and haplotype pairs shown in Table 3 and the haplotypes shown in Table 4.

30. A genome anthology for the N-acetyltransferase l(arylamine N-acetyltransferase) (NATl) gene which comprises NATl isogenes defined by any one of haplotypes 1-7 shown in Table 4.