WO2001079231A2 - Haplotypes of the npr1 gene - Google Patents

Abstract

Novel single nucleotide polymorphisms in the human natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPR1) gene are described. In addition, various genotypes, haplotypes and haplotype pairs for the NPR1 gene that exist in the population are described. Compositions and methods for haplotyping and/or genotyping the NPR1 gene in an individual are also disclosed. Polynucleotides containing one or more of the NPR1 polymorphisms disclosed herein are also described.

Description

HAPLOTYPES OF THE NPRl GENE

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 60/197,330 filed April 14, 2000.

FIELD OF THE INVENTION

This invention relates to variation in genes that encode pharmaceutically-important proteins. In particular, this invention provides genetic variants ofthe human natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) 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 ofthe 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 absoφtion, 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 polymoφhisms 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 polymoφhisms. 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 Ada Psychiatr Scand 96 (Suppl 391): 14-21), in many other cases an individual polymoφhism may be found in a variety of genomic backgrounds, i.e., different haplotypes, and therefore shows no definitive coupling between the polymoφhism and the causative site for the phenotype (Clark AG et al. 1998 Am J Hum 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 hypertension is the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene or its encoded product. NPRl, also known as NPRA, is a receptor that binds to atrial natriuretic peptides (ANP). ANP produced in the heart causes vasodilation and natriuresis, which are important for the regulation of blood pressure. Mice lacking functional NPRl have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease (Oliver et al., Proc. Natl. Acad. Sci. U. S. A 1997; 94:14730-14735). The binding of ANP to the extracellular domain of NPRl activates the receptor guanylate cyclase to synthesize cGMP.(Lowe, Biochemistry 1992; 31 :10421-10425). Increases in the levels of cGMP causes the downregulation of NPRl mRNA, thus allowing NPRl to autoregulate its own transcription (Cao et al., Am. J. Physiol 1998; 275:F119-F125).

Serum testosterone levels tend to be lower in hypertensive males than in normal males. Pandey et al. (Endocrinology 1999; 140:5112-5119) studied the influence of NPRl on serum testosterone levels in male hypertensive rats lacking a functional NPRl gene, wild type animals expressing two copies, and those expressing four copies ofthe NPRl gene. The animals with four copies of NPRl gene had higher levels of testosterone than those with two copies ofthe gene. The NPRl knockout mice had testosterone levels lower than the two-copy mice. Also, Leydig cells lacking NPRl , did not show ANP-stimulated cGMP accumulation and had no ANP-dependent testosterone production. This study establishes the role of NPRl in testicular steridogenesis, and shows a relationship between hypertension associated with decreased NPRl and low testosterone levels.

ANP has also been shown to inhibit the agonist stimulated activity of mitogen-activated protein kinase/extracellular signal regulated kinase 2(MAPK ERK2). This inhibitory effect of ANP was reversed on treatment with NPRl antagonist, suggesting that the ANP/NPRl system negatively regulates MAPK7Erk2 (Pandey et al., Biochem. Biophys. Res. Commun. 2000; 271:374-379).

The natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) gene is located on chromosome Iq21-q22 and contains 22 exons that encode a 1061 amino acid protein. Reference sequences for the NPRl gene (Genaissance Reference No. 1568505; SEQ ID NO:l), coding sequence (GenBank Accession No:NM_000906.1 ), and protein are shown in Figures 1, 2 and 3, respectively.

Because ofthe potential for variation in the NPRl gene to affect the expression and function ofthe encoded protein, it would be useful to know whether polymoφhisms exist in the NPRl gene, as well as how such polymoφhisms are combined in different copies ofthe gene. Such information could be applied for studying the biological function of NPRl 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 21 novel polymoφhic sites in the NPRl gene. These polymoφhic sites (PS) correspond to the following nucleotide positions in Figure 1 : 730 (PSI), 731 (PS2), 811 (PS3), 822 (PS4), 1235 (PS5), 1351 (PS6), 2184 (PS7), 2472 (PS8), 2979 (PS9), 4345 (PS10), 5290 (PS11), 5537 (PS12), 6900 (PS13), 7410 (PS14), 7947 (PS15), 9313 (PS16), 9619 (PS17), 9675 (PS18), 9904 (PS19), 10004 (PS20) and 11062 (PS21). The polymoφhisms at these sites are guanine or adenine at PSI, guanine or cytosine at PS2, cytosine or thymine at PS3, cytosine or adenine at PS4, guanine or cytosine at PS5, cytosine or thymine at PS6, thymine or cytosine at PS7, adenine or guanine at PS8, guanine or cytosine at PS9, thymine or adenine at PS 10, thymine or cytosine at PSI 1, guanine or adenine at PS 12, guanine or adenine at PS13, adenine or thymine at PS14, cytosine or thymine at PS15, guanine or adenine at PS16, guanine or adenine at PS 17, adenine or thymine at PS 18, cytosine or thymine at PS 19, guanine or adenine at PS20 and cytosine or thymine at PS21. In addition, the inventors have determined the identity of the alleles at these sites in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS 1-21 in the NPRl gene, which are shown below in Tables 5 and 4, respectively. Each of these NPRl haplotypes defines a naturally-occurring isoform (also referred to herein as an "isogene") ofthe NPRl gene that exists in the human population.

Thus, in one embodiment, the invention provides a method, composition and kit for genotyping the NPRl gene in an individual. The genotyping method comprises identifying the nucleotide pair that is present at one or more polymoφhic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21 in both copies ofthe NPRl gene from the individual. A genotyping composition ofthe invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel NPRl polymoφhic sites. A genotyping kit ofthe invention comprises a set of oligonucleotides designed to genotype each of these novel NPRl polymoφhic sites. The genotyping method, composition, and kit are useful in determining whether an individual has one ofthe haplotypes in Table 5 below or has one ofthe haplotype pairs in Table 4 below.

The invention also provides a method for haplotyping the NPRl gene in an individual. In one embodiment, the haplotyping method comprises determining, for one copy ofthe NPRl gene, the identity ofthe nucleotide at one or more polymoφhic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS 19, PS20 and PS21. In another embodiment, the haplotyping method comprises determining whether one copy ofthe individual's NPRl gene is defined by one ofthe NPRl haplotypes shown in Table 5, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises determining whether both copies ofthe individual's NPRl gene are defined by one ofthe NPRl haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. The method for establishing the NPRl 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 NPRl activity, e.g., hypertension. For example, the haplotyping method can be used by the pharmaceutical research scientist to validate NPRl as a candidate target for treating a specific condition or disease predicted to be associated with NPRl activity. Determining for a particular population the frequency of one or more ofthe individual NPRl haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue NPRl as a target for treating the specific disease of interest. In particular, if variable NPRl activity is associated with the disease, then one or more NPRl haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls. Conversely, if each ofthe observed NPRl haplotypes are of similar frequencies in the disease and control groups, then it may be inferred that variable NPRl 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 NPRl haplotype or haplotype pair, apply the information derived from detecting NPRl haplotypes in an individual to decide whether modulating NPRl activity would be useful in treating the disease.

The claimed invention is also useful in screening for compounds targeting NPRl to treat a specific condition or disease predicted to be associated with NPRl activity. For example, detecting which of the NPRl haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each ofthe most frequent NPRl isoforms present in the disease population. Thus, without requiring any a priori knowledge ofthe phenotypic effect of any particular NPRl 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 for haplotyping the NPRl gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with NPRl activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which ofthe NPRl haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute NPRl haplotypes and/or haplotype pairs evenly to treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of a NPRl haplotype or haplotype pair that had a previously unknown association with response to the drug being studied in the trial. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any NPRl haplotype or haplotype pair.

In another embodiment, the invention provides a method for identifying an association between a trait and a NPRl genotype, haplotype, or haplotype pair for one or more ofthe novel polymoφhic sites described herein. The method comprises comparing the frequency ofthe NPRl genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency ofthe NPRl genotype or haplotype in a reference population. A higher frequency ofthe NPRl genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the NPRl 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 NPRl haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic treatments for hypertension.

In yet another embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymoφhic variant of a reference sequence for the NPRl gene or a fragment thereof. The reference sequence comprises SEQ ID NO: 1 and the polymoφhic variant comprises at least one polymoφhism selected from the group consisting of adenine at PSI, cytosine at PS2, thymine at PS3, adenine at PS4, cytosine at PS5, thymine at PS6, cytosine at PS7, guanine at PS8, cytosine at PS9, adenine at PS10, cytosine at PSI 1, adenine at PS12, adenine at PS13, thymine at PS14, thymine at PS15, adenine at PS16, adenine at PS17, thymine at PS18, thymine at PS19, adenine at PS20 and thymine at PS21.

A particularly preferred polymoφhic variant is an isogene ofthe NPRl gene. A NPRl isogene ofthe invention comprises guanine or adenine at PSI, guanine or cytosine at PS2, cytosine or thymine at PS3, cytosine or adenine at PS4, guanine or cytosine at PS5, cytosine or thymine at PS6, thymine or cytosine at PS7, adenine or guanine at PS8, guanine or cytosine at PS9, thymine or adenine at PS 10, thymine or cytosine at PSI 1, guanine or adenine at PS 12, guanine or adenine at PS 13, adenine or thymine at PS 14, cytosine or thymine at PS 15, guanine or adenine at PS 16, guanine or adenine at PS 17, adenine or thymine at PS 18, cytosine or thymine at PS 19, guanine or adenine at PS20 and cytosine or thymine at PS21. The invention also provides a collection of NPRl isogenes, referred to herein as a NPRl genome anthology.

In another embodiment, the invention provides a polynucleotide comprising a polymoφhic variant of a reference sequence for a NPRl cDNA or a fragment thereof. The reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymoφhic cDNA comprises at least one polymoφhism selected from the group consisting of thymine at a position corresponding to nucleotide 5, adenine at a position corresponding to nucleotide 16, cytosine at a position corresponding to nucleotide 429, thymine at a position corresponding to nucleotide 545, cytosine at a position corresponding to nucleotide 1023 and thymine at a position corresponding to nucleotide 2406. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of a NPRl isogene defined by haplotypes 1-6 and 8-14.

Polynucleotides complementary to these NPRl genomic and cDNA variants are also provided by the invention. It is believed that polymoφhic variants ofthe NPRl -gene will be useful in studying the expression and function of NPRl, and in expressing NPRl protein for use in screening for candidate drugs to treat diseases related to NPRl activity. In other embodiments, the invention provides a recombinant expression vector comprising one ofthe polymoφhic 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 NPRl for protein structure analysis and drug binding studies.

In yet another embodiment, the invention provides a polypeptide comprising a polymoφhic variant of a reference amino acid sequence for the NPRl protein. The reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymoφhic variant comprises at least one variant amino acid selected from the group consisting of leucine at a position corresponding to amino acid position 2, serine at a position corresponding to amino acid position 6, valine at a position corresponding to amino acid position 182 and isoleucine at a position corresponding to amino acid position 341. A polymoφhic variant of NPRl is useful in studying the effect ofthe variation on the biological activity of NPRl as well as on the binding affinity of candidate drugs targeting NPRl for the treatment of hypertension.

The present invention also provides antibodies that recognize and bind to the above polymoφhic NPRl protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.

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

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

BRIEF DESCRIPTION OF THE DRAWTNGS

Figure 1 illustrates a reference sequence for the NPRl gene (Genaissance Reference No. 1568505; contiguous lines; SEQ ID 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 polymoφhic site(s) and polymoφhism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymoφhic site in the sequence. SEQ ID NO: 109 is equivalent to Figure 1 , with the two alternative allelic variants of each polymoφhic 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; WIPO standard ST.25).

Figure 2 illustrates a reference sequence for the NPRl coding sequence (contiguous lines; SEQ ID NO:2), with the polymoφhic site(s) and polymoφhism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymoφhic site in the sequence.

Figure 3 illustrates a reference sequence for the NPRl protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymoφhism(s) of Figure 2 positioned below the polymoφhic site in the sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the discovery of novel variants ofthe NPRl gene. As described in more detail below, the inventors herein discovered 15 isogenes ofthe NPRl gene by characterizing the NPRl 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 NPRl isogenes present in the human reference population are defined by haplotypes for 21 polymoφhic sites in the NPRl gene, all of which are believed to be novel. The NPRl polymoφhic sites identified by the inventors are referred to as PS 1-21 to designate the order in which they are located in the gene (see Table 3 below), with the novel polymoφhic sites referred to as PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS 19, PS20 and PS21. Using the genotypes identified in the Index Repository for PS 1-21 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the NPRl gene present in individual human members of this repository. The human genotypes and haplotypes found in the repository for the NPRl gene include those shown in Tables 4 and 5, respectively. The polymoφhism and haplotype data disclosed herein are useful for validating whether NPRl is a suitable target for drugs to treat hypertension, 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 polymoφhic 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 polymoφhic 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 ofthe known polymoφhic 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 polymoφhic 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 polymoφhic 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 ofthe known polymoφhic 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 ofthe following for a specific gene: a listing ofthe haplotype pairs in each individual in a population; a listing ofthe different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.

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

Isogene - One ofthe isoforms of a gene found in a population. An isogene contains all ofthe polymoφhisms present in the particular isoform ofthe gene.

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

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

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 polymoφhic site on the two copies of a chromosome from an individual.

Phased - As applied to a sequence of nucleotide pairs for two or more polymoφhic sites in a locus, phased means the combination of nucleotides present at those polymoφhic sites on a single copy ofthe locus is known.

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

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 polymoφhism in the gene.

Polymorphism - The sequence variation observed in an individual at a polymoφhic site. Polymoφhisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.

Polymorphism data - Information concerning one or more ofthe following for a specific gene: location of polymoφhic sites; sequence variation at those sites; frequency of polymoφhisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.

Polymorphism Database - A collection of polymoφhism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.

Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.

Population Group - A group of individuals sharing a common ethnogeographic origin.

Reference Population - A group of subjects or individuals who are predicted to be representative ofthe genetic variation found in the general population. Typically, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.

Single Nucleotide Polymorphism (SNP) - Typically, the specific pair of nucleotides observed at a single polymoφhic 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 polymoφhic sites in a locus, unphased means the combination of nucleotides present at those polymoφhic sites on a single copy ofthe locus is not known.

As discussed above, information on the identity of genotypes and haplotypes for the NPRl 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 NPRl polymoφhisms and haplotypes identified herein.

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

Genotyping oligonucleotides ofthe invention must be capable of specifically hybridizing to a target region of a NPRl polynucleotide, i.e., a NPRl 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-NPRl 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 polymoφhisms in the NPRl gene using the polymoφhism information provided herein in conjunction with the known sequence information for the NPRl gene and routine techniques.

A nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect" or "complete" complement of another nucleic acid molecule if every nucleotide of one ofthe molecules is complementary to the nucleotide at the corresponding position ofthe other molecule. A nucleic acid molecule is "substantially complementary" to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D. et al. in Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). While perfectly complementary oligonucleotides are preferred for detecting polymoφhisms, 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 ofthe 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 ofthe invention are allele-specific oligonucleotides. As used herein, the term allele-specific oligonucleotide (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 polymoφhic 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 Ruano 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 ofthe oligonucleotide probe aligns with the polymoφhic site in the target region (e.g., approximately the 7^th or 8^th position in a 15mer, the 8^th or 9^th position in a 16mer, and the 10^th or 11^th position in a 20mer). An ASO primer ofthe invention has a 3' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present. ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.

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; WIPO standard ST.25) at the position of the polymoφhic site to represent the two alternative allelic variants observed at that polymoφhic site. A preferred ASO probe for detecting NPRl gene polymoφhisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:

GATGCCTRGGACCGG (SEQ ID NO 4) and its complement,

ATGCCTGSGACCGGC (SEQ ID NO 5) and its complement,

GCCATGCYGGGGCCC (SEQ ID NO 6) and its complement,

GCCCCGGMGCCCCGC (SEQ ID NO 7) and its complement,

CCCCGGCSCTGGGCT (SEQ ID NO 8) and its complement,

CGCCAAGYGCTCATG (SEQ ID NO 9) and its complement,

CTGACTCYCCGTCTT (SEQ ID NO 10) and its complement,

CCTCAGCRTCTGAAA (SEQ ID NO 11) and its complement,

TCACCATSGAGGATG (SEQ ID NO 12) and its complement,

CTCCTAC TCCCCCC (SEQ ID NO 13) and its complement,

GCTCCTGYCCCATGC (SEQ ID NO 14) and its complement,

GTGATGTRGGGGGTT (SEQ ID NO 15) and its complement,

ACTTGCTRTGTGACC (SEQ ID NO 16) and its complement,

ATAAGGCWGGATAAG (SEQ ID NO 17) and its complement,

TCGGGGAYGCAAGGG (SEQ ID NO 18) and its complement,

GACTACCRACCTCTG (SEQ ID NO 19) and its complement,

CATTGCTRCCAGTGA (SEQ ID NO 20) and its complement,

CCATCTCWGCTGGTT (SEQ ID NO 21) and its complement,

CGTTGCGYAAATTTA (SEQ ID NO 22) and its complement,

CAGCAGTRGCAGAGG (SEQ ID NO 23) and its complement, and

CACCAGAYCTGCCTT (SEQ ID NO 24) and its complement.

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

GCGCCTGATGCCTRG (SEQ ID NO: 25) ; CAGCGGCCGGTCCYA SEQ ID NO: 26) CGCCTGATGCCTGSG (SEQ ID NO 27) ; TCAGCGGCCGGTCSC SEQ ID NO- 28) GCTGAGGCCATGCYG (SEQ ID NO- 29) ; GCGCCGGGGCCCCRG SEQ ID NO: 30) GCCGGGGCCCCGGMG (SEQ ID NO 31) ; GAGCCAGCGGGGCKC SEQ ID NO 32) CCGGCGCCCCGGCSC (SEQ ID NO 33) ; CACCGAAGCCCAGSG SEQ ID NO 34) TGGGAGCGCCAAGYG (SEQ ID NO 35) ; GTAGAGCATGAGCRC SEQ ID NO 36) CTCTCTCTGACTCYC (SEQ ID NO 37) ; TGGAGAAAGACGGRG SEQ ID NO 38) TGTGTCCCTCAGCRT (SEQ ID NO 39) ; GAATTCTTTCAGAYG SEQ ID NO 40) TCAACTTCACCATSG (SEQ ID NO 41) ; CCAGGCCATCCTCSA SEQ ID NO 42) CTCACCCTCCTAC T (SEQ ID NO 43) ; GCTGTGGGGGGGAWG SEQ ID NO 44) GTAGGTGCTCCTGYC (SEQ ID NO 45); CCCTCAGCATGGGRC SEQ ID NO 46) TGAGCTGTGATGTRG (SEQ ID NO 47) ; TCACTCAACCCCCY - SEQ ID NO 48) GCTTTCACTTGCTRT (SEQ ID NO 49) ; GCTCAAGGTCACAYA SEQ ID NO 50) ACAAAGATAAGGC G (SEQ ID NO 51); CCCTGCCTTATCCWG SEQ ID NO 52) GGGCCCTCGGGGAYG (SEQ ID NO 53) ; CAGTCTCCCTTGCRT SEQ ID NO 54) CTCGGTGACTACCRA (SEQ ID NO 55) ; GTGGGTCAGAGGTYG SEQ ID NO 56) TTGACCCATTGCTRC (SEQ ID NO 57); GACTGGTCACTGGYA SEQ ID NO 58) CCCCTGCCATCTCWG (SEQ ID NO 59); TGGGGCAACCAGC G SEQ ID NO 60) GCCTGACGTTGCGYA (SEQ ID NO 61); ACCTGTTAAATTTRC SEQ ID NO 62) TTATCCCAGCAGTRG (SEQ ID NO 63) ; GGTCTCCCTCTGCYA SEQ ID NO 64) GGATCCCACCAGAYC (SEQ ID NO 65) ; and AACCAGAAGGCAC ,RT (SEQ ID NO 66 )

Other genotyping oligonucleotides ofthe invention hybridize to a target region located one to several nucleotides downstream of one ofthe novel polymoφhic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one ofthe novel polymoφhisms 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 deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymoφhic site.

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

CCTGATGCCT (SEQ ID NO: 67) CGGCCGGTCC (SEQ ID NO: 68)

CTGATGCCTG (SEQ ID NO: 69) GCGGCCGGTC (SEQ ID NO: 70)

GAGGCCATGC (SEQ ID NO: 71) CCGGGGCCCC (SEQ ID NO: 72)

GGGGCCCCGG (SEQ ID NO: 73) CCAGCGGGGC (SEQ ID NO: 74)

GCGCCCCGGC (SEQ ID NO. 75) CGAAGCCCAG (SEQ ID NO: 76)

GAGCGCCAAG (SEQ ID NO. 77) ' GAGCATGAGC (SEQ ID NO: 78)

TCTCTGACTC (SEQ ID NO 79) ^■ AGAAAGACGG (SEQ ID NO: 80)

GTCCCTCAGC (SEQ ID NO 81) ^■ TTCTTTCAGA (SEQ ID NO: 82)

ACTTCACCAT (SEQ ID NO 83) ^■ GGCCATCCTC (SEQ ID NO- 84)

ACCCTCCTAC (SEQ ID NO 85) ^• GTGGGGGGGA (SEQ ID NO- 86)

GGTGCTCCTG (SEQ ID NO 87) ^• TCAGCATGGG (SEQ ID NO 88)

GCTGTGATGT (SEQ ID NO 89) ^■ CTCAACCCCC (SEQ ID NO 90)

TTCACTTGCT (SEQ ID NO 91) CAAGGTCACA (SEQ ID NO 92)

AAGATAAGGC (SEQ ID NO: 93) TGCCTTATCC (SEQ ID NO: 94) ,

CCCTCGGGGA (SEQ ID NO: 95) ^■ TCTCCCTTGC (SEQ ID NO: 96)

GGTGACTACC (SEQ ID NO: 97) ^■ GGTCAGAGGT (SEQ ID NO: 98)

ACCCATTGCT (SEQ ID NO 99) ^■ TGGTCACTGG (SEQ ID NO: 100) ;

CTGCCATCTC (SEQ ID NO 101 ; GGCAACCAGC (SEQ IE NC :102)

TGACGTTGCG (SEQ ID NO 103 ;TGTTAAATTT (SEQ IE NC :104)

TCCCAGCAGT (SEQ ID NO 105 ;CTCCCTCTGC (SEQ IE NC :106)

TCCCACCAGA (SEQ ID NO 107 ; and CAGAAGGCAG (SEQ ID NO 108 )

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

NPRl genotyping oligonucleotides ofthe invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized genotyping oligonucleotides may be used in a variety of polymoφhism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized NPRl genotyping oligonucleotides ofthe 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 NPRl gene in an individual. As used herein, the terms "NPRl genotype" and "NPRl haplotype" mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more ofthe 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 NPRl gene. The additional polymoφhic sites may be currently known polymoφhic sites or sites that are subsequently discovered.

One embodiment ofthe genotyping method involves isolating from the individual a nucleic acid sample comprising the two copies ofthe NPRl gene, or a fragment thereof, that are present in the individual, and determining the identity ofthe nucleotide pair at one or more polymoφhic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21 in the two copies to assign a NPRl 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 particularly preferred embodiment, the genotyping method comprises determining the identity ofthe nucleotide pair at each of PS 1-21.

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 NPRl 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 NPRl gene fragment is isolated, it must contain the polymoφhic site(s) to be genotyped.

One embodiment ofthe haplotyping method comprises isolating from the individual a nucleic acid sample containing only one ofthe two copies ofthe NPRl gene, or a fragment thereof, that is present in the individual and determining in that copy the identity ofthe nucleotide at one or more polymoφhic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl l, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21 in that copy to assign a NPRl haplotype to the individual. The nucleic acid may be isolated using any method capable of separating the two copies ofthe NPRl gene or fragment such as one ofthe methods described above for preparing NPRl 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 information on one ofthe two NPRl gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional NPRl 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 ofthe NPRl gene in an individual. In a particularly preferred embodiment, the nucleotide at each of PSl-21 is identified.

In another embodiment, the haplotyping method comprises determining whether an individual has one or more ofthe NPRl haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies ofthe individual's NPRl gene, the phased sequence of nucleotides present at each of PSl-21. The present invention also contemplates that typically only a subset of PSl-21 will need to be directly examined to assign to an individual one or more ofthe haplotypes shown in Table 5. 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 NPRl 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 PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl l, PS12, PS13, PS14, PS 15, PS 16, PS 17, PS 18, PS 19, PS20 and PS21 in each copy ofthe NPRl 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 PSl-21 in each copy ofthe NPRl gene. When haplotyping both copies ofthe gene, the identifying step is preferably performed with each copy ofthe 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 ofthe 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 ofthe first and third dyes would identify the polymoφhism in the first gene copy while detecting a combination ofthe 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 ofthe NPRl gene, or a fragment thereof, and the sequence ofthe amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a 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 guanine and cytosine, if the individual is heterozygous at that site. Alternatively, the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/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 ofthe pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant. 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 ofthe 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 ofthe invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads. The solid support may be treated, coated or derivatized to facilitate the immobilization ofthe allele-specific oligonucleotide or target nucleic acid.

The genotype or haplotype for the NPRl gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies ofthe gene, or fragment(s) thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995. The arrays would contain a battery of allele-specific oligonucleotides representing each ofthe 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. Patent No. 5,605,798. Another primer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res. 17:8392, 1989; Ruaήo 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 ofthe nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).

In addition, the identity ofthe allele(s) present at any ofthe novel polymoφhic sites described herein may be indirectly determined by genotyping another polymoφhic 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 ofthe 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 ofthe above-mentioned methods for detecting the identity ofthe allele at a polymoφhic site.

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

Generally, the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups ofthe world. A preferred reference population for use in the methods ofthe 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 ofthe four population groups named above. A particularly preferred reference population includes a 3- generation family representing"one or more ofthe four population groups to serve as controls for checking quality of haplotyping procedures.

In a preferred embodiment, the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L. 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, / H₂ is equal to p_H__w(H_ IH₂) = 2p(H_l)p(H₂) if H, ≠ H₂ and p_H__w(H_λ / H₂) = p(H )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 NPRl haplotype pair for an individual, the assigning step involves performing the following analysis. First, each ofthe possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one ofthe haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair. 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 NPRl genotype, haplotype, or haplotype pair in a population. The method comprises, for each member ofthe population, determining the genotype or the haplotype pair for the novel NPRl polymoφhic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pan- 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 ofthe invention, frequency data for NPRl 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 NPRl genotype, haplotype, or haplotype pair. The trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment. The method involves obtaining data on the frequency ofthe genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait. Frequency data for one or both ofthe reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one ofthe 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 ofthe 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 NPRl 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 NPRl genotype, haplotype or haplotype pair. Preferably, the NPRl 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 ofthe method, the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting NPRl 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 ofthe following: a quantitative measure ofthe response, no response, and adverse response (i.e., side effects).

In order to deduce a correlation between clinical response to a treatment and a NPRl 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 III 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 ofthe medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment ofthe underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome. This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.

The therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the NPRl 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 NPRl genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their NPRl 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 NPRl 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/USOO/ 17540, entitled "Methods for Obtaining and Using Haplotype Data".

A second method for finding correlations between NPRl 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/USOO/17540.

Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much ofthe variation in the clinical data is explained by different subsets ofthe polymoφhic sites in the NPRl gene. As described in PCT Application Serial No. PCT/USOO/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, supra, 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 NPRl 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 NPRl 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 ofthe polymoφhic sites in the NPRl 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 NPRl 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 ofthe NPRl gene or a fragment ofthe gene which contains at least one ofthe novel polymoφhic sites described herein. The nucleotide sequence of a variant NPRl gene is identical to the reference genomic sequence for those portions ofthe gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more ofthe novel polymoφhic sites PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21. Similarly, the nucleotide sequence of a variant fragment ofthe NPRl gene is identical to the corresponding portion ofthe reference sequence except for having a different nucleotide at one or more ofthe novel polymoφhic sites described herein. Thus, the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence ofthe NPRl gene, which is defined by haplotype 7, (or other reported NPRl sequences) or to portions ofthe reference sequence (or other reported NPRl 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: 1. The polymoφhism is selected from the group consisting of adenine at PSI, cytosine at PS2, thymine at PS3, adenine at PS4, cytosine at PS5, thymine at PS6, cytosine at PS7, guanine at PS8, cytosine at PS9, adenine at PS10, cytosine at PSI 1, adenine at PS12, adenine at PS13, thymine at PS14, thymine at PS15, adenine at PS16, adenine at PS17, thymine at PS18, thymine at PS 19, adenine at PS20 and thymine at PS21. In a preferred embodiment, the polymoφhic variant comprises a naturally-occurring isogene ofthe NPRl gene which is defined by any one of haplotypes 1-6 and 8-14 shown in Table 5 below.

Polymoφhic variants ofthe invention may be prepared by isolating a clone containing the NPRl 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.

NPRl isogenes may be isolated using any method that allows separation ofthe two "copies" ofthe NPRl 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 (TTVC) 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 NPRl genome anthologies, which are collections of NPRl 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 NPRl genome anthology may comprise individual NPRl isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups ofthe NPRl 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 NPRl genome anthology ofthe invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.

An isolated polynucleotide containing a polymoφhic variant nucleotide sequence ofthe invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded NPRl 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, adeno virus, 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 ofthe 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 NPRl sequences ofthe invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art. The recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). 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 embryonic 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 ofthe NPRl gene will produce NPRl 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 NPRl cDNA comprising a nucleotide sequence which is a polymoφhic variant ofthe NPRl reference coding sequence shown in Figure 2. Thus, the invention also provides NPRl mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig. 2), or its corresponding RNA sequence, except for having one or more polymoφhisms selected from the group consisting of thymine at a position corresponding to nucleotide 5, adenine at a position corresponding to nucleotide 16, cytosine at a position corresponding to nucleotide 429, thymine at a position corresponding to nucleotide 545, cytosine at a position corresponding to nucleotide 1023 and thymine at a position corresponding to nucleotide 2406. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of a NPRl isogene defined by haplotypes 1-6 and 8-14. Fragments of these variant mRNAs and cDNAs are included in the scope ofthe invention, provided they contain the novel polymoφhisms described herein. The invention specifically excludes polynucleotides identical to previously identified and characterized NPRl 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 NPRl 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 ofthe 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 NPRl polymoφhic sites identified herein, reference is made to the sense strand ofthe gene for convenience. However, as recognized by the skilled artisan, nucleic acid molecules containing the NPRl 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 ofthe NPRl 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 NPRl protein isoform, an expression vector encoding the isoform may be administered to the patient. The patient may be one who lacks the NPRl 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 NPRl isogene. Expression of a NPRl 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) ofthe 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) ofthe isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). Antisense oligonucleotides may also be designed to block translation of NPRl mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of NPRl 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. Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life. Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.

The invention also provides an isolated polypeptide comprising a polymoφhic variant ofthe reference NPRl amino acid sequence shown in Figure 3. The location of a variant amino acid in a NPRl polypeptide or fragment ofthe invention is identified by aligning its sequence against SEQ ID NO:3 (Fig. 3). A NPRl protein variant ofthe invention comprises an amino acid sequence identical to SEQ ID NO: 3 except for having one or more variant amino acids selected from the group consisting of leucine at a position corresponding to amino acid position 2, serine at a position corresponding to amino acid position 6, valine at a position corresponding to amino acid position 182 and isoleucine at a position corresponding to amino acid position 341. The invention specifically excludes amino acid sequences identical to those previously identified for NPRl, including SEQ ID NO:3, and previously described fragments thereof. NPRl protein variants included within the invention comprise all amino acid sequences based on SEQ ID NO: 3 and having the combination of amino acid variations described in Table 2 below. In preferred embodiments, a NPRl protein variant ofthe invention is encoded by an isogene defined by one ofthe observed haplotypes shown in Table 5.

Table 2. Novel Polymoφhic Variants of NPRl Polymoφhic Amino Acid Position and Identities

Variant

Number 2 6 182 341

1 P R A I

2 P R V M

3 P R V I

4 P S A M

5 P S A I

6 P S V M

7 P S V I

8 L R A M

9 L R A I

10 L R V M

11 L R V I

12 L S A M

13 L S A I

14 L S V M

15 L S V I

The invention also includes NPRl peptide variants, which are any fragments of a NPRl protein variant that contain one or more ofthe amino acid variations shown in Table 2. A NPRl peptide variant is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long. Such NPRl peptide variants may be useful as antigens to generate antibodies specific for one ofthe above NPRl isoforms. In addition, the NPRl peptide variants may be useful in drug screening assays.

A NPRl variant protein or peptide ofthe invention may be prepared by chemical synthesis or by expressing one ofthe variant NPRl genomic and cDNA sequences as described above. Alternatively, the NPRl protein variant may be isolated from a biological sample of an individual having a NPRl isogene which encodes the variant protein. Where the sample contains two different NPRl isoforms (i.e., the individual has different NPRl isogenes), a particular NPRl isoform ofthe invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular NPRl isoform but does not bind to the other NPRl isoform.

The expressed or isolated NPRl protein may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isoform ofthe NPRl protein as discussed further below. NPRl variant proteins can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Current Protocols in Molecular Biology John Wiley and Sons, New York, New York). In the case of immunoaffinity chromatography, antibodies specific for a particular polymoφhic variant may be used.

A polymoφhic variant NPRl gene ofthe invention may also be fused in frame with a heterologous sequence to encode a chimeric NPRl protein. The non-NPRl portion ofthe chimeric protein may be recognized by a commercially available antibody. In addition, the chimeric protein may also be engineered to contain a cleavage site located between the NPRl and non-NPRl portions so that the NPRl protein may be cleaved and purified away from the non-NPRl portion.

An additional embodiment ofthe invention relates to using a novel NPRl protein isoform in any of a variety of drug screening assays. Such screening assays may be performed to identify agents that bind specifically to all known NPRl protein isoforms or to only a subset of one or more of these isoforms. The agents may be from chemical compound libraries, peptide libraries and the like. The NPRl protein or peptide variant may be free in solution or affixed to a solid support. In one embodiment, high throughput screening of compounds for binding to a NPRl variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the NPRl protein(s) of interest and then washed. Bound NPRl protein(s) are then detected using methods well-known in the art.

In another embodiment, a novel NPRl protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the NPRl protein.

In yet another embodiment, when a particular NPRl haplotype or group of NPRl haplotypes encodes a NPRl protein variant with an amino acid sequence distinct from that of NPRl protein isoforms encoded by other NPRl haplotypes, then detection of that particular NPRl haplotype or group of NPRl haplotypes may be accomplished by detecting expression ofthe encoded NPRl protein variant using any ofthe methods described herein or otherwise commonly known to the skilled artisan.

In another embodiment, the invention provides antibodies specific for and immunoreactive with one or more ofthe novel NPRl variant proteins described herein. The antibodies may be either monoclonal or polyclonal in origin. The NPRl protein or peptide variant used to generate the antibodies may be from natural or recombinant sources or produced by chemical synthesis using synthesis techniques known in the art. If the NPRl protein variant is of insufficient size to be antigenic, it may be conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity ofthe peptide. Examples of carrier molecules, include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).

In one embodiment, an antibody specifically immunoreactive with one ofthe novel protein isoforms described herein is administered to an individual to neutralize activity ofthe NPRl isoform expressed by that individual. The antibody may be formulated as a pharmaceutical composition which includes a pharmaceutically acceptable carrier.

Antibodies specific for and immunoreactive with one ofthe novel protein isoforms described herein may be used to immunoprecipitate the NPRl protein variant from solution as well as react with NPRl protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates. In another preferred embodiment, the antibodies will detect NPRl protein isoforms in paraffin or frozen tissue sections, or in cells which have been fixed or unfixed and prepared on slides, coverslips, or the like, for use in immunocytochemical, immunohistochemical, and immunofluorescence techniques.

In another embodiment, an antibody specifically immunoreactive with one ofthe novel NPRl protein variants described herein is used in immunoassays to detect this variant in biological samples. In this method, an antibody ofthe present invention is contacted with a biological sample and the formation of a complex between the NPRl protein variant and the antibody is detected. As described, suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Press, New York, New York; Current Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York). Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and Campbell et al., 1984, Methods in Immunology, W.A. Benjamin, Inc.). Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Pres, NY, NY; and Oellirich, M., 1984, J. Clin. Chem. Clin. Biochem., 22:895-904). Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Current Protocols in Molecular Biology, supra.

Exemplary antibody molecules for use in the detection and therapy methods ofthe present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site. Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam). The antibodies or antigen binding fragments thereof may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication number WO 901443, WO 901443 and WO 9014424 and in Huse et al., 1989, Science, 246: 1275-1281. The antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci.USA 86; 10029). Effect(s) ofthe polymoφhisms identified herein on expression of NPRl may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymoφhic variant ofthe NPRl gene. As used herein, "expression" includes but is not limited to one or more ofthe following: transcription ofthe gene into precursor mRNA; splicing and other processing ofthe precursor mRNA to produce mature mRNA; mRNA stability; translation ofthe mature mRNA into NPRl protein (including codon usage and tRNA availability); and glycosylation and/or other modifications ofthe translation product, if required for proper expression and function.

To prepare a recombinant cell ofthe invention, the desired NPRl 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 NPRl isogene is introduced into a cell in such a way that it recombines with the endogenous NPRl gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired NPRl 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 ofthe skilled practitioner. Examples of cells into which the NPRl isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells ofthe relevant tissue type, i.e., they express the NPRl isogene. Such recombinant cells can be used to compare the biological activities ofthe different protein variants.

Recombinant nonhuman organisms, i.e., transgenic animals, expressing a variant NPRl 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 ofthe animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage. Transgenic animals carrying the constructs ofthe invention can be made by several methods known to those having skill in the art. One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes 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 NPRl 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 NPRl isogene and producing human NPRl protein can be used as biological models for studying diseases related to abnormal NPRl expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.

An additional embodiment ofthe invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel NPRl isogene described herein. The pharmaceutical composition may comprise any ofthe following active ingredients: a polynucleotide comprising one of these novel NPRl isogenes; an antisense oligonucleotide directed against one of the novel NPRl isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel NPRl 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 ofthe symptoms relating to disorders affected by expression or function of a novel NPRl 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 ofthe pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

For any composition, determination ofthe 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 ofthe disease state, general health, age, weight and gender ofthe patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.

Any or all analytical and mathematical operations involved in practicing the methods ofthe present invention may be implemented by a computer. 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 NPRl 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 NPRl 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 ofthe invention are described in the following examples. Other embodiments within the scope ofthe claims herein will be apparent to one skilled in the art from consideration ofthe specification or practice ofthe invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit ofthe 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 ofthe 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 ofthe NPRl gene for polymoφhic sites.

Amplification of Target Regions

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

PCR Primer Pairs

Fragment No. Forward Primer Reverse Primer PCR Product

Fragment 1 263-285 complement of 958-937 696 nt Fragment 2 538-558 complement of 1261-1240 724 nt Fragment 3 911-930 complement of 1498-1478 588 nt Fragment 4 911-930- complement of 1744-1722 834 nt Fragment 5 2026-2047 complement of2681-2660 656 nt Fragment 6 2681-2702 complement of 3178-3155 498 nt Fragment 7 4022-4043 complement of 4471-4450 450 nt Fragment 8 4857-4882 complement of 5438-5415 582 nt Fragment 9 5254-5275 complement of 5674-5651 421 nt Fragment 10 6450-6473 complement of 6992-6970 543 nt Fragment 11 7304-7327 complement of 7809-7787 506 nt Fragment 12 7650-7672 complement of 8071-8047 422 nt Fragment 13 8529-8550 complement of 8960-8939 432 nt Fragment 14 9148-9170 complement of 9701-9679 554 nt Fragment 15 9565-9588 complement of l0193-10171 629 nt Fragment 16 10349-10371 complement of 11094-1 1072 746 nt Fragment 17 10734-10755 complement of 11190-11166 457 nt Fragment 18 10954-10976 complement of 11406-11384 453 nt Fragment 19 11329-11351 complement of 11917-11895 589 nt Fragment 20 11668-11689 complement of 12281-12303 636 nt Fragment 21 14605-14627 complement of 15074-15053 470 nt Fragment 22 14858-14880 complement of 15340-15320 483 nt

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

Reaction volume = lO μl

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

100 ng of human genomic DNA = l μl lO mM 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

97°C - 15 sec. 70°C - 45 sec. 10 cycles 72°C - 45 sec.

97°C - 15 sec. 64°C - 45 sec. 35 cycles 72°C - 45 sec. Sequencing of PCR Products

The PCR products were purified using a Whatman/Polyfiltronics 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 1 334-353 complement of 865-846 Fragment 2 638-656 complement of 1135-1116 Fragment 3 942-961 complement of 1422-1404 Fragment 4 1111-1130 complement of 1648-1629 Fragment 5 2051-2070 complement of 2570-2551 Fragment 6 2740-2759 complement of 3149-3130 Fragment 7 4051-4070 complement of 4409-4389 Fragment 8 4919-4938 complement of 5337-5316 Fragment 9 5289-5308 complement of 5645-5626 Fragment 10 6509-6528 complement of 6914-6895 Fragment 11 7331-7350 complement of 7740-7721 Fragment 12 7702-7721 complement of 8033-8014 Fragment 13 8576-8595 complement of 8905-8885 Fragment 14 9181-9200 complement of 9617-9598 Fragment 15 9605-9624 complement of 10064-10045 Fragment 16 10514-10533 complement of 10978-10959 Fragment 17 10758-10777 complement of 11151-11131 Fragment 18 11023-11041 complement of 11338-11319 Fragment 19 11377-11396 complement of 11869- 11849 Fragment 20 11850-11871 complement of 12194- 12175 Fragment 21 14659-14678 complement of 15021-15002 Fragment 22 14894-14913 complement of 15237-15218

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 NPRl gene are listed in Table 3 below. Table 3. Polymoφhic Sites Identified in the NPRl Gene

Polymoφhic Nucleotide Reference Variant CDS Variant AA

Site Number PolyId^a Position Allele Allele Position Variant.

PSI 8199638 730 G A

PS2 8199735 731 G C

PS3 8199831 811 C T 5 P2L

PS4 8199927 822 C A 16 R6S

PS5 8203209 1235 G C 429 A143A

PS6 8203306 1351 C T 545 A182V

PS7 1568564 2184 T C

PS8 1568566 2472 A G

PS9 1568570 2979 G C 1023 M341I

PS10 8214019 4345 T A

PSl l 1568572 5290 T C

PS12 1568576 5537 G A

PS13 1568584 6900 G A

PS14 1568586 7410 A T

PS15 8206656 7947 C T

PS16 1568590 9313 G A

PS17 1568596 9619 G A

PS18 1568598 9675 A T

PS19 1568600 9904 C T 2406 R802R

PS20 1568606 10004 G A

PS21 1568620 11062 C T ^aPolyId is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc.

EXAMPLE 2

This example illustrates analysis ofthe NPRl 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 4 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below. In Table 4, homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 4 were inferred based on linkage disequilibrium and/or Mendelian inheritance.

The haplotype pairs shown in Table 4 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 1, 111-122) for assigning haplotypes to unrelated 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 ofthe 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 14 human NPRl haplotypes shown in Table 5 below.

In Table 6 below, the number of chromosomes in unrelated individuals characterized by a given haplotype is shown, arranged by ethnic background ofthe subjects in the Index Repository. In Table 7 below, the number of unrelated subjects characterized by a given haplotype is shown, again arranged by ethnic background ofthe subjects in the Index Repository. In Tables 6 and 7, the following abbreviations are used: AF, African or African-American; AS, Asian; CA, Caucasian; HL, Hispanic-Latino; and AM, Native Americans.

In view ofthe 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 ofthe 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 ofthe cited references.

Claims

What is Claimed is:

1. A method for haplotyping the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene of an individual which comprises determining whether the individual has one ofthe NPRl haplotypes shown in Table 5 or one ofthe haplotype pairs shown in Table 4.

2. The method of claim 1, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PSl-21 on at least one copy ofthe individual's NPRl gene.

3. The method of claim 1 , wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PSl-21 on both copies ofthe individual's NPRl gene.

4. A method for genotyping the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene of an individual, comprising determining for the two copies ofthe NPRl gene present in the individual the identity ofthe nucleotide pair at one or more polymoφhic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl l, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21.

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

(a) isolating from the individual a nucleic acid mixture comprising both copies ofthe NPRl 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 ofthe 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 ofthe reaction, wherein said terminators are complementary to the alternative nucleotides present at the selected polymoφhic site; and

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

6. The method of claim 4, which comprises determining for the two copies ofthe NPRl gene present in the individual the identity ofthe nucleotide pair at each of PSl-21.

7. A method for haplotyping the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene of an individual which comprises determining, for one copy ofthe NPRl gene present in the individual, the identity ofthe nucleotide at two or more polymoφhic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21.

8. 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 ofthe NPRl 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 ofthe 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 ofthe reaction, wherein said terminators are complementary to the alternative nucleotides present at the selected polymoφhic site; and

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

9. A method for predicting a haplotype pair for the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene of an individual comprising:

(a) identifying a NPRl genotype for the individual, wherein the genotype comprises the nucleotide pair at two or more polymoφhic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl l, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21;

(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.

10. The method of claim 9, wherein the identified genotype ofthe individual comprises the nucleotide pair at each of PSl-21.

11. A method for identifying an association between a trait and at least one haplotype or haplotype pair ofthe natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene which comprises comparing the frequency ofthe haplotype or haplotype pair in a population exhibiting the trait with the frequency ofthe haplotype or haplotype pair in a reference population, wherein the haplotype is selected from haplotypes 1-14 shown in Table 5 and the haplotype pair is selected from the haplotype pairs shown in Table 4, wherein a higher frequency ofthe haplotype or haplotype pair in the trait population than in the reference population indicates the trait is associated with the haplotype or haplotype pair.

12. The method of claim 11 , wherein the trait is a clinical response to a drug targeting NPRl .

13. A composition comprising at least one genotyping oligonucleotide for detecting a polymoφhism in the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene at a polymoφhic site selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl l, PS12, PS13, PS14, PS15, PS16, PS 17, PS 18, PS 19, PS20 and PS21.

14. The composition of claim 13, wherein the genotyping oligonucleotide is an allele-specific oligonucleotide that specifically hybridizes to an allele ofthe NPRl gene at a region containing the polymoφhic site.

15. The composition of claim 14, wherein the allele-specific oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:4 -24, the complements of SEQ ID NOS:4-24, and SEQ ID NOS:25-66.

16. The composition of claim 13, wherein the genotyping oligonucleotide is a primer-extension oligonucleotide.

17. The composition of claim 16, wherein the primer extension oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:67-108.

18. A kit for genotyping the NPRl gene of an individual, which comprises a set of oligonucleotides designed to genotype each of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20 and PS21.

19. 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 natriuretic peptide receptor A guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene or a fragment thereof, wherein the reference sequence comprises SEQ ID NO: 1 and the polymoφhic variant comprises a NPRl isogene defined by a haplotype selected from the group consisting of haplotypes 1-14 in Table 5; and

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

20. The isolated polynucleotide of claim 19, 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.

21. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 19, wherein the organism expresses a NPRl protein encoded by the first nucleotide sequence.

22. The recombinant organism of claim 21 , which is a nonhuman transgenic animal.

23. The isolated polynucleotide of claim 19, wherein the first nucleotide sequence is a polymoφhic variant of a fragment ofthe NPRl gene, the fragment comprising one or more polymoφhisms selected from the group consisting of adenine at PSI, cytosine at PS2, thymine at PS3, adenine at PS4, cytosine at PS5, thymine at PS6, cytosine at PS7, guanine at PS8, cytosine at PS9, adenine at PS10, cytosine at PSI 1, adenine at PS12, adenine at PS13, thymine at PS14, thymine at PS 15, adenine at PS 16, adenine at PS 17, thymine at PS 18, thymine at PS 19, adenine at PS20 and thymine at PS21.

24. An isolated polynucleotide comprising a nucleotide sequence which is a polymoφhic variant of a reference sequence for the NPRl cDNA or a fragment thereof, wherein the reference sequence comprises SEQ ID NO:2 and the polymoφhic variant comprises the coding sequence of a NPRl isogene defined by one ofthe haplotypes shown in Table 5.

25. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 24, wherein the organism expresses a natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) protein encoded by the polymoφhic variant sequence.

26. The recombinant organism of claim 25, which is a nonhuman transgenic animal.

27. An isolated polypeptide comprising an amino acid sequence which is a polymoφhic variant of a reference sequence for the NPRl protein or a fragment thereof, wherein the reference sequence comprises SEQ ID NO:3 and the polymoφhic variant is encoded by an isogene defined by one ofthe haplotypes shown in Table 5.

28. An isolated antibody specific for and immunoreactive with the isolated polypeptide of claim 27.

29. A method for screening for drugs targeting the isolated polypeptide of claim 27 which comprises contacting the NPRl polymoφhic variant with a candidate agent and assaying for binding activity.

30. A computer system for storing and analyzing polymoφhism data for the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) 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 4 and the haplotypes shown in Table 5.

31. A genome anthology for the natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPRl) gene which comprises NPRl isogenes defined by any one of haplotypes 1-14 shown in Table 5.