WO2002040503A2 - Haplotypes of the plau gene - Google Patents

Abstract

Novel genetic variants of the Plasminogen Activator, Urokinase (PLAU) gene are described. Various genotypes, haplotypes, and haplotype pairs that exist in the general United States population are disclosed for the PLAU gene. Compositions and methods for haplotyping and/or genotyping the PLAU gene in an individual are also disclosed. Polynucleotides defined by the haplotypes disclosed herein are also described.

Description

HAPLOTYPES OF THE PLAU GENE

RELATED APPLICATIONS

This application ^'claims the benefit of U.S. Provisional Application Serial No. 60/249,703 fιled November l7, 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 plasminogen activator, urokinase (PLAU) 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 absorption, disposition, metabolism and toxicological profiles. Typically, this testing involves use of cell lines and animal models with limited, if any, genetic diversity.

What this approach fails to consider, however, is that natural genetic variability exists between individuals in any and every population with respect to pharmaceutically-important proteins, including the protein targets of candidate drugs, the enzymes that metabolize these drugs and the proteins whose activity is modulated by such drug targets. Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a pharmaceutically-important protein may be manifested as significant variation in expression, structure and/or function o the protein. Such alterations may explain the relatively high degree of uncertainty inherent in the treatment of individuals with a drug whose design is based upon a single representative example 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 ofthe 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 L999 Mol. Med. Today 5:538-43; Davidson S 2000 Nature Biotech 18:1134-5).

The standard for measuring genetic variation among individuals is the haplotype, which is the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymorphisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses AD supra; Ulbrecht M et al. 2000 Am JRespir Crit Care Med 161: 469-74) and drug response (Wolfe CR et al. 2000 BMJ 320:987-90; Dahl BS 1997 Acta Psychiatr Scand 96 (Suppl 391): 14-21), in many other cases an individual polymorphism may be found in a variety of genomic backgrounds, i.e., different haplotypes, and therefore shows no definitive coupling between the polymorphism and the causative site for the phenotype (Clark AG et al. 1998 Am JHum Genet 63:595-612; Ulbrecht M et al. 2000 supra; Drysdale et al. 2000 PNAS 97: 10483-10488). Thus, there is an unmet need in the pharmaceutical industry for infoπnation 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 thrombolytic disorders and cancer is the plasminogen activator, urokinase (PLAU) gene or its encoded product. PLAU is a serine protease that belongs to peptidase family si . PLAU plays an important role in the physiological and pathological processes in which cell adhesion, migration, or tissue remodeling is required. It converts plasminogen to plasmin and degrades fibrin and other protein components ofthe extracellular matrix (e.g., fibronectin). Clinically, PLAU activator is given to patients suffering from thrombolytic disorders and is used in cases of pulmonary embolism to initiate fibrinolysis (Gene Cards: PLAU).

Through proteolysis, PLAU also activates pro-hepatocyte growth factor and vascular endothelial growth factor, and, via generation of plasmin, it activates transforming growth factor-beta (Bhat et al., Am JPhysiol 1999 Aug;277(2 Pt l):L301-9). Recent studies have shown that PLAU is commonly overexpressed in many human cancers (Jankun et -jl., Cancer Res 1997 Feb 15;57(4):559- 63). Thus, drugs that affect the expression ofthe PLAU gene or its encoded product may be of use in the treatment of thrombolytic disorders and cancer.

The plasminogen activator, urokinase gene is located on chromosome 10q24-qter and contains 11 exons that encode a 431 amino acid protein. A reference sequence for the PLAU gene is shown in the contiguous lines of Figure 1 (Genaissance Reference No. 4874587; SEQ ID NO: 1). Reference sequences for the coding sequence (GenBank Accession No. NM_002658.1) and protein are shown in Figures 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3), respectively.

Because ofthe potential for variation in the PLAU gene to affect the expression and function of the encoded protein, it would be useful to know whether polymorphisms exist in the PLAU gene, as well as how such polymorphisms are combined in different copies ofthe gene. Such information could be applied for studying the biological function of PLAU 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 18 novel polymo hic sites in the PLAU gene. These polymorphic sites (PS) correspond to the following nucleotide positions in Figure 1 : 3186 (PS1), 3419 (PS2), 4030 (PS3), 4032 (PS4), 4134 (PS5), 4611 (PS6), 4795 (PS7), 4966 (PS8), 5697 (PS9), 5837 (PS10), 6332 (PS11), 6484 (PS12), 6615 (PS13), 7476 (PS14), 7822 (PS15), 7955 (PS 16), 8900 (PS 17) and 9199 (PS 18). The polymorphisrύs at these sites are cytosine or thymine at . PS1, guanine or thymine at PS2, adenine or guanine at PS3, cytosine or adenine at PS4, cytosine or adenine at PS5, guanine or adenine at PS6, guanine or adenine at PS7, cytosine or thymine at PS8, guanine or adenine at PS9, cytosine or thymine at PS 10, cytosine or thymine at PS 11 , adenine or cytosine at PS 12, cytosine or thymine at PS 13, cytosine or thymine at PS 14, thymine or cytosine at PS15, thymine or cytosine at PS16, cytosine or thymine at PS17 and thymine or cytosine at PS18. In addition, the inventors have determined the identity ofthe alleles at these sites in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS 1 -PS 18 in the PLAU gene, which are shown below in Tables 5 and 4, respectively. Each of these PLAU haplotypes constitutes a code that defines the variant nucleotides that exist in the human population at this set of polymorphic sites in the PLAU gene. Thus each PLAU haplotype also represents a naturally-occurring isoform (also referred to herein as an "isogene") ofthe PLAU gene. The frequency of each haplotype and haplotype pair within the total reference population and within each ofthe four major population groups included in the reference population was also determined.

Thus, in one embodiment, the invention provides a method, composition and kit for genotyping the PLAU gene in an individual. The genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17 and PS 18 in both copies ofthe PLAU 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 PLAU polymorphic sites. A genotyping kit ofthe invention comprises a set Of oligonucleotides designed to genotype each of these novel PLAU polymorphic 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 PLAU gene in an individual. In one embodiment, the haplotyping method comprises determining, for one copy ofthe PLAU gene, the identity ofthe nucleotide at one or more polymorphic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17 and PS 18. In another embodiment, the haplotyping method comprises determining whether one copy of the individual's PLAU gene is defined by one ofthe PLAU 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 PLAU gene are defined by one ofthe PLAU haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. Establishing the PLAU 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 PLAU activity, e.g., thrombolytic disorders and cancer.

For example, the haplotyping method can be used by the pharmaceutical research scientist to validate PLAU as a candidate target for treating a specific condition or disease predicted to be associated with PLAU activity. Defe-mining for a particular population the frequency of one or more ofthe individual PLAU haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue PLAU as a target for treating the specific disease of interest. In particular, if variable PLAU activity is associated with the disease, then one or more PLAU 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 PLAU haplotypes are of similar frequencies in the disease and control groups, then it may be inferred that variable PLAU 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 PLAU haplotype or haplotype pair, apply the information derived from detecting PLAU haplotypes in an individual to decide whether modulating PLAU activity would be useful in treating the disease.

The claimed invention is also useful in screening for compounds targeting PLAU to treat a specific condition or disease predicted to be associated with PLAU activity. For example, detecting which ofthe PLAU 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 PLAU isoforms present in the disease population, or for only the most frequent PLAU isoforms present in the disease population. Thus, without requiring any a priori knowledge ofthe phenotypic effect of any particular PLAU 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.

Haplotyping the PLAU 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 PLAU 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 PLAU haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute PLAU 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 PLAU haplotype or haplotype pair that is associated with response to the drug being studied in the trial, even if this association was previously unknown. 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 PLAU haplotype or haplotype pair.

In another embodiment, the invention provides a method for identifying an association between a trait and a PLAU genotype, haplotype, or haplotype pair for one or more ofthe novel polymorphic sites described herein. The method comprises comparing the frequency ofthe PLAU genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency ofthe PLAU genotype or haplotype in a reference population. A higher frequency ofthe PLAU genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the PLAU 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 PLAU 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 thrombolytic disorders and cancer.

In yet another embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the PLAU gene or a fragment thereof. The reference sequence comprises the contiguous sequences shown in Figure 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of thymine at PS1, thymine at PS2, gu-mine at PS3, adenine at PS4, adenine at PS5, adenine at PS6, adenine at PS7, thymine at PS8, adenine at PS9, thymine at PS10, thymme at PS11, cytosine at PS12, thymine at PS13, thymine at PS14, cytosine at PS15, cytosine at PS16, thymine at PS17 and cytosine at PS18. A particularly preferred polymorphic variant is an isogene ofthe PLAU gene. A PLAU isogene ofthe invention comprises cytosine or thymine at PS1, guanine or thymine at PS2, adenine or guanine at PS3, cytosine or adenine at PS4, cytosine or adenine at PS5, guanine or adenine at PS6, guanine or adenine at PS7, cytosine or thymine at PS8, guanine or adenine at PS9, cytosine or thymine at PS10, cytosine or thymine at PS11, adenine or cytosine at PS12, cytosine or thymine at PS13, cytosine or thymine at PS14, thymine or cytosine at PS15, thymine or cytosine at PS16, cytosine or thymine at PS 17 and thymine or cytosine at PS 18. The invention also provides a collection of PLAU isogenes, referred to herein as a PLAU genome anthology.

In another embodiment, the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a PLAU cDNA or a fragment thereof. The reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymorphic cDNA comprises at least one polymoφhism selected from the group consisting of adenine at a position corresponding to nucleotide 172, thymine at a position corresponding to nucleotide 422, cytosine at a position corresponding to nucleotide 691, thymine at a position corresponding to nucleotide 822, cytosine at a position corresponding to nucleotide 1048 and thymine at a position corresponding to nucleotide 1137. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of a PLAU isogene defined by haplotypes 3,6-8,11,14-16 and 19.

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

In other embodiments, the invention provides a recombinant expression vector comprising one ofthe polymoφhic genomic and cDNA variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector. The recombinant vector and host cell may be used to express PLAU 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 PLAU 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 arginine at a position corresponding to amino acid position 58, leucine at a position corresponding to amino acid position 141, glutamine at a position corresponding to amino acid position 231 and histidine at a position corresponding to ammo acid position 350. A polymoφhic variant of PLAU is useful in studying the effect ofthe variation on the biological activity of PLAU as well as on the binding affinity of candidate drugs targeting PLAU for the treatment of thrombolytic disorders and cancer.

The present invention also provides antibodies that recognize and bind to the above polymoφhic PLAU 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 or more of the PLAU polymoφhic genomic variants described herein and methods for producing such animals. The transgenic. animals are useful for studying expression ofthe PLAU isogenes in vivo, for in vivo screening and testing of drugs targeted against PLAU protein, and for testing the efficacy of therapeutic agents and compounds for thrombolytic disorders and cancer in a biological system.

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

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a reference sequence for the PLAU gene (Genaissance Reference No.

4874587; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the 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: 1 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). SEQ ID NO:94 is a modified version of SEQ ID NO: 1 that shows the context sequence of each polymoφhic site, PS 1 -PS 18, in a uniform format to facilitate electronic searching. For each polymoφhic site, SEQ ID NO: 94 contains a block of 60 bases ofthe nucleotide sequence encompassing the centrally-located polymoφhic site at the 30^th position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein.

Figure 2 illustrates a reference sequence for the PLAU 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 PLAU 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 of the PLAU gene. As described in more detail below, the inventors herein discovered 20 isogenes ofthe PLAU gene by characterizing the PLAU gene found in genomic DNAs isolated from an Index Repository that contains immortalized cell lines from one chimpanzee and 93 human individuals. The human individuals included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (21 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic/Latino (18 individuals). To the extent possible, the members of this reference population were organized into population subgroups by their self-identified ethnogeographic origin as shown in Table 1 below. 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.

Table 1. Po ulation Grou s in the Index Re ositor

The PLAU isogenes present in the human reference population are defined by haplotypes for 18 polymoφhic sites in the PLAU gene, all of which are believed to be novel. The novel PLAU polymoφhic sites identified by the inventors are referred to as PS1-PS18 to designate the order in which they are located in the gene (see Table 3 below). Using the genotypes identified in the Index Repository for PS 1 -PS 18 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the PLAU gene present in individual human members of this repository. The human genotypes and haplotypes found in the repository for the PLAU gene include those shown in Tables 4 and 5, respectively. The polymoφhism and haplotype data disclosed herein are useful for validating Whether PLAU is a suitable target for drugs to treat thrombolytic disorders and cancer, screening for such drugs and reducing bias in clinical trials of such drugs. In the context of this disclosure, the following terms shall be defined as follows unless otherwise indicated:

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

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

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

Genotype — An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at one or more 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 polymoφhic sites examined herein 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 polymoφhic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.

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

Haplotype - A 5' to 3' sequence of nucleotides found at one or more 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 polymoφhic sites examined herein in a locus on a single chromosome from a single individual.

Sub-haplotype - The 5 ' to 3 ' sequence of nucleotides seen at a subset ofthe polymoφhic sites examined herein in a locus on a single chromosome from a single individual. Haplotype pair - The two haplotypes found for a locus in a single individual.

Haplotyping - A process for deteπnn-ing one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference. Haplotype data - Information concerning one or more ofthe following for a specific gene: a listing ofthe haplotype pairs in each individual' in a population; a listing ofthe different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait. Isoform - A particular form of a gene, mRNA, cDNA, coding sequence or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.

Isogene - One ofthe isoforms (e.g., alleles) of a gene found in a population. An isogene (or allele) 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, where physical features include polymoφhic sites.

Naturally-occurring - A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man. Nucleotide pair - The nucleotides found at a 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 on a chromosome or DNA molecule at which at least two alternative sequences are found in a population.

Polymorphic variant (variant)- A gene, mRNA, cDNA, polypeptide, protein or peptide whose nucleotide or amino acid sequence varies from a reference sequence due to the presence of a 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 of the locus is not known.

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

The compositions comprise at least one oligonucleotide for detecting the variant nucleotide or nucleotide pair located at a novel PLAU polymoφhic site in one copy or two copies ofthe PLAU gene. Such oligonucleotides are referred to herein as PLAU haplotyping oligonucleotides or genotyping oligonucleotides, respectively, and collectively as PLAU oligonucleotides. In one embodiment, a PLAU haplotyping or genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, one ofthe novel 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.

Haplotyping or genotyping oligonucleotides ofthe invention must be capable of specifically hybridizing to a target region of a PLAU polynucleotide. Preferably, the target region is located in a PLAU 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 another region in the PLAU polynucleotide or with a non-PLAU 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 PLAU gene using the polymoφhism information provided herein in conjunction with the known sequence information for the PLAU 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 probe o primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.

Preferred haplotyping or 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 ofthe invention include ASO probes and ASO primers. ASO probes which usually provide good discrimination between different alleles are those in which a central position ofthe oligonucleotide probe aligns with the 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 noncod ng 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 ofthe polymoφhic site to represent that the ASO contains either ofthe two alternative allelic variants observed at that polymoφhic site. A preferred ASO probe for detecting PLAU gene polymoφhisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:

TTTGTCGYGTTGATG SEQ ID NO: 4 and its complement, GCACGGAKAATTTAC SEQ ID NO: 5 and its complement, GCCGTCTRGCGCCCC SEQ ID NO: 6 and its complement, CGTCTAGMGCCCCGA SEQ ID NO: 7 and its complement, ACTGATGMTGCCCAA SEQ ID^' NO: 8 and its complement, TGGGAAGRCTTCAGG SEQ ID NO: 9 and its complement, GAAATTCRGAGGGCA SEQ ID NO:10 and its complement, ACGCTCAYGAAACAG SEQ ID NO : 11 and its complement, GAAGTGCRGCCTCTG SEQ ID NO: 12 and its complement, CTAAAGCYGCTTGTC SEQ ID NO:13 and its complement, AGTGTTCYGCCTCAT SEQ ID NO:14 and its complement, TTACCCAMAGAAGGA SEQ ID NO:15 and its complement, ACCACAAYGACATTG SEQ ID NO:16 and its complement, AGCCCAGYGTGATCA SEQ ID NO: 17 and its complement, GCCCCACYACTACGG SEQ ID NO:18 and its complement, GGCTTGTYCCAGCCA SEQ ID NO: 19 and its complement, GGGGACCYCTCGTCT SEQ ID NO:20 and its complement, and ACCAGGGYGAACGAC SEQ ID NO:21 and its complement.

A preferred ASO primer for detecting PLAU gene polymoφhisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of: CGTACATTTGTCGYG (SEQ ID NO 22) AGTCTTCATCAACRC (SEQ ID NO : 23 ) ; GAGGAAGCACGGAKA (SEQ ID NO 24) AGGCTTGTAAATTMT (SEQ ID NO : 25 ) ; AGAGCCGCCGTCTRG (SEQ ID NO 26) GAGGTCGGGGCGCYA (SEQ ID NO : 27 ) ; AGCCGCCGTCTAGMG (SEQ ID NO 28) GCGAGGTCGGGGCKC (SEQ ID NO : 29 ) ; GCTTTGACTGATG T (SEQ ID NO 30) AGGTCCTTGGGCAKC (SEQ ID NO - 31 ) ; ACAAGTTGGGAAGRC (SEQ ID NO 32) ATGTCCCCTGAAGYC (SEQ ID NO : 33 ) CCCAAAGAAATTCRG (SEQ ID NO 34) ■CAGTGCTGCCCTCYG (SEQ ID NO : 35 ) ; ACACACACGCTCAYG (SEQ ID NO 36) TGGCCACTGTTTCRT (SEQ ID NO : 37 ; ACAAGAGAAGTGCRG (SEQ ID NO 38) CTCAACCAGAGGCYG (SEQ ID NO : 39 ) ; GTGGGCCTAAAGCYG (SEQ ID NO 40) CTCTTGGACAAGCRG (SEQ ID NO : 41 ) CAGCAAAGTGTTCYG (SEQ ID NO 42) GGAGAAATGAGGCRG (SEQ ID NO : 43 ) CAGTGATTACCCAMA (SEQ ID NO 44) TAGTCCTCCTTCTKT (SEQ ID NO : 45) TTGCTCACCACAAYG (SEQ ID NO 46) CCTCACCAATGTCRT (SEQ ID NO : 47 ) AAAATGAGCCCAGYG (SEQ ID NO 48) TTCCCTTGATCACRC (SEQ ID NO : 49 ) TCAGCAGCCCCACYA (SEQ ID NO 50) TCAGAGCCGTAGTRG (SEQ ID NO : 51 ) ; CTCCTGGGCTTGTYC (SEQ ID NO 52) TTAAGCTGGCTGGRA (SEQ ID NO : 53 ) ; ACTCAGGGGGACCYC (SEQ ID NO 54) GGGAACAGACGAGRG (SEQ ID NO : 55) ; GGCACCACCAGGGYG (SEQ ID NO 56) and GCTATTGTCGTTCRC (SEQ ID NO : 57 )

Other 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 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 PLAU gene polymoφhisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:

ACATTTGTCG SEQ ID NO 58) ; CTTCATCAAC(SEQ ID NO: 59);

GAAGCACGGA SEQ ID NO 60) ; CTTGTAAATT(SEQ ID NO: 61)

GCCGCCGTCT SEQ ID NO 62) ; GTCGGGGCGC(SEQ ID NO: 63)

CGCCGTCTAG SEQ ID NO 64); AGGTCGGGGC(SEQ ID NO: 65)

TTGACTGATG SEQ ID NO 66) ; TCCTTGGGCA(SEQ ID NO: 67)

AGTTGGGAAG SEQ ID NO 68) ; TCCCCT.GAAG(SEQ ID NO: 69)

AAAGAAATTC SEQ ID NO 70); TGCTGCCCTC(SEQ ID NO: 71)

CACACGCTCA SEQ ID NO 72) ; CCACTGTTTC(SEQ ID NO: 73)

AGAGAAGTGC SEQ ID NO 74); AACCAGAGGC ( SEQ ID NO: 75)

GGCCTAAAGC SEQ ID NO 76); TTGGACAAGC(SEQ ID NO: 77)

CAAAGTGTTC SEQ ID NO 78); GAAATGAGGC(SEQ ID NO: 79)

TGATTACCCA SEQ ID NO 80) ; TCCTCCTTCT(SEQ ID NO: 81)

CTCACCACAA SEQ ID NO 82) ; CACCAATGTC(SEQ ID NO: 83)

ATGAGCCCAG SEQ ID NO 84) ; CCTTGATCAC (SEQ ID NO: 85)

GCAGCCCCAC SEQ ID NO 86) ; GAGCCGTAGT(SEQ ID NO: 87)

CTGGGCTTGT SEQ ID NO 88) ; AGCTGGCTGG(SEQ ID NO: 89)

CAGGGGGACC SEQ ID NO 90) ; AACAGACGAG (SEQ ID NO: 91)

ACCACCAGGG SEQ ID NO 92); and ATTGTCGTTC(SEQ ID NO: 93)

In some embodiments, a composition contains two or more differently labeled PLAU oligonucleotides for simultaneously probing the identity of nucleotides or nucleotide pairs 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. PLAU oligonucleotides ofthe invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized oligonucleotides may be used in a variety of polymoφhism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized PLAU 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 PLAU 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 PLAU gene in an individual. As used herein, the terms "PLAU genotype" and "PLAU 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 PLAU gene. The additional polymoφhic sites may be currently known polymoφhic sites or sites that are subsequently discovered.

One embodiment of a genotyping method ofthe invention involves isolating from the individual a nucleic acid sample comprising the two copies ofthe PLAU gene, mRNA transcripts thereof or cDNA copies thereof, or a fragment of any ofthe foregoing, that are present in the individual, and dete--m--ning the identity ofthe nucleotide pair at one or more polymoφhic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17 and PS18 in the two copies to assign a PLAU genotype to the individual. As will be readily understood by the skilled artisan, the two "copies" of a gene, mRNA or cDNA (or fragment of such PLAU molecules) in an individual may be the same allele or may be different alleles. In another embodiment, a genotyping method ofthe invention comprises determining the identity ofthe nucleotide pair at each of PS 1 -PS 18.

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 PLAU 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 not present in the mRNA or cDNA. If a PLAU gene fragment is isolated, it must contain the polymoφhic site(s) to be genotyped. One embodiment of a haplotyping method ofthe invention comprises isolating from the individual a nucleic acid sample containing only one ofthe two copies ofthe PLAU gene, mRNA or cDNA, or a fragment of such PLAU molecules, 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 PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS 16, PS 17 and PS 18 in that copy to assign a PLAU haplotype to the individual.

The nucleic acid used in the above haplotyping methods ofthe invention may be isolated using any method capable of separating the two copies ofthe PLAU gene or fragment such as one of the methods described above for preparing PLAU 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 typically only provide haplotype information on one ofthe two PLAU gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional PLAU clones will usually need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies ofthe PLAU gene in an individual. In some cases, however, once the haplotype for one PLAU allele is directly determined, the haplotype for the other allele may be inferred if the individual has a known genotype for the polymoφhic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known. In a particularly preferred embodiment, the nucleotide at each of PS 1 -PS 18 is identified.

In another embodiment, the haplotyping method comprises determining whether an individual has one or more ofthe PLAU haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies ofthe individual's PLAU gene, the phased sequence of nucleotides present at each of PS 1 -PS 18. This identifying step does not necessarily require that each of PS 1 -PS 18 be directly examined. Typically only a subset of PS 1 -PS 18 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 another embodiment of a haplotyping method ofthe invention, a PLAU haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymoφhic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17 and PS 18 in each copy of the PLAU 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 PS1-PS18 in each copy ofthe PLAU 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 PLAU 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 guariine/gu-mine).

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-biot n, 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 PLAU gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies ofthe gene, mRNA, cDNA or fragment(s) thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995. The arrays would contain a battery of allele-specific oligonucleotides representing each ofthe 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; Ruaiio 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 haplotyping or 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. Detection ofthe allele(s) present at 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 PLAU haplotype pair is predicted from its PLAU genotype using information on haplotype pairs known to exist in a reference population. In its broadest embodiment, the haplotyping prediction method comprises identifying a PLAU genotype for the individual at two or more PLAU polymoφhic sites described herein, accessing data containing PLAU haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the genotype data. In one embodiment, the reference haplotype pairs include the PLAU haplotype pairs shown in Table 4. The PLAU haplotype pair can be assigned by comparing the individual's genotype with the genotypes corresponding to the haplotype pairs known to exist in the general population or in a specific population group, and determining which haplotype pair is consistent with the genotype ofthe individual. In some embodiments, the comparing step may be performed by visual inspection (for example, by consulting Table 4). When the genotype ofthe individual is consistent with more than one haplotype pair, frequency data (such as that presented in Table 7) may be used to determine which of these haplotype pairs is most likely to be present in the individual. This determination may also be performed in some embodiments by visual inspection, for example by consulting Table 7. If a particular PLAU haplotype pair consistent with the genotype of the individual is more frequent in the reference population than others consistent with the genotype, then that haplotype pair with the highest frequency is the most likely to be present in the individual. In other embodiments, the comparison may be made by a computer-implemented algorithm with the genotype ofthe individual and the reference haplotype data stored in computer-readable formats. For example, as described in PCT US01/12831, filed April 18, 2001, one computer-implemented algorithm to perform this comparison entails enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing PLAU haplotype pairs frequency data determined in a reference population to determine a probability that the individual has a possible haplotype pair, and analyzing the determined probabilities to assign a haplotype pair to the individual. 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-descent, 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 umelated individuals from each ofthe four population groups named above. A particularly preferred reference population includes a 3-generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures. .

In a preferred embodiment, the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L. Haiti et al., Principles of Population Genomics, Sinauer Associates (Sunderland, MA), 3^rd Ed., 1997) postulates that the frequency of finding the haplotype pair H_x /H₂is equal to p_H__v(H- IH ) = 2p(H₁)p(H₂) if H- ≠ H₂ and p_H__w(H_x IH₂) = ^(#X#₂) 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 PLAU 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 contai-r-ing 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 l -.111-22; copending PCT/USO 1/12831 filed April 18, 2001 ) 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 PLAU 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 PLAU polymoφhic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population. The population may be e.g., a reference population, a family population, a same gender population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment). hi another aspect ofthe invention, frequency data for PLAU 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 PLAU 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. In one embodiment, the method involves obtaining data on the frequency ofthe genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait. Frequency data for one or both ofthe reference and trait populations may be obtained by genotyping or haplotyping each individual, in the populations using one or more ofthe methods described above. The haplotypes for the trait population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as described above. 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 PLAU 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 PLAU genotype, haplotype or haplotype pair. Preferably, the PLAU 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 PLAU 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/or adverse response (i.e., side effects).

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

The therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the PLAU 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 PLAU genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their PLAU 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 PLAU gene give the most significant contribution to the differences in phenotype. One regression model useful in the invention is described in WO 01/01218, entitled "Methods for Obtaining and Using Haplotype Data".

A second method for finding correlations between PLAU 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 WO 01/01218.

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 PLAU gene. As described in WO 01/01218, 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 PLAU 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 PLAU 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 PLAU 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 PLAU 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 PLAU gene or a fragment ofthe gene which contains at least one ofthe novel polymoφhic sites described herein. The nucleotide sequence of a variant PLAU 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 PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS 10, PS11, PS 12, PS 13, PS 14, PS15, PS16, PS17 and PS18. Similarly, the nucleotide sequence of a variant fragment ofthe PLAU 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 PLAU gene, which is defined by haplotype 10, (or other reported PLAU sequences) or to portions ofthe reference sequence (or other reported PLAU sequences), except for the haplotyping and genotyping oligonucleotides described above.

^* The location of a polymoφhism in a variant PLAU gene or fragment is preferably identified by aligning its sequence against SEQ ID NO: 1. The polymoφhism is selected from the group consisting of thymine at PSl, thymine at PS2, guanine at PS3, adenine at PS4, adenine at PS5, adenine at PS6, adenine at PS7, thymine at PS8, adenine at PS9, thymine at PS10, thymine at PS11, cytosine at PS12, thymine at PS13, thymine at PS14, cytosine at PS15, cytosine at PS.16, thymine at PS17 and cytosine at PS 18. In a preferred embodiment, the polymoφhic variant comprises a naturally-occurring isogene ofthe PLAU gene which is defined by any one of haplotypes 1- 9 and 11 - 20 shown in Table 5 below.

Polymoφhic variants ofthe invention may be prepared by isolating a clone containing the PLAU 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 or fragment thereof, that is claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art. Any particular PLAU variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.

PLAU isogenes, or fragments thereof, may be isolated using any method that allows ^■ separation ofthe two "copies" ofthe PLAU gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles. Separation methods include targeted in vivo cloning (TIVC) in yeast as described in WO 98/01573, U.S. Patent No.

5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets. Yet other methods are single molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruano et al., 1989, supra; Ruano et al., 1991, supra; Michalatos-Beloin et al., supra).

The invention also provides PLAU genome anthologies, which are collections of at least two

PLAU isogenes found in a given population. The population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same gender population. A PLAU genome anthology may comprise individual PLAU isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups ofthe PLAU isogenes in the anthology may be stored in separate containers. Individual isogenes or groups of such isogenes in a genome anthology may be stored in any convenient and stable form, including but not limited to in buffered solutions, as DNA precipitates, freeze-dried preparations and the like. A preferred PLAU 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 PLAU protein in a prokaryotic or a eukaryotic host cell. Examples of expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SV40. Other regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation 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 PLAU 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 PLAU gene will produce PLAU RNAs 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 PLAU cDNA comprising a nucleotide sequence which is a polymoφhic variant ofthe PLAU reference coding sequence shown in Figure 2. - Thus, the invention also provides PLAU mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig. 2) (or its corresponding RNA sequence) for those regions of SEQ ID NO:2 that correspond to the examined portions ofthe PLAU gene (as described in the Examples below), except for having one or more polymoφhisms selected from the group consisting of adenine at a position corresponding to nucleotide 172, thymine at a position corresponding to nucleotide 422, cytosine at a position corresponding to nucleotide 691, thymine at a position corresponding to nucleotide 822, cytosine at a position corresponding to nucleotide 1048 and thymine at a position corresponding to nucleotide 1137. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of a PLAU isogene defined by any one of haplotypes 3,6-8,11,14-16 and 19. Fragments of these variant mRNAs and cDNAs are included in the scope ofthe invention, provided they contain one or more ofthe novel polymoφhisms described herein. The invention specifically excludes polynucleotides identical to previously identified PLAU mRNAs or cDNAs, and previously described fragments thereof. Polynucleotides comprising a variant PLAU 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 PLAU gene, mRNA or cDNA 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 PLAU 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 PLAU gene or cDNA may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand. 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 PLAU genomic, mRNA and cDNA variants described herein.

Polynucleotides comprising a polymoφhic gene variant or fragment ofthe invention may be useful for therapeutic puφoses. For example, where a patient could benefit from expression, or increased expression, of a particular PLAU protein isoform, an expression vector encoding the isoform may be administered to the patient. The patient may be one who lacks the PLAU 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 PLAU isogene. Expression of a PLAU isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA or antisense RNA for the isogene or fragment thereof. 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 PLAU mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of PLAU mRNA transcribed from a particular isogene.

The untranslated mRNA, antisense RNA or antisense oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo. Alternatively, such molecules may be formulated as a pharmaceutical composition for administration to the patient. Oligoribonucleotides and or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life. Possible modifications include, but are not limited to phosphorothioate or 2' 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 of (a) the reference PLAU amino acid sequence shown in Figure 3 or (b) a fragment of this reference sequence. The location of a variant amino acid in a PLAU polypeptide or fragment ofthe invention is preferably identified by aligning its sequence against SEQ ID NO:3 (Fig. 3). A PLAU protein variant ofthe invention comprises an amino acid sequence identical to SEQ ID NO: 3 for those regions of SEQ ID NO: 3 that are encoded by examined portions ofthe PLAU gene (as described in the Examples below), except for having one or more variant amino acids selected from the group consisting of arginine at a position corresponding to amino acid position 58, leucine at a position corresponding to amino acid position 141, glutamine at a position corresponding to amino acid position 231 and histidine at a position corresponding to amino acid position 350. Thus, a PLAU fragment ofthe invention, also referred to herein as a PLAU peptide variant, is any fragment of a PLAU protein variant that contains one or more ofthe amino acid variations shown in Table 2. The invention specifically excludes amino acid sequences identical to those previously identified for PLAU, including SEQ ID NO:3, and previously described fragments thereof. PLAU 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

PLAU protein variant ofthe invention is encoded by an isogene defined by one ofthe observed haplotypes, 3,6-8,11,14-16 and 19, shown in Table 5.

Table 2. Novel Polymoφhic Variants of PLAU

Polymoφhic Amino Acid Position and Identities

Variant Number 58 141 231 350 1 G P K H 2 G P Q Y 3 G P Q H 4 G L K Y 5 G L K H 6 G L Q Y 7 G L Q H 8 R P K Y 9 R P K H 10 R P Q Y 11. R p ^• Q H 12 R L K Y 13 R L K H 14 R L Q Y 15 R L Q H A PLAU peptide variant ofthe invention 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 PLAU peptide variants may be useful as antigens to generate antibodies specific for one ofthe above PLAU isoforms. In addition, the PLAU peptide variants may be useful in drug screening assays. A PLAU variant protein or peptide ofthe invention may be prepared by chemical synthesis or by expressing an appropriate variant PLAU genomic or cDNA sequence described above. Alternatively, the PLAU protein variant may be isolated from a biological sample of an individual having a PLAU isogene which encodes the variant protein. Where the sample contains two different PLAU isoforms (i.e., the individual has different PLAU isogenes), a particular PLAU isoform ofthe invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular PLAU isoform but does not bind to the other PLAU isoform.

The expressed or isolated PLAU protein or peptide 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 PLAU protein or peptide as discussed further below. PLAU variant proteins and peptides 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 immimoaffinity chromatography, antibodies specific for a particular polymoφhic variant may be used.

A polymoφhic variant PLAU gene ofthe invention may also be fused in frame with a heterologous sequence to encode a chimeric PLAU protein. The non-PLAU portion ofthe chimeric 5 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 PLAU and non-PLAU portions so that the PLAU protein may be cleaved and purified away from the non-PLAU portion.

An additional embodiment ofthe invention relates to using a novel PLAU protein isoform, or a fragment thereof, in any of a variety of drug screening assays. Such screening assays may be

10 performed to identify agents that bind specifically to all known PLAU 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 PLAU 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 PLAU variant may be accomplished using the method described in PCT application WO84/03565, in

15 which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the PLAU protein(s) of interest and then washed. Bound PLAU protein(s) are then detected using methods well-known in the art.

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

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

25 artisan.

In another embodiment, the invention provides antibodies specific for and immunoreactive with one or more ofthe novel PLAU protein or peptide variants described herein. The antibodies may be either monoclonal or polyclonal in origin. The PLAU protein or peptide variant used to generate the antibodies may be from natural or recombinant sources (in vitro or in vivo) or produced by chemical

30. synthesis or semi-synthetic synthesis using synthesis techniques known in the art. If the PLAU protein or peptide variant is of insufficient size to be antigenic, it may be concatenated or 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,

35 and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).

In one embodiment, an antibody specifically immunoreactive with one ofthe novel protein or peptide variants described herein is administered to an individual to neutralize activity ofthe PLAU 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 PLAU protein variant from solution as well as react with PLAU protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates. In another preferred embodiment, the antibodies will detect PLAU 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 PLAU 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 PLAU 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, 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 PLAU may be investigated by various means known in the art, such as by in vitro translation of mRNA transcripts ofthe PLAU gene, cDNA or fragment thereof, or by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymoφhic variant ofthe PLAU gene. As used herein, "expression" includes but is not limited to one or more ofthe following: transcription of the gene into precursor mRNA; splicing and other processing ofthe precursor mRNA to produce mature mRNA; mRNA stability; translation ofthe mature mRNA(s) into PLAU protein(s) (including effects of polymoφhisms on codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.

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

Recombinant nonhuman organisms, i.e., transgenic animals, expressing a variant PLAU gene, cDNA or coding sequence are prepared using standard procedures known in the art. Preferably, a construct comprising the variant gene, cDNA or coding sequence is introduced into a nonhuman animal or an ancestor ofthe animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage. Transgenic ammals carrying the constructs ofthe invention can be made by several methods known to those having skill in the art. One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes (or cDNA or coding sequence) of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent

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 PLAU isogene, cDNA or coding sequences may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice" and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M. Gilman, J. Witkowski, and M. Zoller; W.H. Freeman and Company, New York, pages 254-272). Transgenic animals stably expressing a human PLAU isogene, cDNA or coding sequence and producing the encoded human PLAU protein can be used as biological models for studying diseases related to abnormal PLAU 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 PLAU isogene described herein. The pharmaceutical composition may comprise any ofthe following active ingredients: a polynucleotide comprising one of these novel PLAU isogenes (or cDNAs or coding sequences); an antisense oligonucleotide directed against one ofthe novel PLAU isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel PLAU 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 PLAU 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 PLAU 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 PLAU 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 PLAU gene for polymoφhic sites.

Amplification of Target Regions The following target regions ofthe PLAU 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 SEQ ID NO:l (Figure 1). PCR Primer Pairs

Fragment No. Forward Primer Reverse Primer PCR Product

Fragment 1 2937-2958 complement of 3637-3617 701 nt

Fragment 2 3212-3232 complement of 3879-3861 668 nt

Fragment 3 3893-3914 complement of 4239-4219 347 nt

Fragment 4 4334-4356 complement of 4733-4709 400 nt

Fragment 5 4492-4513 complement of 5016-4996 ^• 525 nt

Fragment 6 5624-5645 complement of 6037-6015 414 nt

Fragment 7 5814-5836 complement of 6439-6417 626 nt

Fragment 8 6311-6332 complement of 6845-6823 535 nt

Fragment 9 7119-7140 complement of 7568-7544 450 nt

Fragment 10 7498-7520 complement of 8206-8186 709 nt

Fragment 11 8625-8647 complement of 9341-9319 717 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 = 10 μl

10 x Advantage 2 Polymerase reaction buffer (Clontech) = 1 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 represented below by the nucleotide positions of their initial and final nucleotides, which correspond to positions in SEQ ID NO: 1 (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 2997-3018 complement of 3559-3540 Fragment 2 3233-3254 complement of 3736-3719 Fragment 3 3908-3927 complement of 4211 -4192 Fragment 4 4413-4432 complement of 4689-4669 Fragment 5 4575-4595 complement of 4968-4948 Fragment 6 5651-5670 complement of 6005-5985 Fragment 7 5905-5924 complement of 6394-6376 Fragment 8 6341-6360 complement of 6778-6759 Fragment 9 7144-7163 complement of 7531-7513 Fragment 10 7568-7587 complement of 8019-7998 Fragment 11 8710-8728 complement of 9244-9226

Analysis of Sequences for Polymoφhic Sites

Sequence information for a minimum of 80 humans was 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 PLAU reference genomic sequence (SEQ ID NO:l) are listed in Table 3 below.

Table 3. Polymoφhic Sites Identified in the PLAU Gene

Polymoφhic Nucleotide Reference Variant CDS Variant AA

Site Number Polyld(a) Position Allele Allele Position Variant

PSl 5435813 3186 C T

PS2 5435811 3419 G T

PS3 5435805 4030 A G

PS4 5435803 4032 C A

PS5 ^' 5435801 . 4134 C A

PS6 5435797 ^• 4611 G A

PS7 5435793 4795 G A 172 G58R

PS8 5435791 4966 C T

PS9 5435787 5697 G A

PS10 5435785 5837 C T 422 P141L

PS11 5435777 6332 C T

PS12 5435771 6484 A C 691 K231Q

PS13 5435769 6615 C T 822 N274N

PS14 5435767 7476 C T

PS15 5435759 7822 T C 1048 Y350H

PS16 5435757 7955 T C

PS17 5435755 8900 C T 1137 P379P

PS18 5435751 9199 T C

(a) Polyld is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc

EXAMPLE 2

This example illustrates analysis ofthe PLAU polymoφhisms identified in the Index

Repository for human genotypes and haplotypes.

The different genotypes containing these polymoφhisms that were observed in unrelated members ofthe 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.

Table 4 (Part 1). Genotypes and Haplotype Pairs Observed for PLAU Gene

Genotype Polymoφhic Sites

Number HAP Pah PSl PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10

1 12 12 C G A C C G G C G C

2 8 8 C G A C C G G C A C

3 14 14 C G A C C G . G C G T

4 9 9 C G A C C G G C G C

5 12 17 C G A/G C C G/A G C G C

6 12 7 C G A C C G G C G/A C

7 8 5 C G A C C G/A G C AG C

8 12 6 C G A C C G G/A C G/A C

9 3 2 C G A C A G G T/C G C

10 3 16 C G A C A/C G G T G C

11 12 18 C G/T A C C G G C . G C

12 ^•14 4 C G A C C G/A G C G T/C

13 12 3 C G A C C/A G G C/T G C

14 12 5 C G A C C G/A G C G C

15 12 20 C/T G A C C G G C G C

16 14 10 C G A C C G G C G T/C

17 8 18 C G/T A C C G G C A/G C

18 14 13 C G A C C G G C G T/C

19 9 19 C. G/T A C C G G C/T G C

20 12 10 C G A C C G G C G C

21 8 10 C G A C C G G C A/G C

22 9 10 C G A C C G G C G C

23 8 11 C G A C C G G C A/G C

24 12 14 C G A C C G G C G C/T

25 14 9 C G A C C G G C G T/C

26 8 9 C G A C C G G C A/G C

27 14 5 C G A C C G/A G C G T/C

28 12 15 C G A C C G G C/T G/A C

29 12 8 C G A C C G G C G/A C

30 14 8 C G A C C G G C G/A T/C

31 9 1 C G A C/A C G G C G C

32 12 9 C G A C C G G C G C

33 12 13 C G A C C G G C G C Table 4 (Part 2). Genotypes and Haplotype Pairs Observed for PLAU Gene

Genotype Polymoφhic Sites

Number HAP Pair PS11 PS12 PS13 PS14 PS15 PS16 PS17 PS18

1 12 12 C A C T T T C T

2 8 8 C A T C T T C C

3 14 14 C A C C T T C C

4 9 9 C A C C T T C C

5 12 17 C A C T T T C T

6 12 7 C A ^' C/T T/C T/C T C T/C

7 8 5 C A T/C C/T T T C C/T

8 12 6 C A C/T T/C T T C T/C

9 3 2 C C/A C C T T C C/T

10 3 16 C C C C T T C C

11 12 18 C A C T/C T T C T/C

12 14 4 C A C C T T C C/T

13 12 3 C A/C C T/C T T C T/C

14 12 5 C A C T T T C T

15 12 20 C A C T/C T T/C C T

16 14 10 C A C C T T C C/T

17 8 18 C A T/C C T T C C

18 14 13 C/T A C C T T C C/T

19 9 19 C A/C C C T T C C

20 12 10 C A C T/C T T C T

21 8 10 C A T/C C T T C C/T

22 9 10 C A C C T T C C/T

23 8 11 C A T/C C T T C/T C

24 12 14 C A C T/C T T C T/C

25 14 9 C A C C T T C C

26 8 9 C A T/C C T T C C

27 14 5 C A C C/T T T C C/T

28 12 15 C A C/T T/C T T C T/C

29 12 8 C A C/T T/C T T C T/C

30 14 8 C A C/T C T T C C

31 9 1 C A C C T T C C

32 12 9 C A C T/C T T C T/C

33 12 13 C/T A C T/C T T C T

The haplotype pahs 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 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in

PCT/US01/12831, filed April 18, 2001. 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 then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals. In the present analysis, the list of haplotypes was augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation

African- American family).

By following this protocol, it was determined that the Index Repository examined herein and, by extension, the general population contains the 20 human PLAU haplotypes shown in Table 5 below.

A PLAU isogene defined by a full-haplotype shown in Table 5 below comprises the regions of the SEQ ID NOS indicated in Table 5, with theh corresponding set of polymoφhic locations and identities, which are also set forth in Table 5.

Table 5 (Part 1). Haplotypes ofthe PLAU gene.

Regions PS PS Haplotype Number(d)

Examined(a) No.(b). Position(c) 1 2 3 4 5 6 7 8 9 10

2937-3879 1 3186/30 C C C C C C C C C C

2937-3879 2 3419/150 G G G G G G G G G G

3893-4239 3 4030/270 ^■ A A A Ά A A A A A A

3893-4239 4 ^' 4032/390 A C C C C C C C C C

3893-4239 5 4134/510 C A A C C C C C C C

4334-5016 6 4611/630 G G G A A G G G G G

4334-5016 7 4795/750 G G G G G A G G G G

4334-5016 8 4966/870 . C C T . C C C C C C C

5624-6845 9 5697/990 G G G G G A A A G G

5624-6845 10 5837/1110 C C C C C C C C C C

5624-6845 . 11 6332/1230 C C C C C C C C C C

5624-6845 12 6484/1350 A A C A A A A A A A

5624-6845 13 6615/1470 C C C C C T T T C C

7119-8206 14 7476/1590 C C C C T C C C C C

7119-8206 ^' 15 7822/1710 T T T T T T C T T T

7119-8206 16 7955/1830 T T T T T T T T T T

8625-9341 17 8900/1950 C C C C C C C C C C

8625-9341 18 9199/2070 C T C T T C C C C T

Table 5 (Part 2). Haplotypes ofthe PLAU gene.

Regions PS PS Haplotype Number(d)

Examined(a) No.(b) Position(c) 11 12 13 14 15 16 17 18 19 20

2937-3879 1 3186/30 C C C C C C C C C T

2937-3879 2 3419/150 G G G G G G G T T G

3893-4239 3 4030/270 A A A A A A G A A A

3893-4239 4 4032/390 C C C C C C C C C C

3893-4239 5 4134/510 C C C C C C C C C C

4334-5016 6 4611/630 G G G G G G A G G G

4334-5016 7 4795/750 G G G G G G G G G G

4334-5016 8 4966/870 C C C C T T C C T C

5624-6845 9 5697/990 G G G G A G G G G G

5624-6845 10 5837/1110 C C C T C C C C C C

5624-6845 11 6332/1230 C C T C C C . C C C C

5624-6845 12 6484/1350 A A A A A C A A C A

5624-6845 13 6615/1470 C C C C T C C C C C

7119-8206 14 7476/1590 C T C C C C T C C C

7119-8206 15 7822/1710 T T T T T T T T T T

7119-8206 16 7955/1830 T T T T T T T T T C

8625-9341 17 8900/1950 T C C C C C C C C C

8625-9341 18 9199/2070 C T T C C C T C C T

(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 ofthe regions sequenced;

(b) PS = polymoφhic site;

(c) Position of PS within the indicated SEQ ID NO, with the Imposition number referring to SEQ ID NO:l and the 2^nd position number referring to SEQ ID NO:94, a modified version of SEQ ID NO:l that comprises the context sequence of each polymoφhic site, PS1-PS18, to facilitate electronic se-irching ofthe haplotypes; (d) Alleles for PLAU haplotypes are presented 5' to 3' in each column.

SEQ ID NO:l refers to Figure 1, with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol. SEQ ID NO: 94 is a modified version of SEQ ID

NO:l that shows the context sequence of each of PSl -PS 18 in a uniform format to facilitate electronic se_-rching ofthe PLAU haplotypes. For each polymoφhic site, SEQ ID NO: 94 contains a block of 60 bases ofthe nucleotide sequence encompassing the centrally-located polymoφhic site at the 30^th -position, followed by 60 bases of unspecified sequence to represent that each polymoφhic site is separated by genomic sequence whose composition is defined elsewhere herein.

Table 6 below shows the percent of chromosomes characterized by a given PLAU haplotype for all unrelated individuals in the Index Repository for which haplotype data was obtained. The percent of these unrelated individuals who have a given PLAU haplotype pair is shown in Table 7. In Tables 6 and 7, the "Total" column shows this frequency data for all of these unrelated individuals, while the other columns show the frequency data for these unrelated individuals categorized according to their self-identified ethnogeographic origin. Abbreviations used in Tables 6 and 7 are AF = African Descent, AS = Asian, CA = Caucasian, HL = Hispanic-Latino, and AM = Native American.

Table 6. Frequency of Observed PLAU Haplotypes In Unrelated Individuals

HAP No. HAP ID Total CA AF AS HL AM

1 5438798 0.61 0.0 2.5 0.0 0.0 0.0

2 5438799 0.61 0.0 2.5 0.0 0.0 0.0

3 5438789 2.44 0.0 7.5 0.0 2.78 0.0

4 5438793 0.61 2.38 0.0 0.0 0.0 0.0

5 5438791 1.83 2.38 0.0 0.0 5.56 0.0

6 5438801 0.61 0.0 0.0 0.0 0.0 16.67

7 . 5438795 0.61 0.0 0.0 0.0 2.78 0.0

8 5438786 18.29 16.67 10.0 35.0 11.11 16.67

9 5438787 10.37 0.0 22.5 15.0 2.78 16.67

10 5438788 3.66 2.38 10.0 0.0 2.78 0.0

11 5438802 0.61 0.0 0.0 2.5 0.0 0.0

12 5438784 31.71 40.48 30.0 10.0 47.22 33.33

13 5438792 1.22 0.0 5-0 ^' 0.0 0.0 0.0

14 5438785 21.95 28.57 2.5 35.0 22.22 16.67

15 5438800 0.61 2.38 0.0 0.0 0.0 0.0

16 5438794 0.61 0.0 2.5 0.0 0.0 0.0

17 5438796 0.61 0.0 0.0 0-0 2.78 0.0

18 5438790 1.83 2.38 2.5 2.5 0.0 0.0

19 5438803 0.61 ^" 0.0 2.5 0.0 0.0 0.0

20 5438797 0.61 2.38 0.0 0.0 0.0 0.0 Table 7. Frequency of Observed PLAU Haplotype Pairs In Unrelated Individuals

HAP1 HAP2 Total CA AF AS HL AM

12 12 10.98 14.29 15.0 0.0 16.67 0.0

8 8 4.88 4.76 0.0 15.0 0.0 0.0

14 14 6.1 9.52 0.0 15.0 0.0 0.0

9 9 1.22 0.0 5.0 0.0 0.0 0.0

12 17 1.22 0.0 0.0 0.0 5.56 0.0

12 7 1.22 0.0 0.0 0.0 5.56 0.0

8 5 1.22 0.0 0.0 0.0 5.56 0.0

12 6 1.22 0.0 0.0 0.0 0.0 33.33

3 2 1.22 0.0 5.0 0.0 0.0 0.0

3 16 1.22 0.0 5.0 0.0 0.0 0.0

12 18 1.22 0.0 0.0 5.0 0.0 0.0

14 4 1.22 4.76 0.0 0.0 0.0 0.0

12 3 2.44 0.0 5.0 0.0 5.56 0.0

12 5 1.22 0.0 0.0 0.0 5.56 0.0

12 20 1.22 4.76 0:0 0.0 0.0 0.0

14 10 2.44 4.76 0.0 0.0 5.56 0.0

8 18 2.44 4.76 5.0 0.0 0.0 0.0

14 13 1.22 0.0 5.0 0.0 0.0 0.0

9 19 . 1.22 0.0 5.0 0.0 0.0 0.0

12 10 1.22 0.0 5.0 0.0 0.0 0.0

8 10 1.22 0.0 5.0 0.0 0.0 0.0

9 10 . 2.44 0.0 10.0 0.0 0.0 0.0

8 11 1.22 0.0 , 0.0 5.0 0.0 0.0

12 14 13.41 23.81 0.0 5.0 27.78 0.0

14 9 6.1 0.0 0.0 15.0 5.56 33.33

8 9 3.66 0.0 5.0 10.0 0.0 0.0

14 5 1.22 4.76 0.0 0.0 0.0 0.0

12 15 1.22 4.76 0.0 0.0 . 0.0 0.0

12 8 10.98 19.05 5.0 5.0 11.11 33.33

14 8 6.1 0.0 0.0 20.0 5.56 0.0

9 1 1.22 0.0 5.0 0.0 0.0 0.0

12 9 3.66 0.0 10.0 5.0 0.0 0.0

12 13 1.22 0.0 5.0 0.0 0.0 0.0

The size and composition ofthe Index Repository were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population. For example, as described in Table 1 above, this repository contains approximately equal sample sizes of African-descent, Asian- American, European- American, and Hispanic-Latino population groups. Almost all individuals representing each group had all four grandparents with the same ethnogeographic background. The number of unrelated individuals in the Index Repository provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population. Similarly, a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group. In addition, the size and composition ofthe Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pahs ofthe PLAU gene are likely to be similar to the relative frequencies of these PLAU haplotypes and haplotype pahs in the general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity observed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.

In view ofthe above, it will be seen that the several advantages ofthe 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 o the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be inteφreted as illustrative and not in a limiting sense.

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

Claims

What is Claimed is:

1. A method for haplotyping the plasminogen activator, urokinase (PLAU) gene of an individual, which comprises determining which ofthe PLAU haplotypes shown in the table immediately below defines one copy ofthe individual's PLAU gene, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PSl -PS 18 on at least one copy ofthe individual's PLAU gene, and wherein each ofthe PLAU haplotypes comprises a sequence of polymoφhisms whose positions and identities are set forth in the table immediately below:

PS PS Haplotype Number(c) (Part 1) 1

No.(a) Position(b) 1 2 3 4 5 6 7 8 9 10

1 3186 C C C C C C C C C C

2 3419 G G G G G G G G G G

3 4030 A A A A A A A A A A

4 4032 A C C C C C C C C C

5 4134 C A A C C C C C C C

6 4611 G G G A A G G G G G

7 4795 G G G G G A . G G G G

8 4966 C C T C C C C C C C

9 5697 G G G G G A A A G G

10 5837 C C C C C . C C C C C

11 6332 C C C C C C C C C C

12 6484 A A C A A A A A A A

13 6615 C C C C C T T T C C

14 7476 C C C C T C C C C C

15 7822 T T T T T T C T T T

16 7955 T T T T T T T T T T

17 8900 C C C C C C C C C C

18 9199 C T C T T C C C C T

PS PS Haplotype Number(c) (Part 2) 1

No.(a) Position(b) 11 12 13 14 15 16 17 18 19 20

1 3186 C C C C C C C C C T

2 3419 G G G G G G G T T G

3 4030 A A A A A A G A A A

4 4032 C C C C C C C C C C

5 4134 C C C C C C C C C C

6 4611 G G G G G G A G G G

7 4795 G G G G G G Q G G G

8 4966 C C C C T T C C T C

9 5697 G G G G A G G G G G

10 5837 C C C T C C C C C C

11 6332 C C , T C C C C C C C

12 6484 A A A A A C A A C A

13 6615 C C C C T C C C C C

14 7476 C T C C C C T C C C

15 7822 T T T T T T T T T T

16 7955 T T T T T T T T T C

17 8900 T C C C C C C C C C

18 9199 C T T C C C T C C T

(a) PS = polymoφhic site;

(b) Position of PS within SEQ ID NO: 1 ;

(c) Alleles for haplotypes are presented 5 ' to 3 ' in each column.

A method for haplotyping the plasminogen activator, urokinase (PLAU) gene of an individual, which comprises determining which, of the PLAU haplotype pahs shown in the table immediately below defines both copies ofthe individual's PLAU gene, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PSl -PS 18 on both copies ofthe individual's PLAU gene, and wherein each ofthe PLAU haplotype pahs consists of first and second haplotypes which comprise first and second sequences of polymoφhisms whose positions and identities are set forth in the table immediately below:

PS PS Haplotype Pair(c) (Part 1)

No.(a) Position(b) 12/12 8/8 14/14 9/9 12/17 12/7 8/5 12/6

1 3186 C/C C/C C/C C/C C/C C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/G A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/G G/G G/A G/G G/A G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/A

8 4966 C/C C/C C/C C/C C/C C/C C/C C/C

9 5697 G/G A/A G/G G/G G/G G/A A/G G/A

10 5837 C/C C/C T/T C/C C/C C/C C/C C/C

11 • 6332 C/C C/C C/C CIC C/C C/C ^' C/C C/C

12 6484 A/A A/A A/A A A A/A A/A A/A A/A

13 6615 C/C T/T C/C C/C C/C C/T T/C C/T

14 7476 T/T ■ C/C C/C C/C T/T T/C C/T T/C

15 7822 T/T T/T T/T T/T T/T T/C T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 T/T C/C C/C C/C T/T T/C C/T T/C

PS PS Haplotype Pah(c) (Part 2)

No.(a) Position(b) 3/2 3/16 12/18 14/4 12/3 12/5 12/20 14/10

1 3186 C/C C/C C/C C/C C/C C/C C/T C/C

2 . 3419 G/G G/G G/T G/G G/G G/G G/G G/G

3 4030 AA A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 A/A A/C C/C C/C C/A C/C C/C C/C

6 4611 G/G G/G G/G G/A G/G G/A G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 T/C T/T C/C C/C C/T C/C C/C C/C

9 5697 G/G G/G . G/G G/G G/G G/G G/G G/G

10 5837 C/C C/C C/C T/C C/C C/C C/C T/C

11 6332 C/C C/C C/C C/C C/C C/C C/C ■ C/C

12 6484 C/A C/C A/A A/A A/C A/A A/A A/A

13 6615 C/C C/C C/C C/C C/C C/C C/C C/C

14 7476 C/C C/C T/C C/C T/C T/T T/C C/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/C T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 C/T C/C T/C C/T T/C T/T T/T C/T

PS PS Haplotype Pah(c) (Part 3)

No.(a) Position(b) 8/18 14/13 9/19 12/10 8/10 9/10 8/11 12/14

1 3186 C/C C/C C/C C/C C/C C/C C/C C/C

2 3419 G/T G/G G/T G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 . 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/G G/G G/G G/G G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 C/C C/C C/T C/C C/C C/C C/C C/C

9 5697 A/G G/G G/G G/G A/G G/G AG G/G

10 5837 ^'C/C T/C C/C C/C C/C C/C C/G C/T

11 6332 C/C C/T C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/C A/A A/A A/A A/A A/A

13 6615 T/C C/C C/C C/C T/C .CC T/C C/C

14 7476 C/C C/C C/C T/C C/C C/C C/C ^" T/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/T ^' C/C

18 9199 C/C C/T C/C T/T C/T C/T C/C T/C

PS PS Haplotype Pair(c) (Part 4)

No.(a) Position(b) 14/9 8/9 14/5 12/15 12/8 14/8 9/1 12/9

1 3186 C/C • C/C C/C C/C C/C C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/A C/C

5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/A G/G G/G G/G G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 C/C C/C C/C C/T C/C C/C C/C C/C

9 5697 G/G A/G G/G G/A G/A G/A G/G G/G

10 5837 T/C C/C T/C C/C C/C T/C C/C C/C

11 6332 C/C C/C C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/A A/A A/A A/A A/A A/A

13 6615 C/C T/C C/C C/T C/T C/T C/C C/C

14 7476 C/C C/C C/T T/C T/C C/C C/C T/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 C/C C/C C/T T/C T/C C/C. C/C T/C

PS PS HHaappllcotype Pah(c) (Part 5)

No.(a) Position(b) 12/13

1 3186 C/C

2 3419 G/G

3 4030 A/A

4 4032 C/C

5 4134 C/C

6 4611 G/G

7 4795 G/G

8 4966 C/C

9 5697 G/G

10 5837 C/C

11 6332 C/T

12 6484 A/A

15 7822 T/T

16 7955 T/T

17 8900 C/C

13 6615 C/C

14 7476 T/C

18 9199 T/T

(a) PS = polymoφhic site;

(b) Position of PS in SEQ ID NO:l;

(c) Haplotype pahs are represented as 1^st haplotype/2^nd haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column.

3. A method for genotyping the plasminogen activator, urokinase (PLAU) gene of an individual, comprising dete-rmining for the two copies ofthe PLAU gene present in the individual the identity ofthe nucleotide pah at one or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17 and-PS18, wherein the one or more polymoφhic sites (PS) have the position and alternative alleles shown in SEQ ID NO: 1.

4. The method of claim 3, wherein the determining step comprises:

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

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

(c) hybridizing a primer extension oligonucleotide to one allele ofthe amplified target region, wherein the oligonucleotide is designed for genotyping the selected polymoφhic site in the target region;

(d) performing a nucleic acid template-dependent, primer extension reaction on the hybridized oligonucleotide in the presence of at least one terminator ofthe reaction, wherein the terminator is complementary to one ofthe alternative nucleotides present at the selected polymoφhic site; and

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

5. The method of claim 3, which comprises determining for the two copies ofthe PLAU gene present in the individual the identity ofthe nucleotide pair at each of PS1-PS18.

6. A method for haplotyping the plasminogen activator, urokinase (PLAU) gene of an individual which comprises determining, for one copy ofthe PLAU gene present in the individual, the identity ofthe nucleotide at two or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17 and PS18, wherein the selected PS have the position and alternative alleles shown in SEQ ID NO:l.

7. The method of claim 6, wherein the determining step comprises:

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

(b) amplifying from the nucleic acid sample a target region containing one ofthe selected polymoφhic sites;

(c) hybridizing a primer extension oligonucleotide to one allele ofthe amplified target region, wherein the oligonucleotide is designed for haplotyping the selected polymoφhic site in the target region;

(d) performing a nucleic acid template-dependent, primer extension reaction on the hybridized oligonucleotide in the presence of at least one terminator of the reaction, wherein the terminator is complementary to one ofthe alternative nucleotides present at the selected polymoφhic site; and

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

8. A method for predicting a haplotype pah for the plasminogen activator, urokinase (PLAU) gene of an individual comprising:

(a) identifying a PLAU genotype for the individual, wherein the genotype comprises the nucleotide pair at two or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15,

PS16, PS17 and PS18, wherein the selected PS have the position and alternative alleles shown in SEQ ID NO: 1;

(b) comparing the genotype to the haplotype pah data set forth in the table immediately below; and (c) determining which haplotype pah is consistent with the genotype ofthe individual and with the haplotype pah data PS PS Haplotype Pah(c) (Part 1)

No.(a) Position(b) 12/12 8/8 14/14 9/9 12/17 12/7 8/5 12/6

1 3186 C/C . C/C C/C C/C C/C C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/G A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/G G/G G/A G/G G/A G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/A

8 4966 C/C C/C C/C C/C C/C C/C C/C C/C

9 5697 G/G A/A G/G G/G G/G G/A A/G G/A

10 5837 C/C C/C T/T C/C C/C C/C C/C C/C

11 6332 c/c^". C/C C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/A A/A A/A A/A A/A A/A

13 6615 C/C T/T C/C C/C C/C C/T T/C C/T

14 7476 T/T C/C C/C C/C T/T T/C C/T T/C

15 ' 7822 T/T T/T T/T T/T T/T T/C T/T T/T

16 7955 T/T T/T T/T ^' T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 T/T C/C C/C C/C T/T T/C C/T^' T/C

PS PS Haplotype Pah(c) (Part 2)

No.(a) Position(b) 3/2 3/16 12/18 14/4 12/3 12/5 12/20 14/10

1 3186 C/C C/C C/C C/C C/C C/C C/T C/C

2 3419 G/G G/G G/T G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 A/A A/C C/C C/C C/A C/C C/C C/C

6 4611 G/G G/G G/G G/A G/G G/A G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 T/C T/T C/C C/C C/T C/C C/C C/C ^'

9 5697 G/G G/G G/G G/G G/G G/G G/G G/G

10 5837 • C/C C/C C/C T/C C/C C/C C/C T/C .

11 6332 C/C C/C C/C C/C C/C C/C C/C C/C

12 6484 C/A C/C A/A A/A A/C A/A A/A A/A

13 6615 C/C C/C C/C C/C C/C C/C C/C C/C

14 7476 C/C C/C T/C C/C T/C T/T T/C C/C

15 7822 T/T T/T T/T T/T T/T T/T T/T . T/T

16 7955 T/T T/T . T/T T/T T/T . T/T T/C T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 C/T C/C T/C C/T ^'T/C T/T T/T C/T

PS PS Haplotype Pah(c) (Part 3)

No.(a) Position(b) 8/18 14/13 9/19 12/10 8/10 9/10 8/11 12/14

1 3186 C/C C/C C/C C/C C/C C/C C/C C/C

2 3419 G/T G/G G/T G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/G G/G G/G G/G G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 C/C C/C C/T C/C C/C C/C C/C C/C

9 5697 A/G G/G G/G G/G A/G G/G A/G G/G

10 5837 C/C T/C C/C C/C C/C C/C C/C C/T

11 6332 C/C C/T C/C C/C C/C C/C C/C C/C

12 6484 A/A. A/A A/C A/A A/A A/A A/A A/A

13 6615 T/C C/C C/C C/C T/C C/C T/C C/C

14 7476 C/C C/C C/C T/C C/C C/C C/C T/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C ^' C/C C/C C/C C/T C/C

18 9199 C/C C/T C/C T/T C/T C/T C/C T/C

PS PS Haplotype Pah(c) (Part 4)

No.(a) Position(b) 14/9 8/9 14/5 12/15 12/8 14/8 9/1 12/9

1 3186 C/C C/C C/C C/C C/C C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

3^' 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 . 4032 C/C C/C C/C C/C C/C C/C C/A C/C

5 4134 C/C C/C C/C C/C ^' C/C C/C C/C C/C

6 4611 G/G G/G G/A G/G G/G G/G G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 C/C C/C C/C C/T C/C C/C C/C C/C

9 5697 G/G A/G G/G G/A G/A G/A G/G G/G

10 5837 T/C C/C T/C C/C C/C T/C C/C C/C

11 6332 C/C C/C C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/A A/A A/A A/A A/A A/A

13 6615 C/C T/C C/C C/T C/T C/T C/C C/C

14 7476 C/C C/C C/T T/C T/C ^• C/C C/C T/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 C/C C/C C/T T/C T/C C/C C/C T/C

PS PS HHaappllcotype Pah(c) .(Part 5)

No.(a) Position(b) 12/13

1 3186 C/C

2 3419 G/G

3 4030 A/A

4 4032 C/C

5 4134 C/C

6 4611 GIG

7 4795 G/G

8 4966 C/C

9 5697 G/G

10 5837 C/C

11 6332 C/T

12 6484 A/A

13 6615 C/C

14 7476 T/C

15 7822 T/T

16 7955 T/T

17 8900 ' C/C

18 9199 T/T

(a) PS = polymoφhic site;

(b) Position of PS in SEQ ID NO:l; (c) Haplotype pahs are represented as 1^st haplotype/2^nd haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column.

9. The method of claim 8, wherein the identified genotype ofthe individual comprises the nucleotide pah at each of PS1-PS18, which have the position and alternative alleles shown in SEQ ID NO: 1.

10. A method for identifying an association between a trait and at least one haplotype or haplotype pair ofthe plasminogen activator, urokinase (PLAU) gene which comprises comparing the frequency ofthe haplotype or haplotype pah in a population exhibiting the trait with the frequency ofthe haplotype or haplotype pah in a reference population, wherein the haplotype is selected from haplotypes 1-20 shown in the table presented immediately below, wherein each of the haplotypes comprises a sequence of polymoφhisms whose positions and identities are set forth in the table immediately below:

PS PS Haplotype Number(c) (Part V )

No.(a) Position(b) 1 2 3 4 5 6 7 8 9 10

1 3186 C C C C C C C C C C

2 3419 G G G G G G G G G G

3 4030 A A A A A A A A A A

4 4032 A C C C C C C C C C

5 4134 C A A C C C C C C C

6 4611 G G G A A G G G G G

7 4795 G G G G G A G G G G

8 4966 C C T C C C C C C C

9 5697 G G G G G A A A G G

10 5837 C C C C C C C C C C

11 6332 C C C C C C C C C C

12 6484 . A A C A A A A A A A 13 6615 C C C C C T T T C C

14 7476 C C C C T C C C C C

15 7822 T T T T T T C T T T .

16 7955 T T T T T T T T T T

17 8900 C C C C C C C C C C

18 9199 C T C T T C C C C T

PS PS Haplotype Number(c) (Part 2)

No.(a) Position(b) 11 12 13 14 15 16 17 18 19 20

1 3186 C C C C C C C C C T

2 3419 G G G G G G G T T G

3 4030 ^" A A A A A A G A A A

4 4032 C C C C C C C C C C

5 4134 C C C C C C C C C C

6 4611 G G G G G G A G G G

7 4795 G G G G G G G G G G

8 4966 C C C C T T C C T C

9 5697 G G G G A G G G G G

10 5837 C C C T C C C C C C

11 6332 C C T C C C C C C C

12 6484 A A A A A C A A C A

13 6615 C C C C T C C C C C

14 7476 C T C C C C T C C C

15 7822 T T T T T T T T T T

16 7955 T T T T T T T T T C

17 8900 T C C C C C C C C C

18 9199 C T T C C C T C C T

(a) PS = polymoφhic site;

(b) Position of PS within SEQ ID NO: 1 ;

(c) Alleles for haplotypes are presented 5' to 3' in each column; and wherein the haplotype pair is selected from the haplotype pahs shown in the table immediately below, wherein each ofthe PLAU haplotype pahs consists of first and second haplotypes which comprise first and second sequences of polymoφhisms whose positions in SEQ ID NO: 1 and identities are set forth in the table immediately below: PS PS Haplotype Pah(c) (Part 1)

No.(a) Position(b) 12/12 8/8 14/14 9/9 12/17 12/7 8/5 12/6

1 3186 C/C C/C .C/C C/C C/C C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/G A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/G G/G G/A G/G G/A G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/A

8 4966 C/C C/C C/C C/C C/C . C/C C/C C/C

9 5697 G/G A/A G/G G/G G/G .G/A A/G G/A

10 5837 C/C C/C T/T C/C C/C C/C C/C ^' C/C

11 6332 C/C C/C C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/A A/A A/A A/A A/A A/A

13 6615 • C/C T/T C/C C/C C/C C/T T/C C/T

14 7476 T/T C/C C/C C/C T/T T/C C/T T/C

15 7822 T/T T/T T/T T/T T/T T/C T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 T/T C/C C/C C/C' T/T T/C C/T T/C

PS PS Haplotype Pah(c) (Part 2)

No.(a) Position(b) 3/2 3/16 12/18 14/4 12/3 12/5 12/20 14/10

1 3186 C/C C/C C/C C/C C/C C/C C/T ^' C/C

2 3419 G/G G/G G/T G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 A/A A/C C/C C/C C/A C/C C/C C/C

6 4611 G/G G/G G/G G/A G/G G/A G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G- G/G G/G

8 4966 T/C T/T C/C C/C C/T C/C C/C C/C

9 5697 G/G G/G G/G G/G G/G G/G G/G G/G

10 5837 C/C C/C C/C T/C C/C C/C C/C T/C

11 6332 C/C C/C C/C C/C • C/C C/C C/C C/C

12 6484 C/A C/C A/A A/A A/C A/A A/A A/A

13 6615 C/C C/C C/C C/C C/C C/C C/C C/C

14 7476 C/C C/C T/C C/C T/C T/T T/C C/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/C T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C ^•

18 9199 C/T C/C T/C C/T T/C T/T T/T C/T

PS PS Haplotype Pair(c) (Part 3)

No.(a) Position(b) 8/18 14/13 9/19 12/10 8/10 9/10 8/11 12/14

105 1 3186 C/C C/C C/C C/C C/C C/C C/C C/C

2 3419 G/T G/G G/T G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

110 6 4611 G/G G/G G/G G/G G/G G/G G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 C/C C/C C/T C/C C/C C/C C/C C/C

9 5697 A/G G/G G/G G/G A/G G/G A/G G/G

10 5837 C/C T/C C/C C/C C/C C/C C/C C/T

115 11 6332 . C/C C/T C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/C A/A A/A A/A A/A A/A

13 6615 T/C C/C C/C C/C T/C C/C T/C C/C

14 . 7476 C/C C/C C/C T/C C/C C/C C/C . T/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

120 16 7955 T/T T/T . T/T T/T T/T T/T T/T T/T

17 8900 C/C ■ C/C C/C C/C C/C C/C C/T C/C

18 9199 C/C C/T C/C T/T ■C/T C/T C/C T/C

PS PS Haplotype Pah(c) (Part 4)

125 No.(a) Position(b) 14/9 8/9 14/5 12/15 12/8 14/8 9/1 12/9

1 3186 C/C C/C C/C C/C C/C. C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A •A/A A/A A/A A/A A/A

4 . 4032 C/C C/C C/C C/C C/C C/C C/A C/C

130 5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/A G/G G/G G/G G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 C/C C/C C/C C/T C/C C/C C/C C/C

9 5697 G/G A/G G/G G/A G/A G/A G/G G/G

135 10 5837 T/C C/C T/C C/C C/C T/C C/C C/C

11 6332 C/C C/C C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/A A/A A/A A/A A/A A/A

13 6615 C/C T/C C/C C/T C/T C/T C/C C/C

14 7476 C/C C/C C/T T/C T/C C/C C/C T/C

140 15 7822 T/T T/T T/T T/T .T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 C/C C/C C/T T/C T/C C/C C/C T/C

145 145 PPSS PPSS HHaapplloctype Pair(c) (Part 5)

No.(a) Position(b) 12/13

1 3186 C/C

2 3419 G/G

3 4030 A/A

150 4 4032 C/C

5 4134 C/C

6 4611 G/G

7 4795 G/G

8 4966 C/C

155 9 5697 G/G

10 5837 C/C

11 6332 C/T.

12 6484 A/A

13 6615 C/C

160 14 7476 T/C

15 7822 T/T

16 7955 T/T

17 8900 C/C

18 9199 T/T

165

(a) PS = polymoφhic site;

(b) Position of PS in SEQ ID NO: 1;

(c) Haplotype pahs are represented as 1^st haplotype/2^nd haplotype; with alleles of each haplotype shown 5' to 3' as 1^st polymoφhism/2^nd polymoφhism in each column;

170 wherein a higher frequency ofthe haplotype or haplotype pah in the trait population than in the reference population indicates the trait is associated with the haplotype or haplotype pair.

11. The method of claim 10, wherein the trait is a clinical response to a drug targeting PLAU or to a drug for treating a condition or disease associated with PLAU activity.

12. An isolated oligonucleotide designed for detecting a polymoφhism in the plasminogen activator, urokinase (PLAU) gene at a polymoφhic site (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS 10, PS11, PS12, PS13, PS14, PS15, PS16, PS 17 and PS 18, wherein the selected PS have the position and alternative alleles shown in SEQ ID NO:l.

13. The isolated oligonucleotide of claim 12, which is an allele-specific oligonucleotide that specifically hybridizes to an allele ofthe PLAU gene at a region containing the polymoφhic site.

14. The allele-specific oligonucleotide of claim 13, which comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:4-21, the complements of SEQ ID NOS:4-21, and SEQ ID NOS:22-57.

15. The isolated oligonucleotide of claim 12, which is a primer-extension oligonucleotide.

16. The primer-extension oligonucleotide of claim 15,which comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS :58-93.

17. A kit for haplotyping or genotyping the plasminogen activator, urokinase (PLAU) gene of an individual, which comprises a set of oligonucleotides designed to haplotype or genotype each of polymoφhic sites (PS) PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17 and PS18, wherein the selected PS have the position and alternative alleles shown in SEQ ID NO:l.

An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence which comprises a plasminogen activator, urokinase (PLAU) isogene, wherein the PLAU isogene is selected from the group consisting of isogenes 1- 9 and 11 - 20 shown in the table immediately below and wherein each ofthe isogenes comprises the regions of SEQ ID NO: 1 shown in the table immediately below and wherein each ofthe isogenes 1- 9 and 11 - 20 is further defined by the corresponding sequence of polymoφhisms whose positions and identities are set forth in the table immediately below; and

Region PS PS Isogene Number(d) (Part 1)

Examined(a) No.(b) Position(c) 1 2 3 4 5 6 7 8 9

2937-3879 1 3186 C C C C C C C C C

2937-3879 2 3419 G G G G G G G G G

3893-4239 ^•3 4030 A A A A A A A A A

3893-4239 4 4032 A C C C C C C C C

3893-4239 5 4134 C A A C C C C C C

4334-5016 6 4611 G G G A A G G G G

4334-5016 7 4795 G G G G G A G G G

4334-5016 8 4966 C C T C C C C C C

5624-6845 9 5697 G G G G G A A A G

5624-6845 10 5837 C C C C C C C C C

5624-6845 11 6332 C C C C C C C C C

5624-6845 12 6484 A A C A A A A A A

5624-6845 13 661.5 C C C C C T T T C

7119-8206 14 7476 C C C C T C C C C

7119-8206 15 7822 T T T T T T C T T

7119-8206 16 7955 T T T T T T T T T

8625-9341 17 8900 C C C C C C C C C

8625-9341 18 9199 C T C T T C C C C

Region PS PS Isogene Number(d) (Part 2)

Examined(a) No.(b) Position(c) 11 12 13 14 15 16 17 18 19 20

2937-3879 1 3186 C C C C C C C C C T

2937-3879 2 3419 G G G G G G G T T G

3893-4239 3 4030 A A A A A A G A A A

3893-4239 4 4032 C C C C C C C C C C

3893-4239 5 4134 C C C C C C C C C C

4334-5016 6 4611 G G G G G G A G G G

4334-5016 7 4795 G G G G G G G G G G

4334-5016 8 4966 C C C C T T C C T C

5624-6845 9 5697 G G G G A G G G G G

5624-6845 10 5837 C C C T C C C C C C

5624-6845 11 6332 C C T C C C C C C C

5624-6845 12 6484 A A A A A . C A A C A

5624-6845 13 6615 C C C C T C C C C C

7119-8206 14 7476 C T C C C C T C C C

7119-8206 15 7822 T T T T T T T T T T

7119-8206 16 7955 T T T T T T T T T C

8625-9341 17 8900 T C C C C C C C C C

8625-9341 18 9199 C T T C C C T C C T

(a) Region examined represents the nucleotide positions defining the start and stop positions within the 1^st SEQ ID NO ofthe sequenced region;

(b) PS = polymoφhic site;

(c) Position of PS in SEQ ED NO:l;

(d) Alleles for isogenes are presented 5 ' to 3 ' in each column;

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

19. The isolated polynucleotide of claim 18, 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.

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

21. The recombinant nonhuman organism of claim 20, which is a transgenic animal. 22. An isolated fragment of a plasminogen activator, urokinase (PLAU) isogene, wherein the fragment comprises at least 10 nucleotides in one ofthe regions of SEQ ID NO: 1 shown in the table immediately below and wherein the fragment comprises one or more polymoφhisms selected from the group consisting of thymine at PSl, thymine at PS2, guanine at PS3, adenine at PS4, adenine at PS5, adenine at PS6, adenine at PS7, thymine at PS8, adenine at PS9, thymine at PS10, thymine at PSl 1, cytosine at PS12, thymine at PS13, thymine at PS14, cytosine at PS15, cytosine at PS16, thymme at PS17 and cytosine at PS18, wherein the selected polymoφhism has the position set forth in the table immediately below: Region PS PS Isogene Number(d) (Part 1)

Examined(a) No.(b) Position(c) 1 2 3 4 5 6 7 8 9

2937-3879 1 3186 C C C C C C C C C

2937-3879 2 . 3419 G G G G G G G G G

^•3893-4239 3 4030 A A A A A A A A A

3893-4239 4 4032 A C C C C C C C C

3893-4239 5 4134 C A A C C C C C c

4334-5016 6 4611 G G G A A G G G G

4334-5016 7 4795 G G G G G A G G G

4334-5016 8 4966 C C T C C C C C G

5624-6845 9 5697 G G G G G A A A G

5624-6845 10 5837 C C C C C C C C C

5624-6845 11 6332 C C C C C C C C C

5624-6845 12 6484 A A C A A A A A A

5624-6845 13 6615 C C C C C T T T C

7119-8206 14 7476 C C C C T C C C C

7119-8206 15 7822 T T T T T T C T T

7119-8206 16 , 7955 T T T T T T T T T

8625-9341 17 8900 C C C C C C C C C

8625-9341 18 9199 C T C T T C C C C

Region PS PS Isogene Number(d) (Part 2)

Examined(a) No.(b) Position(c) 11 12 13 14 15 16 17 18 19 20

2937-3879 1 3186 C C C C C C C C C T

2937-3879 2 3419 G G G G G G G T T G

3893-4239 3 4030 A A A A A A G A A A

3893-4239 4 4032 C C C C C C C C C C

3893-4239 5 4134 C C C C C C C C C C

4334-5016 6 4611 G G G G G G A G G G

4334-5016 7 4795 G G G G G G G G G G

4334-5016 8 4966 C C C C T T . C C T C

5624-6845 9 5697 G G G G A G G G G G

5624-6845 10 5837 C C C T C C C C C C

5624-6845 11 6332 C C T C C C C C C C

5624-6845 12 6484 A A ^' A A A C A A C A

5624-6845 13 ^■ 6615 C C C C T C C C C C

7119-8206 14 7476 C T C C C C T C C C

7119-8206 15 7822 T T T T T T T T T T

7119-8206 16 7955 T T T T T T T T T C

8625-9341 17 8900 T C C C C C C C C C

8625-9341 18 9199 C T T C C C T C C T

(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO:l ofthe regions sequenced;

(b) PS = polymoφhic site;

(c) Position of PS within SEQ ID NO:l;

(d) Alleles for PLAU isogenes are presented 5 ' to 3 ' in each column.

23. An isolated polynucleotide comprising a coding sequence for a PLAU isogene, wherein the coding sequence comprises the regions of SEQ ID NO:2, covered by exons 1-4 and 6-11, except at each ofthe polymoφhic sites which have the positions in SEQ ID NO: 2 and polymoφhisms set forth in the table immediately below: PS PS Isogene Coding Sequence Number(c)

No.(a) Position(b) 3c 6c 7c 8c l ie 14c 15c 16c 19c

7 172 G A G G G G G G G

10 422 C C C C C T C C C

12 691 C A A A A A A C C

13 822 C T T T C C T C C

15 1048 T T C T T T T T T

17 1137 C C C C T C C C C

(a) PS = polymoφhic site;

(b) Position of PS in SEQ ID NO:2;

(c) Alleles for the isogene coding sequence are presented 5 ' to 3 ' in each column; the numerical portion ofthe isogene coding sequence number represents the number ofthe parent full PLAU isogene.

24. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 23, wherein the organism expresses a plasminogen activator, urokinase (PLAU) protein that is encoded by the polymoφhic variant sequence.

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

26. An isolated fragment of a PLAU coding sequence, wherein the fragment comprises one or more polymoφhisms selected from the group consisting of adenine at a position corresponding to nucleotide 172, thymine at a position corresponding- to nucleotide 422, cytosine at a position corresponding to nucleotide 691, thymine at a position corresponding to nucleotide 822, cytosine at a position corresponding to nucleotide 1048 and thymine at a position corresponding to nucleotide 1137 in SEQ ED NO:2.

27 An isolated polypeptide comprising an amino acid sequence which is a polymoφhic variant of a reference sequence for the plasminogen activator, urokinase (PLAU) protein, wherein the reference sequence comprises SEQ ID NO:3, covered by exons 1-4 and 6-11, except the polymoφhic variant comprises one or more variant amino acids selected from the group consisting of -trginine at a position corresponding to amino acid position 58, leucine at a position corresponding to amino acid position 141, glutamine at a position corresponding to amino acid position 231 and histidine at a position corresponding to amino acid position 350.

28. An isolated monoclonal 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 PLAU polymoφhic variant with a candidate agent and assaying for binding activity.

30. An isolated fragment of a PLAU protein, wherein the fragment comprises one or more variant amino acids selected from the group consisting of --xgh-ine at a position corresponding to amino acid position 58, leucine at a position corresponding to amino acid position 141, glutamine at a position corresponding to amino acid position 231 and histidine at a position corresponding to amino acid position 350 in SEQ ID NO:3. A computer system for storing and analyzing polymoφhism data for the plasminogen activator, urokinase 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 poiymoφhism data; wherein the polymoφhism data comprises any one or more ofthe haplotypes set forth in the table immediately below:

PS PS Haplotype Number(c) (Part 1) 1

No.(a) Position(b) 1 2 3 4 5 6 7 8 9 10

1 3186 C C C C C C C C C C

2 3419 G G G G G G G G G G

3 4030 A A A A A A A A A A

4 4032 A C C C C C C C C C

5 ^' . 4134 C A A C C C C C C C

6 4611 G G G A A G G G G G

7 4795 G G G G G A G G G G

8 4966 C C T C C C C C C C

9 5697 G G G G G A A A G G

10 5837 C C C C C C C C C C

11 6332 C C C C C C C C C C

12 6484 A A C A A A A A A A

13 6615 C C C C C T T T C C

14 7476 C C C C T C C C C C

15 7822 T T T T T T C T T T

16 7955 T T T T T T T T ^' T T

17 8900 C C C C C C C C C C

18 9199 C T C T T C C C C T

PS PS . Haplotype Number(c) (Part 2) )

No.(a) Position(b) 11 12 13 14 15 16 17 18 19 20

1 3186 C C C C C C C C C T

2 3419 G G G G G G G T T G

3 4030 A A A A A A G ^' A A A

4 4032 C C C C C C C C C C

5 4134 C C C C C C C C C C

6 4611 ■ G G G G G G A G G G

7 4795 G G G G G G G G G G

8 4966 C C C C T T C C T C

9 5697 G G G G A G G G G G

10 5837 C C C T C C C C C C

11 6332 C C T C C C C C C C

12 6484 A A A A A C A A C A

13 6615 C C C C T C C C C C

14 7476 C T C C C C T C C C

15 7822 T T T T T T T T T T

16 7955 T T T T T T T T T C

17 8900 T C C C C C C C C C

18 9199 C T T C C C T C C T

(a) PS = polymoφhic site;

(b) Position of PS within SEQ ID NO: 1;

(c) Alleles for haplotypes are presented 5 ' to 3 ' in each column; the haplotype pahs set forth in the table immediately below: PS PS Haplotype Pah(c) (Part 1)

No.(a) Position(b) 12/12 8/8 14/14 9/9 12/17 12/7 8/5 12/6

1 3186 C/C C/C C/C C/C C/C C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

3 4030 A/A A/A A/A A/A A/G A/A A/A A/A

4 4032 C/C C/C ^■c/c C/C C/C C/C C/C C/C

5- 4134 C/C ^"C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/G G/G G/A G/G G/A G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/A

8 4966 C/C C/C ^■ C/C C/C C/C C/C C/C C/C

9 5697 G/G A/A G/G G/G ^• G/G G/A A/G G/A

10 5837 C/C C/C T/T C/C C/C C/C C/C C/C

11 6332 C/C C/C C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/A A/A A/A A/A A/A A/A

13 6615 C/C T/T C/C C/C C/C C/T^' T/C C/T

14 7476 T/T C/C C/C C/C T/T T/C C/T T/C

15 7822 T/T T/T T/T T/T T/T T/C T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

18 9199 T/T C/C C/C C/C T/T T/C C/T T/C

PS PS Haplotype Pah(c) (Part 2)

No.(a) Position(b) 3/2 3/16 12/18 14/4 12/3 12/5 12/20 14/10

1 3186 C/C C/C C/C C/C C/C C/C C/T C/C

2 3419 G/G G/G G/T G/G G/G G/G G/G G/G •

3 4030 A/A A/A A/A A/A A/A A/A A/A A/A

4 4032 C/C C/C C/C C/C C/C C/C C/C C/C

5 . 4134 .A/A A/C C/C C/C C/A C/C C/C C/C

6 4611 G/G G/G G/G G/A G/G G/A G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

8 4966 T/C T/T C/C C/C C/T C/C C/C C/C

9 5697 G/G G/G G/G G/G G/G G/G G/G G/G

10 5837 C/C C/C C/C T/C C/C C/C C/C T/C

11 6332 C/C C/C C/C C/C C/C C/C C/C C/C

12 6484 C/A C/C A/A A/A A/C A/A A/A A/A

13 6615 C/C C/C C/C C/C C/C C/C C/C C/C

14 7476 C/C C/C T/C C/C T/C T/T T/C C/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/C T/T

17 8900 C/C C/C. C/C C/C C/C C/C C/C C/C

18 9199 C/T C/C T/C C/T T/C T/T T/T C/T

1 .00 PS PS Haplotype Pah(c) (Part 4)

No.(a) Position(b) 14/9 8/9 14/5 12/15 12/8 14/8 9/1 12/9

1 3186 C/C C/C C/C C/C C/C C/C C/C C/C

2 3419 G/G G/G G/G G/G G/G G/G G/G G/G

1 3 4030 A/A^' A/A A/A A/A A/A A/A A/A A/A

.05 4 4032 C/C C/C C/C C/C C/C C/C C/A C/C

5 4134 C/C C/C C/C C/C C/C C/C C/C C/C

6 4611 G/G G/G G/A G/G G/G G/G G/G G/G

7 4795 G/G G/G G/G G/G G/G G/G G/G G/G

] 8 4966 C/C C/C C/C C/T C/C C/C C/C C/C

.10 9 5697 G/G A/G G/G G/A G/A G/A G/G G/G

10 5837 T/C C/C T/C C/C C/C T/C C/C C/C

11 6332 C/C . C/C C/C C/C C/C C/C C/C C/C

12 6484 A/A A/A A/A A/A A/A A/A A/A A/A

] 13 6615 ^• C/C T/C . C/C C/T C/T C/T C/C C/C '

.15 14 7476 C/C C/C C/T T/C T/C C/C C/C T/C

15 7822 T/T T/T T/T T/T T/T T/T T/T T/T

16 7955 T/T T/T T/T T/T T/T T/T T/T T/T

17 8900 C/C C/C C/C C/C C/C C/C C/C C/C

] 18 9199 C/C C/C C/T T/C T/C C/C C/C T/C

[20

PS PS Haplotype Pah(c) (Part 5)

No.(a) Position(b) 12/13

1 3186 C/C

] 2 3419 G/G

125 3 4030 A/A 4 4032 C/C 5 4134 C/C 6 4611 G/G ] 7 4795 G/G

[30 8 4966 C/C

9 5697 G/G

10 5837 C/C

11 6332 C/T

] 12 6484 A/A

[35 13 6615 C/C 14 7476 T/C 15 7822 T/T 16^' 7955 T/T

] 17 8900 C/C

L40 18 9199 T/T

(a) PS = polymoφhic site;

(b) Position of PS in SEQ ID NO: 1 ;

] (c) Haplotype pairs are represented as 1^st haplotype/2^nd haplotype; with alleles of each

L45 haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column;

and the frequency data in Tables 6 and 7. 32. A genome anthology for the plasminogen activator, urokinase (PLAU) gene which comprises two or more PLAU isogenes selected from the group consisting of isogenes 1-20 shown in the table immediately below, and wherein each ofthe isogenes comprises the regions of SEQ ID NO: 1 shown in the table immediately below and wherein each ofthe isogenes 1-20 is further defined by the corresponding sequence of polymoφhisms whose positions and identities are set forth in the table immediately below:

Region PS PS IsogeneNumber(d) (Part 1)

Examined(a) No.(b) Position(c) 1 2 3 4 5 6 7 8 9 10

2937-3879 1 3186 C C C C C C C C C C

2937-3879 2 3419 G G G G G G G G G G

3893-4239 3 4030 A A A A A A A A A A

3893-4239 4 4032 A C C C C C C C C C

3893-4239 5 4134 C A A C C C C C C C

4334-5016 6 4611 G G G A A G G G G G

4334-5016 7 4795 G G G G G A G G G G

4334-5016 8 4966 C C T C C C C C C C

5624-6845 9 5697 G G G G G A A A G G

5624-6845 10 5837 C C C C C C C C C C

5624-6845 11 6332 C C C C C C C C C C

5624-6845 12 6484 A A C A A A A A A A

5624-6845 13 6615 C C C C C T T T C C

7119-8206 14 7476 C C C C T C C C C C

7119-8206 15 7822 T T T T T T C T T T

7119-8206 16 7955 T T T T T T T T T T

8625-9341 17 8900 C C C C C C C C C c

8625-9341 18 9199 C T C . T T C C C C T

Region PS PS IsogeneNumber(d) (Part 2)

Examined(a) No.(b) Position(c) 11 12 13 14 15 16 17 18 19 20

2937-3879 1 3186 C C C C C C C C C T

2937-3879 2 3419 G G G G G G G T T G

3893-4239 3 4030 A A A A A A G A A A

3893-4239 4 4032 C C C C C C C C C C

3893-4239 5 4134 C C C C C C C C C C

4334-5016 6 4611 G G G G G G A G G G

4334-5016 7 4795 G G G G G G G G G G

4334-5016 8 4966 C C C C T T C C T C

5624-6845 9 5697 G G G G A G G G G G

5624-6845 10 5837 C C C T C C C C C C

5624-6845 11 6332 C C T C C C C C C C

5624-6845 12 6484 A A A A A C A A C A

5624-6845 13 6615 C C C C T C C C C C

7119-8206 14 7476 C T C C C C T C C C

7119-8206 15 7822 T T T T T T T T T T

7119-8206 16 7955 T T T T T T. T T T C

8625-9341 17 8900 T C C C C C C C C C

8625-9341 18 9199 C T T C C C T C C T

(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 ofthe regions sequenced;

(b) PS = polymoφhic site;

(c) Position of PS within SEQ ED NO:l;

(d) Alleles for PLAU isogenes are presented 5 ' to 3 ' in each column.