WO2002016398A2 - Haplotypes of the bmpr2 gene - Google Patents

Abstract

Novel genetic variants of the Bone Morphogenetic Protein Receptor, Type II (Serine/Threonine Kinase) (BMPR2) gene are described. Various genotypes, haplotypes, and haplotype pairs that exist in the general United States population are disclosed for the BMPR2 gene. Compositions and methods for haplotyping and/or genotyping the BMPR2 gene in an individual are also disclosed. Polynucleotides defined by the haplotypes disclosed herein are also described.

Description

HAPLOTYPES OF THE BMPR2 GENE

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 60/228,272 filed August 25, 2000.

FIELD OF THE INVENTION

This invention relates to variation in genes that encode pharmaceutically-important proteins. In particular, this invention provides genetic variants of the human bone morphogenetic protein receptor, type II (serine/threoniαe kinase) (BMPR2) 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 drags. The desired outcome of this process is a lead compound that is specific for the target, thereby reducing the incidence of the undesired side effects usually caused by activity at non-intended targets. The lead compound identified in this screening process then undergoes further in vitro and in vivo testing to determine its absorption, disposition, metabolism and toxicological profiles. Typically, this testing involves use of cell lines and animal models with limited, if any, genetic diversity.

What this approach fails to consider, however, is that natural genetic variability exists between individuals in any and every population with respect to pharmaceutically-important proteins, including the protein targets of candidate drags, the enzymes that metabolize these drags and the proteins whose activity is modulated by such drug targets. Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a pharmaceutically-important protein may be manifested as significant variation in expression, structure and/or function of the protein. Such alterations may explain the relatively high degree of uncertainty inherent in the treatment of individuals with a drag whose design is based upon a single representative example of the target or enzyme(s) involved in metabolizing the drag. For example, it is well-established that some drags 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 drags 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 drags ineffective or even dangerous in certain groups of the population, leading to the failure of such drugs in clinical trials or their early withdrawal from the market even though they could be highly beneficial for other groups in the population. This problem significantly increases the time and cost of drug discovery and development, which is a matter of great public concern.

It is well-recognized by pharmaceutical scientists that considering the impact of the genetic variability of pharmaceutically-important proteins in the early phases of drag discovery and development is likely to reduce the failure rate of candidate and approved drags (Marshall A 1997 Nature Biotech 15:1249-52; Kleyn PW et al. 1998 Science 281: 1820-21; Kola 1 1999 Curr Opin Biotech 10:589-92; Hill AVS et al. 1999 in Evolution in Health and Disease Stearns SS (Ed.) Oxford University Press, New York, pp 62-76; Meyer U.A. 1999 in Evolution in Health and Disease Stearns SS (Ed.) Oxford University Press, New York, pp 41-49; Kalow W et al. 1999 Clin. Pharm. Therap.

66:445-7; Marshall, E 1999 Science 284:406-7; Judson R et al. 2000 Pharmacogenomics 1:1-12; Roses AD 2000 Nature 405:857-65). However, in practice this has been difficult to do, in large part because of the time and cost required for discovering the amount of genetic variation that exists in the population (Chakravarti A 1998 Nature Genet 19:216-7; Wang DG et al 1998 Science 280:1077-82; Chakravarti A 1999 Nat Genet 21:56-60 (suppl); Stephens JC 1999 Mol. Diagnosis 4:309-317; Kwok PY and Gu S 1999 Mol. Med. Today 5:538-43; Davidson S 2000 Nature Biotech 18: 1134-5).

The standard for measuring genetic variation among individuals is the haplotype, which is the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymorphisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses AD supra; Ulbrecht M et al. 2000 Am JRespir Crit Care Med 161: 469-74) and drag 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 defmitive coupling between the polymorphism and the causative site for the phenotype (Clark AG et al. 1998 Am JHum Genet 63:595-612; Ulbrecht M et al. 2000 supra; Drysdale et al. 2000 PNAS 97: 10483-10488). Thus, there is an unmet need in the pharmaceutical industry for information on what haplotypes exist in the population for pharmaceutically-important genes. Such haplotype information would be useful in improving the efficiency and output of several steps in the drug discovery and development process, including target validation, identifying lead compounds, and early phase clinical trials (Marshall et al., supra).

One pharmaceutically-important gene for the treatment of primary pulmonary hypertension (PPH) and bone disorders is the bone morphogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) gene or its encoded product. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) family that play an important role in bone formation at extraskeletal sites. BMPs act on osteoblasts, chondrocytes, and neural cells, as well as other cell types, and regulate embryonic development (Rosenzweig et al., Proc. Nat. Acad. Sci. 1995 7632-7636). BMPR2 and BMPR1 are two closely related serine-threonine BMP receptors that form a hetero- origomeric complex that mediates BMP signaling. (Astrom et al., Mamm Genome 1999 10:299-302). BMPR2 alone can bind to BMP2 and BMP7, however, the formation of complex with BMPR1 is essential to mediate signaling. Studies in mammahan cells have shown that the binding of BMP2 and BMP7 to BMPR2 is enhanced by the coexpression of BMPR1, and a transcriptional response occurs only when both receptors are expressed (Liu et al., Mol Cell Biol. 1995 3479-3486).

Primary pulmonary hypertension (PPH) is a rare autosomal disorder characterized by monoclonal plexiform lesions of proliferating endothelial cells in the pulmonary arterioles. These lesions lead to elevated pulmonary-artery pressures, right- ventricular failure, and death. Mutations in the BMPR2 gene have been identified in patients suffering from PPH, which indicates a primary defect in the BMPR2 signaling pathway (Deng et al., Am JHum Genet 2000 Sep;67(3):737-744). Additionally, the International PPH Consortium (Nat Genet. 2000; 26(l):3-4) has also reported several mutations in the BMPR2 gene that cause PPH. These mutations affect the expression of BMPR2 on the cell surface, formation of the hetero-oligomeric receptor complex and ligand binding.

The bone morphogenetic protein receptor, type II (serme/threonine kinase) gene is located on chromosome 2q33-q34 and contains 13 exons that encode a 1038 amino acid protein (The International PPH Consortium, supra). Currently, there is no published genomic sequence for exon 2. Although a complete genomic sequence for BMPR2 has not been published, a partial reference sequence for the BMPR2 gene comprises the non-contigous sequences shown in the contiguous lines of Figure 1

(Genaissance Reference No. 9525971; SEQ ID NO: 1), the contiguous lines of Figure 2 (Genaissance Reference No. 9525989; SEQ ID NO: 2), the contiguous lines of Figure 3 (Genaissance Reference No. 9525996; SEQ ED NO: 3), the contiguous lines of Figure 4 (Genaissance Reference No. 9526005; SEQ ID NO: 4), the contiguous lines of Figure 5 (Genaissance Reference No. 9526013; SEQ TD NO: 5), the contiguous lines of Figure 6 (Genaissance Reference No. 9526022; SEQ TD NO: 6), the contiguous lines of Figure 7 (Genaissance Reference No. 9526029; SEQ ID NO: 7), the contiguous lines of Figure 8 (Genaissance Reference No. 9526038; SEQ ID NO: 8). Reference sequences for the coding sequence (GenBank Accession No. NM_001204.2) and protein are shown in Figures 9 (SEQ TD NO: 9) and 10 (SEQ ID NO: 10), respectively. Because of the potential for variation in the BMPR2 gene to affect the expression and function of the encoded protein, it would be useful to know whether polymorphisms exist in the BMPR2 gene, as well as how such polymorphisms are combined in different copies of the gene. Such information could be applied for studying the biological function of BMPR2 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 8 novel polymorphic sites in the BMPR2 gene. These polymorphic sites (PS) correspond to the following nucleotide positions: 1240 (PSl) and 1971 (PS2) in Figure 1; 1321 (PS3) and 1452 (PS4) in Figure 6; 4773 (PS5), 4901 (PS6) and 5260 (PS7) in Figure 7; and 1857 (PS8) in Figure 8. The polymorphisms at these sites are guanine or adenine at PSl, cytosine or thymine at PS2, adenine or cytosine at PS3, adenine or guanine at PS4, guanine or adenine at PS5, thymine or guanine at PS6, guanine or adenine at PS7 and cytosine or thymine at PS8. In addition, the inventors have determined the identity of the alleles at these sites in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS1-PS8 in the BMPR2 gene, which are shown below in Tables 5 and 4, respectively. Each of these BMPR2 haplotypes constitutes a code that defines the variant nucleotides that exist in the human population at this set of polymorphic sites in the BMPR2 gene. Thus each BMPR2 haplotype also represents a naturally- occurring isoform (also referred to herein as an "isogene") of the BMPR2 gene. The frequency of each haplotype and haplotype pair within the total reference population and within each of the 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 BMPR2 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 PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8 in both copies of the BMPR2 gene from the individual. A genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel BMPR2 polymorphic sites. A genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel BMPR2 polymorphic sites. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below.

The invention also provides a method for haplotyping the BMPR2 gene in an individual. In one embodiment, the haplotyping method comprises determining, for one copy of the BMPR2 gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8. In another embodiment, the haplotyping method comprises deteπnining whether one copy of the individual's BMPR2 gene is defined by one of the BMPR2 haplotypes shown in Table 5, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises determining whether both copies of the individual's BMPR2 gene are defined by one of the BMPR2 haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. Establishing the BMPR2 haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drags for treating diseases associated with BMPR2 activity, e.g., primary pulmonary hypertension (PPH) and bone disorders. For example, the haplotyping method can be used by the pharmaceutical research scientist to validate BMPR2 as a candidate target for treating a specific condition or disease predicted to be associated with BMPR2 activity. Determining for a particular population the frequency of one or more of the individual BMPR2 haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue BMPR2 as a target for treating the specific disease of interest. In particular, if variable BMPR2 activity is associated with the disease, then one or more BMPR2 haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls. Conversely, if each of the observed BMPR2 haplotypes are of similar frequencies in the disease and control groups, then it may be inferred that variable BMPR2 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 BMPR2 haplotype or haplotype pair, apply the information derived from detecting BMPR2 haplotypes in an individual to decide whether modulating BMPR2 activity would be useful in treating the disease.

The claimed invention is also useful in screening for compounds targeting BMPR2 to treat a specific condition or disease predicted to be associated with BMPR2 activity. For example, detecting which of the BMPR2 haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each of the BMPR2 isoforms present in the disease population, or for only the most frequent BMPR2 isoforms present in the disease population. Thus, without requiring any a priori knowledge of the phenotypic effect of any particular BMPR2 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 BMPR2 gene in an individual is also useful in the design of clinical trials of candidate drags for treating a specific condition or disease predicted to be associated with BMPR2 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 of the BMPR2 haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute BMPR2 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 BMPR2 haplotype or haplotype pair that is associated with response to the drag 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 deteimiring the phenotypic effect of any BMPR2 haplotype or haplotype pair.

In another embodiment, the invention provides a method for identifying an association between a trait and a BMPR2 genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein. The method comprises comparing the frequency of the BMPR2 genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the BMPR2 genotype or haplotype in a reference population. A higher frequency of the BMPR2 genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the BMPR2 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 BMPR2 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 primary pulmonary hypertension (PPH) and bone disorders.

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 BMPR2 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 adenine at PSl, thymine at PS2, cytosine at PS3, guanine at PS4, adenine at PS5, guanine at PS6, adenine at PS7 and thymine at PS8. A particularly preferred polymo hic variant is an isogene of the BMPR2 gene. A BMPR2 isogene of the invention comprises guanine or adenine at PSl, cytosine or thymine at PS2, adenine or cytosine at PS3, adenine or guanine at PS4, guanine or adenine at PS5, thymine or guanine at PS6, guanine or adenine at PS7 and cytosine or thymine at PS8. The invention also provides a collection of BMPR2 isogenes, referred to herein as a BMPR2 genome anthology. In another embodiment, the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a BMPR2 cDNA or a fragment thereof. The reference sequence comprises SEQ TD NO:9 (Fig.9) and the polymorphic cDNA comprises at least one polymorphism selected from the group consisting of thymine at a position corresponding to nucleotide 63, adenine at a position corresponding to nucleotide 2324, guanine at a position corresponding to nucleotide 2452 and adenine at a position corresponding to nucleotide 2811. A particularly preferred polymorphic cDNA variant comprises the coding sequence of a BMPR2 isogene defined by haplotypes 2,3, and 6-8.

Polynucleotides complementary to these BMPR2 genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants of the BMPR2 gene will be useful . in studying the expression and function of BMPR2, and in expressing BMPR2 protein for use in screening for candidate drags to treat diseases related to BMPR2 activity.

In other embodiments, the invention provides a recombinant expression vector comprising one of the polymorphic genomic and cDNA variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector. The recombinant vector and host cell may be used to express BMPR2 for protein structure analysis and drug binding studies.

In yet another embodiment, the invention provides a polypeptide comprising a polymorphic variant of a reference amino acid sequence for the BMPR2 protein. The reference amino acid sequence comprises SEQ TD NO: 10 (Fig.10) and the polymorphic variant comprises at least one variant amino acid selected from the group consisting of asparagine at a position corresponding to amino acid position 775 and alanine at a position corresponding to amino acid position 818. A polymorphic variant of BMPR2 is useful in studying the effect of the variation on the biological activity of BMPR2 as well as on the binding affinity of candidate drags targeting BMPR2 for the treatment of primary pulmonary hypertension (PPH) and bone disorders.

The present invention also provides antibodies that recognize and bind to the above polymorphic BMPR2 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 BMPR2 polymorphic genomic variants described herein and methods for producing such animals. The transgenic animals are useful for studying expression of the BMPR2 isogenes in vivo, for in vivo ^■ screening and testing of drugs targeted against BMPR2 protein, and for testing the efficacy of therapeutic agents and compounds for primary pulmonary hypertension (PPH) and bone disorders in a biological system.

The present invention also provides a computer system for storing and displaying polymorphism data determined for the BMPR2 gene. The computer system comprises a computer processing unit; a display; and a database containing the polymorphism data. The polymorphism data includes one or more of the following: the polymorphisms, the genotypes, the haplotypes, and the haplotype pairs identified for the BMPR2 gene in a reference population. In a preferred embodiment, the computer system is capable of producing a display showing BMPR2 haplotypes organized according^' to their evolutionary relationships.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (SEQ ID NO: 1) illustrates a partial reference sequence for the BMPR2 gene

(Genaissance Reference No. 9525971; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence. SEQ ID NO:l is equivalent to Figure 1, with the two alternative allelic variants of each polymorphic site indicated by the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WTPO standard ST.25).

Figure 2 (SEQ ID NO: 2) illustrates a partial reference sequence for the BMPR2 gene (Genaissance Reference No. 9525989; 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.

Figure 3 (SEQ ID NO: 3) illustrates a partial reference sequence for the BMPR2 gene (Genaissance Reference No. 9525996; 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.

Figure 4 (SEQ TD NO:4) illustrates a partial reference sequence for the BMPR2 gene (Genaissance Reference No. 9526005; 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.

Figure 5 (SEQ ID NO:5) illustrates a partial reference sequence for the BMPR2 gene

(Genaissance Reference No. 9526013; 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.

Figure 6 (SEQ TD NO: 6) illustrates a partial reference sequence for the BMPR2 gene

(Genaissance Reference No. 9526022; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.

SEQ ID NO:6 is equivalent to Figure 6, with the two alternative allelic variants of each polymorphic site indicated by the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S=

G or C, and W= A or T; WTPO standard ST.25). Figure 7 (SEQ ID NO: 7) illustrates a partial reference sequence for the BMPR2 gene

(Genaissance Reference No. 9526029; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence. SEQ ID NO:7 is equivalent to Figure 7, with the two alternative allelic variants of each polymorphic site indicated by the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S=

G or C, and W= A or T; WTPO standard ST.25).

Figure 8 (SEQ ID NO: 8) illustrates a partial reference sequence for the BMPR2 gene

(Genaissance Reference No. 9526038; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and poiymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.

SEQ TD NO:8 is equivalent to Figure 8, with the two alternative allelic variants of each polymorphic site indicated by the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WTPO standard ST.25).

^• SEQ TD NO:53 is a modified version of SEQ TD NOS: 1, 6, 7, and 8 that shows the context sequence of each polymorphic site, PS1-PS8, in a uniform format to facilitate electronic searching. For each polymorphic site, SEQ TD NO:53 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymorphic site at the 30^th position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein. Figure 9 illustrates a reference sequence for the BMPR2 coding sequence (contiguous lines;

SEQ ID NO:9), with the polymorphic site(s) and polymorphism(s) identified,by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.

Figure 10 illustrates a reference sequence for the BMPR2 protein (contiguous lines; SEQ ED NO: 10), with the variant amino acid(s) caused by the polymorphism(s) of Figure 2 positioned below the polymorphic site in the sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the discovery of novel variants of the BMPR2 gene. As described in more detail below, the inventors herein discovered 8 isogenes of the BMPR2 gene by characterizing the BMPR2 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.

The BMPR2 isogenes present in the human reference population are defined by haplotypes for 8 polymorphic sites in the BMPR2 gene, all of which are believed to be novel. The novel BMPR2 pplymoφhic sites identified by the inventors are referred to as PS 1 -PS8 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 PS1-PS8 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the BMPR2 gene present in individual human members of this repository. The human genotypes and haplotypes found in the repository for the BMPR2 gene include those shown in Tables 4 and 5, respectively. The polymoφhism and haplotype data disclosed herein are useful for validating whether BMPR2 is a suitable target for drags to treat primary pulmonary hypertension (PPH) and bone disorders, screening for such drags and reducing bias in clinical trials of such drags.

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 of the 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 of the 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 determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference. Haplotype data - Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.

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

Isogene - One of the isoforms (e.g., alleles) of a gene found in a population. An isogene (or allele) contains all of the polymoφhisms present in the particular isoform of the gene.

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

Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature, 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 of the 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 - A gene, mRNA, cDNA, polypeptide or peptide whose nucleotide or amino acid sequence varies from a reference sequence due to the presence of a polyrnoφhism 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 of the 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 associations) 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 of the genetic variation found in the general population. Typically, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.

Single Nucleotide Polymorphism (SNP) - Typically, the specific pair of nucleotides observed at a single 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 BMPR2 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 drag discovery and development applications. Thus, the invention also provides compositions and methods for detecting the novel BMPR2 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 BMPR2 polymoφhic site in one copy or two copies of the BMPR2 gene. Such oligonucleotides are referred to herein as BMPR2 haplotyping oligonucleotides or genotyping oligonucleotides, respectively, and collectively as BMPR2 oligonucleotides. In one embodiment, a BMPR2 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 of the 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 of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan. The oligonucleotide may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives. Alternatively, oligonucleotides may have a phosphate- free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and

Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc. (1995), pages 617-620). Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion. The oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.

Haplotyping or genotyping oligonucleotides of the invention must be capable of specifically . hybridizing to a target region of a BMPR2 polynucleotide. Preferably, the target region is located in a BMPR2 isogene. As used herein, specific hybridization means the oligonucleotide forms an anti- parallel double-stranded stracture with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with another region in the BMPR2 polynucleotide or . with a non-BMPR2 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 BMPR2 gene using the polymoφhism information provided herein in conjunction with the known sequence information for the BMPR2 gene and routine techniques. A nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect" or "complete" complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule. A nucleic acid molecule is "substantially complementary" to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D. et al. in Nucleic Acid Hybridization, A Practical Approach, TRL 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 of the primer being complementary to the target region. Alternatively, non-complementary nucleotides may be interspersed into the probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region. Preferred haplotyping or genotyping oligonucleotides of the mvention are allele-specifϊc oligonucleotides. As used herein, the term allele-specific oligonucleotide (ASO) means an oligonucleotide that is able, under sufficiently stringent conditions, to hybridize specifically to one allele of a gene, or other locus, at a target region containing a polymoφhic site while not hybridizing to the corresponding region in another allele(s). As understood by the skilled artisan, allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps. Examples of hybridization and washing conditions typically used for ASO probes are found in Kogan et al., "Genetic Prediction of Hemophilia A" in PCR Protocols, A Guide to Methods and Applications, Academic Press, 1990 and Ruano et al., 87 Proc. Natl. Acad. Sci. USA 6296-6300, 1990. Typically, an ASO will be perfectly complementary to one allele while containing a single mismatch for another allele.

Allele-specific oligonucleotides of the invention include ASO probes and ASO primers. ASO probes which usually provide good discrimination between different alleles are those in which a central position of the 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 of the invention has a 3 ' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present. ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention. ASO probes and primers listed below use the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WTPO standard ST.25) at the position of the polymoφhic site to represent that the ASO contains either of the two alternative allelic variants observed at that polymoφhic site.

A preferred ASO probe for detecting BMPR2 gene polymoφhisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:

AGCCTTCRCCAGGGC (SEQ ID NO 11 and its complement, TGCTGGTYAGCACTG (SEQ ID NO 12 and its complement, AAAAAAAMTCAT AA (SEQ ID NO 13 and its complement, ATCATTTRTGATAGT (SEQ ID NO 14 and its complement, TTTGGCARCAAGCAC (SEQ ID NO: 15 and its complement, TGTTAACKCCCATGC (SEQ ID NO: 16 and its complement, CACAGAGRCCTAATT (SEQ ID NO 17 and its complement, and. GCACCCCYTCCCACC (SEQ ID NO 18 and its complement,

A preferred ASO primer for detecting BMPR2 gene polymoφhisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:

CTCCTCAGCCTTCRC (SEQ ID NO 19); GGGGAGGCCCTGGYG (SEQ ID NO:20);

CCATCCTGCTGGTYA (SEQ ID NO 21); CAGCCGCAGTGCTRA (SEQ ID NO:22);

AAAAGAAAAAAAAMT (SEQ ID NO 23); GTAGGTTTAATGAKT (SEQ ID NO: 24);

TATTCTATCATTTRT (SEQ ID NO 25); TTTCTAACTATCAYA (SEQ ID NO:26);

CTAAAATTTGGCARC (SEQ ID NO 27); TGATTTGTGCTTGYT (SEQ ID NO: 28)

CCACAGTGTTAACKC (SEQ ID NO 29); GTGGCAGCATGGGMG (SEQ ID NO: 30)

GAAGAGCACAGAGRC (SEQ ID NO 31); CCAGAGAATTAGGYC (SEQ ID NO: 32)

CTGTCAGCACCCCYT (SEQ ID NO 33); and TGCAGGGGTGGGARG (SEQ ID NO

Other oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymoφhic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the 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 BMPR2 gene polymoφhisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:

CTCAGCCTTC (SEQ ID NO 35); GAGGCCCTGG (SEQ ID NO 36);

TCCTGCTGGT (SEQ ID NO 37); CCGCAGTGCT (SEQ ID NO 38);

AGAAAAAAAA (SEQ ID NO 39); GGTTTAATGA (SEQ ID NO 40);

TCTATCATTT (SEQ ID NO 41); CTAACTATCA (SEQ ID NO 42);

AAATTTGGCA (SEQ ID NO 43); TTTGTGCTTG (SEQ ID NO 44);

CAGTGTTAAC (SEQ ID NO 45); GCAGCATGGG (SEQ ID NO 46); GAGCACAGAG (SEQ ID NO: 47) ; GAGAATTAGG (SEQ ID NO: 48); TCAGCACCCC (SEQ ID NO:49);and AGGGGTGGGA (SEQ ID NO:50).

In some embodiments, a composition contains two or more differently labeled BMPR2 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. BMPR2 oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized oligonucleotides may be used in a variety of polymoφhism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized BMPR2 oligonucleotides of the mvention 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 BMPR2 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 BMPR2 gene in an individual. As used herein, the terms "BMPR2 genotype" and "BMPR2 haplotype" mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymoφhic sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymoφhic sites in the BMPR2 gene. The additional polymoφhic sites may be currently known polymoφhic sites or sites that are subsequently discovered.

One embodiment of a genotyping method of the invention involves isolating from the individual a nucleic acid sample comprising the two copies of the BMPR2 gene, mRNA transcripts thereof or cDNA copies thereof, or a fragment of any of the foregoing, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8 in the two copies to assign a BMPR2 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 BMPR2 molecules) in an individual may be the same allele or may be different alleles. In another embodiment, a genotyping method of the invention comprises determining the identity of the nucleotide pair at each of PS1-PS8.

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 BMPR2 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 BMPR2 gene fragment is isolated, it must contain the polymoφhic site(s) to be genotyped.

One embodiment of a haplotyping method of the invention comprises isolating from the individual a nucleic acid sample containing only one of the two copies of the BMPR2 gene, mRNA or cDNA, or a fragment of such BMPR2 molecules, that is present in the individual and determining in that copy the identity of the nucleotide at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8 in that copy to assign a BMPR2 haplotype to the individual. The nucleic acid used in the above haplotyping methods of the invention may be isolated using any method capable of separating the two copies of the BMPR2 gene or fragment such as one of the methods described above for preparing BMPR2 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 of the two BMPR2 gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional BMPR2 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 of the BMPR2 gene in an individual. In some cases, however, once the haplotype for one BMPR2 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 PSl -PS8 is identified.

In another embodiment, the haplotyping method comprises determining whether an individual has one or more of the BMPR2 haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies of the individual's BMPR2 gene, the phased sequence of nucleotides present at each of PS1-PS8. This identifying step does not necessarily require that each of PS1-PS8 be directly examined. Typically only a subset of PS1-PS8 will need to be directly examined to assign to an individual one or more of the 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 of the invention, a BMPR2 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 PS 1 , PS2, PS3, PS4, PS5, PS6, PS7 and PS8 in each copy of the BMPR2 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-PS8 in each copy of the BMPR2 gene.

When haplotyping both copies of the gene, the identifying step is preferably performed with each copy of the gene being placed in separate containers. However, it is also envisioned that if the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be possible in some cases to perform the method in the same container. For example, if first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymoφhic site(s), then detecting a combination of the first and third dyes would identify the polymoφhism in the first gene copy while detecting a combination of the second and third dyes would identify the polymoφhism in the second gene copy.

In both the genotyping and haplotyping methods, the identity of a nucleotide (or nucleotide pair) at a polymoφhic site(s) may be determined by amplifying a target region(s) containing the polymoφhic site(s) directly from one or both copies of the BMPR2 gene, or a fragment thereof, and the sequence of the amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymoφhic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site. The polymoφhism may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification. For example, where a SNP is known to be guanine and cytosine in a reference population, a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site. Alternatively, the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine). The target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241:1077-1080, 1988). Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992). A polymoφhism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art. Typically, allele-specific oligonucleotides are utilized in performing such methods. The allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant. In some embodiments, more than one polymoφhic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs. Preferably, the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymoφhic sites being detected. Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele- specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis. Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads. The solid support may be treated, coated or derivatized to facilitate the immobilization of the allele- specific oligonucleotide or target nucleic acid.

The genotype or haplotype for the BMPR2 gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the 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 of the polymoφhic sites to be included in the genotype or haplotype.

The identity of polymoφhisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variant alleles can be identified by single strand conformation polymoφhism

(SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989). A polymerase-mediated primer extension method may also be used to identify the ρolymoφhism(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 WO91/02087, 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; Ruano et al, Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest.

95:1635-1641, 1995). In addition, multiple polymoφhic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).

In addition, the identity of the allele(s) present at any of the novel polymoφhic sites described herein may be indirectly determined by 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 of the gene or in other genomic regions not examined herein. Detection of the 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 of the above-mentioned methods for detecting the identity of the allele at a polymoφhic site.

In another aspect of the invention, an individual's BMPR2 haplotype pair is predicted from its BMPR2 genotype using information on haplotype pairs known to exist in a reference population. In its broadest embodiment, the haplotyping prediction method comprises identifying a BMPR2 genotype for the individual at two or more BMPR2 polymoφhic sites described herein, accessing data containing BMPR2 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 BMPR2 haplotype pairs shown in Table 4. The BMPR2 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 of the individual. In some embodiments, comparison of the genotype of the individual to the haplotype pairs identified in a reference population and determination of which haplotype pair is consistent with the genotype of the individual may be ^' performed by visual inspection (for example, by consulting Table 4). When the genotype of the individual is consistent with more than one haplotype pair, 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 upon consulting Table 7. If a particular BMPR2 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 of the individual and the reference haplotype data stored in computer- readable formats. For example, as described in PCT/USO 1/12831, 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 BMPR2 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 ethnogeograp ic groups of the world. A preferred reference population for use in the methods of the present invention comprises an approximately equal number of individuals from Caucasian, African-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 unrelated individuals from each of the four population groups named above. A particularly preferred reference population includes a 3-generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures.

In a preferred embodiment, the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L. Hartl et al., Principles of Population Genomics, Sinauer Associates (Sunderland, MA), 3^rd Ed., 1997) postulates that the frequency of finding the haplotype pair H₁ 1 H₂ is equal to p_H__w(Hχ IH₂) = 2p(H_λ)p(H₂) if H_λ ≠ H₂ and p_H__w(H_x I H₂) = p(H_λ)p(H₂) if H_λ = 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 BMPR2 haplotype pair for an individual, the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair. Alternatively, the haplotype pair in an individual may be predicted from the individual's genotype for that gene using reported methods (e.g., Clark et al.. 1990 Mol Bio Evol 7:111-22; 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 BMPR2 genotype, haplotype, or haplotype pair in a population. The method comprises, for each member of the population, determining the genotype or the haplotype pair for the novel BMPR2 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).

In another aspect of the invention, frequency data for BMPR2 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 BMPR2 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 of the genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait. Frequency data for one or both of the reference and trait populations may.be obtained by genotyping or haplotyping each individual in the populations using one or more of the 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 of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared. If a particular BMPR2 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 BMPR2 genotype, haplotype or haplotype pair. Preferably, the BMPR2 genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the full-genotypes and full-haplotypes shown in Tables 4 and 5, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.

In a preferred embodiment of the method, the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drag targeting BMPR2 or response to a therapeutic treatment for a medical condition. As used herein, "medical condition" includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders. As used herein the term "clinical response" means any or all of the following: a quantitative measure of the response, no response, and/or adverse response (i.e., side effects).

In order to deduce a correlation between clinical response to a treatment and a BMPR2 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 aheady 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 HI clinical trials. Standard methods are used to define the patient population and to enroll subjects. It is preferred that the individuals included in the clinical population have been graded for the existence of the medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome. This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity. The therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the BMPR2 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 BMPR2 genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their BMPR2 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 BMPR2 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 BMPR2 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 of the variation in the clinical data is explained by different subsets of the polymoφhic sites in the BMPR2 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 BMPR2 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 BMPR2 gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug. The diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymoφhic sites in the BMPR2 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 BMPR2 genotype or haplotype that is in turn correlated with the clinical response. In a preferred embodiment, this diagnostic method uses the predictive haplotyping method described above.

In another embodiment, the invention provides an isolated polynucleotide comprising a polymoφhic variant of the BMPR2 gene or a fragment of the gene which contains at least one of the novel polymoφhic sites described herein. The nucleotide sequence of a variant BMPR2 gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymoφhic sites PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8. Similarly, the nucleotide sequence of a variant fragment of the BMPR2 gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymoφhic sites described herein. Thus, the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the BMPR2 gene, which is defined by haplotype 4, (or other reported BMPR2 sequences) or to portions of the reference sequence (or other reported BMPR2 sequences), except for the haplotyping and genotyping oligonucleotides described above.

The location of a polymoφhism in a variant BMPR2 gene or fragment is preferably identified by aligning its sequence against SEQ ID NOS: 1-8. The polymoφhism is selected from the group consisting of adenine at PSl, thymine at PS2, cytosine at PS3, guanine at PS4, adenine at PS5, guanine at PS6, adenine at PS7 and thymine at PS8. In a preferred embodiment, the polymoφhic variant comprises a naturally-occurring isogene of the BMPR2 gene which is defined by any one of haplotypes 1- 3 and 5 - 8 shown in Table 5 below.

Polymoφhic variants of the invention may be prepared by isolating a clone containing the BMPR2 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 BMPR2 variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.

BMPR2 isogenes, or fragments thereof, may be isolated using any method that allows separation of the two "copies" of the BMPR2 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 Ruafio 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 BMPR2 genome anthologies, which are collections of at least two BMPR2 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 BMPR2 genome anthology may comprise individual BMPR2 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the BMPR2 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 BMPR2 genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below. A BMPR2 genome anthology is useful in providing control nucleic acids for kits of the invention.

An isolated polynucleotide containing a polymoφhic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded BMPR2 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, adenoviras, retroviruses, or SV40. Other regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence in a given host cell. Of course, the correct combinations of expression regulatory elements will depend on the host system used. In addition, it is understood that the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers. Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current

Protocols in Molecular Biology", John Wiley and Sons, New York, New York). Host cells which may be used to express the variant BMPR2 sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art. The recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE- dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). In a preferred aspect, eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used. Non-limiting examples of such vectors include vaccinia virus vectors, adenoviras vectors, heφes virus vectors, and baculovirus transfer vectors. Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NTH/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 of the BMPR2 gene will produce BMPR2 mRNAs varying from each other at any polymoφhic site retained in the spliced and processed mRNA molecules. These mRNAs can be used for the preparation of a BMPR2 cDNA comprising a nucleotide sequence which is a polymoφhic variant of the BMPR2 reference coding sequence shown in Figure 9. Thus, the invention also provides BMPR2 mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ED NO: 9 (Fig. 9), or its corresponding RNA sequence, for those regions of SEQ ED NO:9 that correspond to the examined portions of the BMPR2 gene (as described in the Examples below), except for having one or more polymoφhisms selected from the group consisting of thymine at a position corresponding to nucleotide 63, adenine at a position corresponding to nucleotide 2324, guanine at a position corresponding to nucleotide 2452 and adenine at a position corresponding to nucleotide 2811. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of a BMPR2 isogene defined by any one of haplotypes 2,3, and 6-8. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain one or more of the novel polymoφhisms described herein. The invention specifically excludes polynucleotides identical to previously identified and characterized BMPR2 mRNAs, cDNAs or fragments thereof. Polynucleotides comprising a variant BMPR2 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 BMPR2 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 of the gene. Preferably, such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length. In describing the BMPR2 polymoφhic sites identified herein, reference is made to the sense strand of the gene for convenience. However, as recognized by the skilled artisan, nucleic acid molecules containing the BMPR2 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 of the BMPR2 genomic, mRNA and cDNA variants described herein.

Polynucleotides comprising a polymoφhic gene variant or fragment of the invention may be useful for therapeutic puφoses. For example, where a patient could benefit from expression, or increased expression, of a particular BMPR2 protein isoform, an expression vector encoding the isoform may be administered to the patient. The patient may be one who lacks the BMPR2 isogene encoding that isoform or may aheady have at least one copy of that isogene.

In other situations, it may be desirable to decrease or block expression of a particular BMPR2 isogene. Expression of a BMPR2 isogene may be turned off by fransforming 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) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred. Similarly, inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). Antisense oligonucleotides may also be designed to block translation of BMPR2 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of BMPR2 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 BMPR2 amino acid sequence shown in Figure 10 or (b) a fragment of this reference sequence. The location of a variant amino acid in a BMPR2 polypeptide or fragment of the invention is identified by aligning its sequence against SEQ TD NO: 10 (Fig. 10). A BMPR2 protein variant of the invention comprises an amino acid sequence identical to SEQ TD NO: 10 for those regions of SEQ ID NO: 10 that are encoded by examined portions of the BMPR2 gene (as described in the Examples below), except for having one or more variant amino acids selected from the group consisting ofasparagine at a position corresponding to amino acid position 775 and alanine at a position corresponding to amino acid position 818. Thus, a BMPR2 fragment of the invention, also referred to herein as a BMPR2 peptide variant, is any fragment of a BMPR2 protein variant that contains one or more of the amino acid variations shown in Table 2. The invention specifically excludes amino acid sequences identical to those previously identified for BMPR2, including SEQ ED NO: 10, and previously described fragments thereof. BMPR2 protein variants included within the invention comprise all amino acid sequences based on SEQ TD NO: 10 and having the combination of amino acid variations described in Table 2 below. In preferred embodiments, a BMPR2 protein variant of the invention is encoded by an isogene defined by one of the observed haplotypes, 2,3, and 6-8, shown in Table 5.

Table 2. Novel Polymoφhic Variants of BMPR2

Polymoφhic Amino Acid Position and Identities Variant

Number 775 818 1 S A

2 N S

3 N A

A BMPR2 peptide variant of the 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 BMPR2 peptide variants may be useful as antigens to generate antibodies specific for one of the above BMPR2 isoforms. In addition, the BMPR2 peptide variants may be useful in drag screening assays.

A BMPR2 variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing an appropriate variant BMPR2 genomic or cDNA sequence described above.

Alternatively, the BMPR2 protein variant may be isolated from a biological sample of an individual having a BMPR2 isogene which encodes the variant protein. Where the sample contains two different BMPR2 isoforms (i.e., the individual has different BMPR2 isogenes), a particular BMPR2 isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular BMPR2 isoform but does not bind to the other BMPR2 isoform.

The expressed or isolated BMPR2 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 of the BMPR2 protein or peptide as discussed further below. BMPR2 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 immunoaffinity chromatography, antibodies specific for a particular polymoφhic variant may be used. A polymoφhic variant BMPR2 gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric BMPR2 protein. The non-BMPR2 portion of the chimeric protein may be recognized by a commercially available antibody. In addition, the chimeric protein may also be engineered to contain a cleavage site located between the BMPR2 and non-BMPR2 portions so that the BMPR2 protein may be cleaved and purified away from the non-BMPR2 portion. An additional embodiment of the invention relates to using a novel BMPR2 protein isoform, or a fragment thereof, in any of a variety of drag screening assays. Such screening assays may be performed to identify agents that bind specifically to all known BMPR2 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 BMPR2 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

BMPR2 variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the BMPR2 protein(s) of interest and then washed. Bound BMPR2 protein(s) are then detected using methods well-known in the art. In another embodiment, a novel BMPR2 protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the BMPR2 protein.

In yet another embodiment, when a particular BMPR2 haplotype or group of BMPR2 haplotypes encodes a BMPR2 protein variant with an amino acid sequence distinct from that of BMPR2 protein isoforms encoded by other BMPR2 haplotypes, then detection of that particular BMPR2 haplotype or group of BMPR2 haplotypes may be accomplished by detecting expression of the encoded BMPR2 protein variant using any of the methods described herein or otherwise commonly known to the skilled artisan.

In another embodiment, the invention provides antibodies specific for and immunoreactive with one or more of the novel BMPR2 variant proteins described herein. The antibodies may be either monoclonal or polyclonal in origin. The BMPR2 protein or peptide variant used to generate the antibodies may be from natural or recombinant sources or produced by chemical synthesis using synthesis techniques known in the art. If the BMPR2 protein variant is of insufficient size to be antigenic, it may be conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity of the peptide. Examples of carrier molecules, include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical

Immunology, 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).

In one embodiment, an antibody specifically immunoreactive with one of the novel protein isoforms described herein is administered to an individual to neutralize activity of the BMPR2 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 of the novel protein isoforms described herein may be used to immunoprecipitate the BMPR2 protein variant from solution as well as react with BMPR2 protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates. In another preferred embodiment, the antibodies will detect BMPR2 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 of the novel BMPR2 protein variants described herein is used in immunoassays to detect this variant in biological samples. In this method, an antibody of the present invention is contacted with a biological sample and the formation of a complex between the BMPR2 protein variant and the antibody is detected. As described, suitable immunoassays include radioimmunoassay, Western blot assay, immunbfluorescent 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 lmmunodiagnosis, 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 OelHrich, 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 of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site. Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam). The antibodies or antigen binding fragments thereof may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication number WO 901443, WO 901443 and WO 9014424 and in Huse et al., 1989, Science, 246:1275-1281. The antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci.USA 86; 10029). Effect(s) of the polymoφhisms identified herein on expression of BMPR2 may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymoφhic variant of the BMPR2 gene. As used herein, "expression" includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into BMPR2 protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function. To prepare a recombinant cell of the invention, the desired BMPR2 isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. In a preferred embodiment, the BMPR2 isogene is introduced into a cell in such a way that it recombines with the endogenous BMPR2 gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired BMPR2 gene polymoφhism. Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner. Examples of cells into which the BMPR2 isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the BMPR2 isogene. Such recombinant cells can be used to compare the biological activities of the different protein variants.

Recombinant nonhuman organisms, i.e., transgenic animals, expressing a variant BMPR2 gene are prepared using standard procedures known in the art. Preferably, a construct comprising the variant gene is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage. Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art. One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053. Another method involves directly injecting a transgene into the embryo. A third method involves the use of embryonic stem cells. Examples of animals into which the BMPR2 isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice" and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M. Gilman, J. Witkowski, and M. Zoller; W.H. Freeman and Company, New York, pages 254-272). Transgenic animals stably expressing a human BMPR2 isogene and producing the encoded human BMPR2 protein can be used as biological models for studying diseases related to abnormal BMPR2 expression and/or activity, and for screening and assaying various candidate drags, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.

An additional embodiment of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel BMPR2 isogene described herein. The pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel BMPR2 isogenes; an antisense oligonucleotide directed against one of the novel BMPR2 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel BMPR2 isogene described herein. Preferably, the composition contains the active ingredient in a therapeutically effective amount. By therapeutically effective amount is meant that one or more of the symptoms relating to disorders affected by expression or function of a novel BMPR2 isogene is reduced and/or eliminated. The composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water. Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist. The pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound. Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

For any composition, determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within the capability of those skilled in the art. For example, the dose can be estimated initially either in cell culture assays or in animal models. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drags being taken by the patient, and tolerance/response to the treatment. Any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer. In addition, the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can • interact to view and analyze large amounts of information relating to the BMPR2 gene and its genomic variation, including chromosome location, gene stracture, 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 BMPR2 polymoφhism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files). These polymoφhism data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network.

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

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

EXAMPLE 1 This example illustrates examination of various regions of the BMPR2 gene for polymoφhic sites.

Amplification of Target Regions

The following target regions were amplified using either the PCR primers represented below or 'tailed' PCR primers, each of which includes a universal sequence forming a noncomplementary 'tail' attached to the 5 ' end of each unique sequence in the PCR primer pairs. The universal 'tail' sequence for the forward PCR primers comprises the sequence 5 '-TGTAAAACGACGGCCAGT-3 ' (SEQ ID NO: 51) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence 5 '- AGGAAACAGCTATGACCAT-3 ' (SEQ ID NO:52). The nucleotide positions of the first and last nucleotide of the forward and reverse primers for each region amplified are presented below and correspond to positions in the indicated Figure.

PCR Primer Pairs

Fragment Figure Forward Primer Reverse Primer PCR Product

Fragment 1 1 1100-1123 complement of 1643-1625 544 nt

Fragment 2 1 1378-1400 complement of 1876-1856 499 nt

Fragment 3 1 1654-1674 complement of 2180-2159 527 nt

Fragment 4 5 161-186 complement of 698-679 538 nt

Fragment 5 5 2042-2061 complement of 2534-2515 493 nt

Fragment 6 6 1287-1303 complement of 1775-1754 489 nt

Fragment 7 7 1356-1377 complement of 1881- 1862 526 nt

Fragment 8 7 4171-4193 complement of 4665-4645 495 nt

Fragment 9 7 4452-4476 ^' complement of 4948-4929 497 nt

Fragment 10 7 4709-4730 complement of 5236-5217 528 nt

Fragment 11 7 4989-5011 complement of 5497-5475 509 nt

Fragment 12 8 1268-1290 complement of 1729-1708 462 nt

Fragment 13 8 1442-1464 complement of 1993-1974 552 nt These primer pairs were used in PCR reactions containing genomic DNA isolated from immortalized cell lines for each member of the Index Repository. The PCR reactions were carried out under the following conditions:

Reaction volume = 10 μl .

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

100 ng of human genomic DNA = l μl

10 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 either the primer sets represented below with the positions of their first and last nucleotide corresponding to positions in the indicated Figure, or the appropriate universal 'tail' sequence as a primer. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.

Sequencing Primer Pairs

Fragment No. Figure Forward Primer Reverse Primer

Fragment 1: 1 . Tailed Seq.

Fragment 2: ^•1 Tailed Seq.

Fragment 3: 1 Tailed Seq.

Fragment 4 5 166-187 complement of 697-678

Fragment 5 5 2043-2062 complement of 2532-2514

Fragment 6 6 1288-1309 complement of 1774-1754

Fragment 7 7 1359-1379 ^' complement of 1880- 1861

Fragment 8 7 4171-4194 complement of 4662-4644

Fragment 9 7 4475-4455 complement of 4947-4928

Fragment 10 7 4710-4731 complement of 5235-5216

Fragment 11 7 4993-5011 complement of 5494-5474

Fragment 12 8 1270-1291 complement of 1727-1707

Fragment 13 8 1444-1464 complement of 1993-1973 Analysis of Sequences for Polymoφhic Sites

Sequence information for a niinimum 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 BMPR2 reference genomic sequence (SEQ TD NOS: 1-8) are listed in Table 3 below.

Table 3. Polymoφhic Sites Identified inthe BMPR2 Gene

Polymoφhic Figure Nucleotide Reference Variant CDS AA

Site Number Polyld³ No.^b Position Allele Allele Variant Variant

PSl 10077965 1 1240 G A

PS2 10077961 1 1971 C T 63 V21V

PS3 10077971 6 1321 A C

PS4 10077967 6 1452 A G

PS5 10078397 7 4773 G A 2324 S775N

PS6 10078395 7 4901 T G 2452 S818A

PS7 10078391 7 5260 G A 2811 R937R

PS8 10078022 8 1857 C T ^aPolyId is a unique-identifier assigned to each PS by Genaissance Pharmaceuticals, Inc. ^b Figure No. refers to SEQ ID NOS in which polymorhi c sites are found

EXAMPLE 2

This example illustrates analysis of the BMPR2 polymoφhisms identified in the Index Repository for human genotypes and haplotypes.

The different genotypes containing these polymoφhisms that were observed in unrelated members of the reference population are shown in Table 4 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below. In Table 4, homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 4 were inferred based on linkage disequilibrium and/or Mendelian inheritance.

Table 4. Genotypes and Haplotype Pairs Observed for BMPR2 Gene Genotype Polymorphic Sites

Number PS l PS2 PS3 PS 4 PS 5 PS 6 PS7 PS8 HAP Pair

1 G C A A G T G C 4 4

2 G C C A G T A ^■ C 6 6

3 G C A A/G G T G C 4 5

4 G C A , A G T/G G C 4 3

5 ^■ G C A/C A/G G T G/A C 4 7

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

7 G C A A G/A T G C 4 2

8 G C/T A A G T G C 4 8

9 G c A/C A G ^' T G/A C 4 6

The haplotype pairs shown in Table 4 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in PCT/USO 1/12831, 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 of the 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 8 human BMPR2 haplotypes shown in Table 5 below.

A BMPR2 isogene defined by a full-haplotype shown in Table 5 below comprises the regions of the SEQ ID NOS indicated in Table 5, with their corresponding set of polymoφhic locations and identities, which are also set forth in Table 5.

Table 5. Haplotypes of the BMPR2 Gene

[aplot YPe Number^a PS PS SEQ ID . Region

1 2 3 4 5 6 7 8 Number⁰ Position⁰ NO. Examined⁶

A G G G G G G G 1 1240/30 1/53 1100-2180

C C C C C C C T 2 1971/150 1/53 1100-2180

5 161-698

5 2042-2534

A A A A A C C A 3 1321/270 6/53 1287-1775

A A A A G A G A 4 1452 /390 6/53 1287-1775 7 1356-1381

G A G G G G G G 5 4773 /510 7/53 4171-5497

T T G T T T T T 6 4901 /630 7/53 4171-5497

G G G G G A A G 7 5260 /750 ^' 7/53 4171-5497

T C C C C C C C 8 1857 /870 8/53 1268-1993 Alleles for BMPR2 haplotypes are presented 5' to 3' in each column ^bPS = polymoφhic site; ^cPosition of PS within the indicated SEQ ED NO, with the Imposition number referring to the first SEQ ID NO and the 2^nd position number referring to the 2^nd SEQ TD NO; ^dl^st SEQ ID NO refers to Figures 1-8, with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol; 2^nd SEQ ED NO is a modified version of Figures 1-8 that comprises the context sequence of each polymoφhic site, PS1-PS8, to facilitate electronic searching of the haplotypes; ^eRegion examined represents the nucleotide positions defining the start and stop positions within the 1^st SEQ DD NO of the sequenced region.

SEQ ED NOS: 1-8 refer to Figures 1-8, with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol. SEQ ED NO:53 is a modified version of SEQ ID NOS:l-8 that shows the context sequence of each of PS1-PS8 in a uniform format to facilitate electronic searching of the BMPR2 haplotypes. For each polymoφhic site, SEQ ED NO:53 contains a block of 60 bases of the 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 BMPR2 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 BMPR2 haplotype pair is shown in Table 7. Ln 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 BMPR2 Haplotypes In Unrelated Individuals

HAP No. HAP ED Total CA AF AS HL AM

1 10082033 0.61 2.38 0.0 0.0 0.0 0.0

2 10082032 1.83 2.38 0.0 0.0 5.56 0.0

3 10082591 0.61 0.0 2.5 0.0 . 0.0 0.0

4 10082030 84.76 83.33 90.0 85.0 77.78 100.0

5 10082034 0.61 0.0 0.0 0.0 2.78 0.0

6 10082031 10.37 11.9 2.5 15.0 13.89 0.0

7 10082120 0.61 0.0 2.5 0.0 0.0 0.0

8 10082371 0.61 0.0 2.5 0.0 0.0 0.0

Table 7. Frequency of Observed BMPR2 Haplotype Pairs In Unrelated . Individ

HAP1 HAP2 Total CA AF AS HL AM

4 4 73.17 66.67 80.0 75.0 66.67 100.0

6 6 3.66 0.0 0.0 5.0 11.11 0.0

4 5 1.22 0.0 0.0 0.0 5.56 0.0

4 3 1.22 , 0,0 5.0 0.0 0.0 0.0

4 7 1.22 0.0 5.0 0.0 0.0 0.0

4 1 1.22 4.76 0.0 0.0 0.0 0.0

4 2 3.66 4.76 0.0 0.0 11.11 0.0

4 8 1.22 0.0 5.0 0.0 0.0 0.0

4 6 13.41 23.81 5.0 20.0 5.56 0.0

The size and composition of the 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% probabihty of being observed in a sample of 20 individuals from that population group. In addition, the size and composition of the Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the BMPR2 gene are likely to be similar to the relative frequencies of these BMPR2 haplotypes and haplotype pairs in the . general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity observed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance. In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained.

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

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

Claims

What is Claimed is:

1. A method for haplotyping the bone moφhogenetic protein receptor, type II (serme/threonine kinase) (BMPR2) gene of an individual, which comprises determining which of the BMPR2 haplotypes shown in the table immediately below defines one copy of the individual's BMPR2 gene, wherein each of the BMPR2 haplotypes comprises a sequence of polymoφhisms whose positions and identities are set forth in the table immediately below:

Eaplotype Number³ PS SEQ. PS

1 2 3 4 5 6. 7 8 Number^b ID NO.^c Position^d

A G ' G G G G G G i 1 1240

C C C C C C C T 2 1 1971

A A^' A A A C c A 3 6 1321

A A A A G A G A 4 6 1452

G A G G G G G G 5 7 4773

T . T G T T T T T 6 7 4901

G G G G G A A G 7 7 5260

T C C C C C C C 8 8 1857 ^aAlleles for haplotypes are presented 5 ' to 3 ' in each column ^bPS = polymoφhic site;

°SEQ ID NO refers to Figures 1-8; Position of PS within indicated SEQ ED NO.

2. The method of claim 1, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PS1-PS8 on at least one copy of the individual's

BMPR2 gene.

3. A method for haplotyping the bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) gene of an individual, which comprises determining which of the BMPR2 haplotype pairs shown in the table immediately below defines both copies of the individual's BMPR2 gene, wherein each of the BMPR2 haplotype pairs 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:

Haplotype Pair PS SEQ PS 4/4 6/6 4/5 4/3 4/7 4/1 4/2 4/8 4/6 No.^b ID NO.^c Position¹³

G/G G/G G/G G/G G/G G/A G/G G/G G/G 1 1 1240

C/C C/C C/C C/C C/C C/C C/C C/T C/C 2 1 1971

A/A C/C A/A A/A A/C A/A A/A A/A A/C 3 6 1321

A/A A/A A/G A/A A/G A/A A/A A/A A/A 4 6 1452 G/G G/G G/G G/G G/G G/G G/A G/G G/G 5 7 4773

T/T T/T T/T T/G T/T T/T T/T T/T T/T 6 7 4901

G/G A/A G/G G/G G/A G/G G/G G/G G/A 7 7 5260

C/C C/C C/C C/C C/C C/T C/C C/C C/C 8 8 1857

Ηaplotype pairs 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; ^bPS = polymoφhic site; ^CSEQ TD NO refers to Figures 1-8; Position of PS within indicated SEQ ED NO.

4. The method of claim 3, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PS1-PS8 on both copies of the individual' S-BMPR2 gene.

5. A method for genotyping the bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) gene of an individual, comprising determining for the two copies of the BMPR2 gene present in the individual the identity of the nucleotide pair at one or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8, wherein the one or more PS have the position and alternative alleles shown in

SEQ ED NOS: 1-8.

6. The method of claim 5, wherein the deteπnining step comprises:

(a) isolating from the individual a nucleic acid mixture comprising both copies of the BMPR2 gene, or a fragment thereof, that are present in the individual;

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

(c) hybridizing a primer extension oligonucleotide to one allele of the 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 of the reaction, wherein the terminator is complementary to one of the alternative nucleotides present at the selected polymoφhic site; and

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

7. The method of claim 5, which comprises determining for the two copies of the BMPR2 gene present in the individual the identity of the nucleotide pair at each of PS1-PS8.

8. A method for haplotyping the bone moφhogenetic protein receptor, type IT (serine/tiireonine kinase) (BMPR2) gene of an individual which comprises determining, for one copy of the BMPR2 gene present in the individual, the identity of the nucleotide at two or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8, wherein the selected PS have the position and alternative alleles shown in SEQ TD NOS:l-8.

9. The method of claim 8, wherein the determining step comprises:

(a) isolating from the individual a nucleic acid sample containing only one of the two copies of the BMPR2 gene, or a fragment thereof, that is present in the individual;

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

(c) hybridizing a primer extension oligonucleotide to one allele of the 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 of the alternative nucleotides present at the selected polymoφhic site; and

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

10. A method for predicting a haplotype pair for the bone moφhogenetic protein receptor, type II

(serme/threonine kinase) (BMPR2) gene of an individual comprising:

(a) identifying a BMPR2 genotype for the individual, wherein the genotype comprises the nucleotide pair at two or more polymoφhic sites (PS) selected from the group consisting of PS 1 , PS2, PS3, PS4, PS5, PS6, PS7 and PS8, wherein the selected PS have the position and alternative alleles shown in SEQ ID NOS: 1-8;

(b) comparing the genotype to the haplotype pair data set forth in the table immediately below; and

(c) determining which haplotype pair is consistent with the genotype of the individual and with the haplotype pair data

Haplc )type Pair PS SEQ PS

4/4 6/6 4/5 4/3 4/7 4/1 4/2 4/8 4/6 No.^b ID NO.^c Position

G/G G/G G/G G/G G/G G/A G/G G/G G/G 1 1 1240

C/C C/C C/C C/C C/C C/C C/C C/T C/C 2 1 1971

A/A C/C A/A A/A A/C A/A A/A A/A A/C 3 6 1321

A/A A/A A/G A/A A/G A/A A/A A/A A/A 4 6 1452

G/G G/G G/G G/G G/G G/G G/A G/G G/G 5 7 4773

T/T T/T T/T T/G T/T T/T T/T T/T T/T 6 7 4901

G/G A/A G/G G/G G/A G/G G/G G/G G/A 7 7 5260

C/C C/C C/C C/C C/C C/T C/C .C/C C/C 8 8 1857 ^aHaplotype pairs are represented as I^s haplotype/2ⁿ haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column; ^bPS = polymoφhic site; ^CSEQ ED NO refers to Figures 1-8;

Position of PS within indicated SEQ ED NO.

11. The method of claim 10, wherein the identified genotype of the individual comprises the

. nucleotide pair at each of PS1-PS8, which have the position and alternative alleles shown in SEQ ED NOS: 1-8.

12. A method for identifying an association between a trait and at least one haplotype or haplotype pair of the bone moφhogenetic protein receptor, type EJ (serine/threonine kinase) (BMPR2) gene which comprises comparing the frequency of the haplotype or haplotype pair in a population exhibiting the trait with the frequency of the haplotype or haplotype pair in a reference population, wherein the haplotype is selected from haplotypes 1-8 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:

Haplotype Number .a PS SEQ. PS

1 2 3 4 5 6 7 8 Number⁰ ID NO.^c Position^d

A G G G G G G G 1 1 1240

C ^' C C C C C C T 2 1 1971

A A A A A C C A 3 6 1321

A A A A G A G A 4 6 1452

G A G G G G G G 5 7 4773

T T G T T T T T ^'6 7 4901

G G G G G A A G 7 7 5260

T C C C C C C C ^'8 8 1857 ^aAlleles for haplotypes are presented 5' to 3' in each column . ^bPS = polymoφhic site; ^CSEQ ED NO refers to Figures 1-8;

Position of PS within indicated SEQ ID NO; and wherein the haplotype pair is selected from the haplotype pairs shown in the table immediately below, wherein each of the BMPR2 haplotype pairs 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:

Haplotype Pair PS SEQ PS

4/4 6/6 4/5 4/3 4/7 4/1 4/2 4/8 4/6 No.^b ID NO.^c Position^d G/G G/G G/G G/G G/G G/A G/G G/G G/G 1 1 1240

C/C C/C C/C C/C C/C C/C C/C C/T C/C 2 1 1971

A/A C/C • A/A A/A A/C A/A A/A A/A A/C 3 6 1321

A/A A/A A/G A/A A/G A/A A/A A/A A/A 4 6 1452

G/G G/G G/G G/G G/G G/G G/A G/G G/G 5 7 4773 T/T T/T T/T T/G T/T T/T T/T T/T T/T 6^' 7 4901

G/G A/A G/G G/G G/A G/G G/G G/G G/A 7 7 5260

C/C C/C C/C C/C C/C C/T C/C C/C C/C 8 8 1857

Ηaplotype pairs are represented as 1^st haplotype/2^nd haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^πd polymoφhism in each column; ^bPS = polymoφhic site; ^CSEQ ED NO refers to Figures 1-8; Position of PS within indicated SEQ ED NO;

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

13. The method of claim 12, wherein the trait is a clinical response to a drug targeting BMPR2.

14. An isolated oligonucleotide designed for detecting a polymoφhism in the bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) gene at a polymoφhic site (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7 and PS8, wherein the selected PS have the position and alternative alleles shown in SEQ ED NOS:l-8.

15. The isolated oligonucleotide of claim 14, which is an allele-specific oligonucleotide that specifically hybridizes to an allele of the BMPR2 gene at a region containing the polymoφhic site.

16. The allele-specific oligonucleotide of claim 15, which comprises a nucleotide sequence selected from the group consisting of SEQ TD NOS:ll-18, the complements of SEQ TD NOS:l 1-18, and SEQ ED NOS: 19-34.

17. The isolated oligonucleotide of claim 14, which is a primer-extension oligonucleotide.

18. The primer-extension oligonucleotide of claim 17,which comprises a nucleotide sequence selected from the group consisting of SEQ ED NOS:35-50.

19. A kit for haplotyping or genotyping the bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) 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 and PS8, wherein the selected PS have the position and alternative alleles shown in SEQ TD NOS: 1-8.

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

(a) a first nucleotide sequence which comprises a bone moφhogenetic protein receptor, type TI (serme/threonine kinase) (BMPR2) isogene, wherein the BMPR2 isogene is selected from the group consisting of isogenes 1- 3 and 5 - 8 shown in the table immediately below and wherein each of the isogenes comprises the regions of SEQ ED NOS: 1-8 shown in the table immediately below and wherein each of the isogenes 1- 3 and 5 - 8 is further defined by the corresponding sequence of polymoφhisms whose positions and identities are set forth in the table immediately below; and

Isogene Number^a PS PS SEQ ID Region

1 2 3 5 6 7 8 No.^b Position⁰ NO.^d Examined⁶

A G G G G G G 1 1240/30 1 1100-2180

C C C C C C T 2 1971/150 1 .1100-21-80 5 161-698 5 2042-2534

A A A A C C A 3 1321/270 6 1287-1775

A A A G A G A 4 1452 /390 6 1287-1775 7 1356-1381

G A G G G G G 5 4773 /510 7 4171-5497

T T G T T T T 6 4901 /630 7 4171-5497

G G G G A A G 7 5260 /750 7 4171-5497

T C C C C C C 8 1857 /870 8 1268-1993 ^aAlleles for isogenes are presented 5' to 3' in each column ^bPS = polymoφhic site;

'Position of PS in SEQ ED NOS 1-8; ^dSEQ ED NO refers to Figures 1-8, with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol;

Region examined represents the nucleotide positions defining the start and stop positions within SEQ ED NOS 1-8 of the sequenced region; (b) a second nucleotide sequence which is complementary to the first nucleotide sequence.

21. The isolated polynucleotide of claim 20, 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.

22. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 20, wherein the organism expresses a BMPR2 protein that is encoded by the first nucleotide sequence.

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

24. An isolated fragment of a bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) isogene, wherein the fragment comprises at least 10 nucleotides in one of the regions of SEQ TD NOS: 1-8 shown in the table immediately below and wherein the fragment comprises one or more polymoφhisms selected from the group consisting of adenine at PSl, thymine at PS2, cytosine at PS3, guanine at PS4, adenine at PS5, guanine at PS6, adenine at PS7 and

. thymine at PS8, wherein the selected polymoφhism has the position set forth in the table immediately below:

Iso gene Numb_' er^a PS PS SEQ ID Region

1 2 . 3 5 6 7 8 No . ^b Position⁰ NO . ^d Examined⁶

A G G G G G G 1 1240 /30 1 1100-2180

C C C C C C T 2 1971 /150 1 1100-2180

, ⁵ 161-698 5 2042-2534

A A A A C C A 3 1321 /270 6 1287-1775

A A A G A G A 4 1452 /390 6 ^• 1287-1775 7 1356-1381

G A G G G G G 5 4773 /510 7 4171-5497

T T G T T T T 6 4901 / 630. 7 4171-5497

G G G G A A G 7 5260 /750 7 4171-5497

T C C C C C C 8 1857 / 870 8 1268-1993 ^aAUeles for isogenes are presented 5' to 3' in each column ^bPS = = pol ymo: φhic site;

'Position of PS in SEQ TD NOS 1-8; ^dSEQ ID NO refers to Figures 1-8, with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol; . 'Region examined represents the nucleotide positions defining the start and stop positions

25. An isolated polynucleotide comprising a BMPR2 coding sequence, wherein the coding sequence is selected from the group consisting of 2,3, and 6-8 shown in the table immediately below, and wherein each of the coding sequences comprises the regions of SEQ ID NO: 9 that are defined by exons 1 and 8-13, except at each of the polymoφhic sites which have the positions in SEQ ED NO: 9 and polymoφhisms set forth in the table immediately below: Coding Sequence Haplotype No . ^a PS PS 2c 3c 6c-7c 8c Number^b Position⁰ C C C T 2 63

A G G G 5 2324

T G T T 6 2452

G G A G 7 2811 ^aAlleles for the isogene coding sequence are presented 5 ' to 3 ' in each column; the numerical portion of the isogene coding sequence number represents the number of the parent full BMPR2 isogene; ^bPS = polymoφhic site;

'Position of PS in SEQ TD NO:9.

26. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 25, wherein the organism expresses a bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) protein that is encoded by the polymoφhic variant sequence.

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

28. An isolated fragment of a BMPR2 cDNA, wherein the fragment comprises one or more polymoφhisms selected from the group consisting of thymine at a position corresponding to nucleotide 63, adenine at a position corresponding to nucleotide 2324, guanine at a position corresponding to nucleotide 2452 and adenine at a position corresponding to nucleotide 2811 in SEQ TD NO:9.

29 An isolated polypeptide comprising an amino acid sequence which is a polymoφhic variant of a reference sequence for the bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) protein, wherein the reference sequence comprises SEQ ED NO: 10 for the regions encoded by exons 1 and 8-13, except the polymoφhic variant comprises one or more variant amino acids selected from the group consisting of asparagine at a position corresponding to amino acid position 775 and alanine at a position corresponding to amino acid position 818.

30. An isolated monoclonal antibody specific for and immunoreactive with the isolated polypeptide of claim 29.

31. A method for screening for drags targeting the isolated polypeptide of claim 29 which comprises contacting the BMPR2 polymoφhic variant with a candidate agent and assaying for binding activity.

32. An isolated fragment of the BMPR2 protein, wherein the fragment comprises one or more variant amino acids selected from the group consisting of asparagine at a position corresponding to amino acid position 775 and alanine at a position corresponding to amino acid position 818 in SEQ ED NO: 10.

33 A computer system for storing and analyzing polymoφhism data for the bone moφhogenetic protein receptor, type Ef (serine/threonine kinase) gene, comprising:

(a) a central processing unit (CPU);

(b) a communication interface; (c) a display device;

(d) an input device; and

(e) a database containing the polymoφhism data; wherein the polymoφhism data comprises any one or more of the haplotypes set forth in the table immediately below:

] Haplotype Number .a PS SEQ. PS

1 2 3 4 5 6 7 8 Number^b ID NO.° Position⁰-

A^' G G G G G G G 1 1 1240 c C C C C C C T 2 1 1971

A A A A A C C A 3 6 ^' 1321

A A A A G A G A 4 6 1452

G A G G G G G G 5 7 4773

T T G T T T T T 6 7 4901

G G G G G A A G 7 7 5260

T C C C C C C C 8 8 1857 ^aAUeles for haplotypes are presented 5' to 3' in each column ^bPS = polymoφhic site;

'SEQ TD NO refers to Figures 1-8;

Position of PS within indicated SEQ TD NO;

the haplotype pairs set forth in the table immediately below:

Haplotype Pair PS SEQ PS 4/4 6/6 4/5 4/3 4/7 4/1 4/2 4/8 4/6 No.^b ID NO.° Posit

G/G G/G G/G G/G G/G G/A G/G G/G G/G 1 1 1240

C/C C/C C/C C/C C/C C/C C/C C/T C/C 2 1 1971

A/A C/C A/A A/A A/C A/A A/A A/A A/C 3 6 1321

A/A A/A A/G A/A A/G A/A A/A A/A A/A 4 6 1452 G/G G/G G/G G/G G/G G/G G/A G/G G/G 5 7 4773

T/T T/T T/T T/G T/T T/T T/T T/T T/T 6 7 4901

G/G A/A G/G G/G G/A G/G G/G G/G G/A 7 7 5260

C/C C/C C/C C/C C/C C/T C/C C/C C/C 8 8 1857 Ηaplotype pairs 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; ^bPS = polymoφhic site; 'SEQ ID NO refers to Figures 1-8; Position of PS within indicated SEQ ED NO; and the frequency data in Tables 6 and 7.

34. A genome anthology for the bone moφhogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) gene which comprises two or more BMPR2 isogenes selected from the group consisting of isogenes 1-8 shown in the table immediately below, and wherein each of the isogenes comprises the regions of SEQ TD NOS: 1-8 shown in the table immediately below and wherein each of the isogenes 1-8 is further defined by the corresponding sequence of polymoφhisms whose positions and identities are set forth in the table immediately below: Haplotype Numbe r^a PS PS . SEQ ID Region

1 2 3 4 5 6 7 8 Number^b Position⁰ NO . ^d Examined⁸

A G G G G G G G 1 1240 /30 1 1100-2180

C C C C C C C T 2 1971 /150 1 1100-2180 5 161-698 5 _. 2042-2534

A A A A A C C A 3 1321/270 6 1287-1775

A A A A G A G A ^• 4 1452 /390 6 1287-1775 7 1356-1381

G A G G G G G G 5 4773 /510 7 4171-5497

T T G T T T T T 6 4901 / 630 7 4171-5497

G G G G ^• G A A G 7 5260 /750 7 4171-5497

T C C C C C C C 8 1857 /870 8 1268-1993 ^aAlleles for isogenes are presented 5' to 3' in each column ^bPS = polymoφhic site; 'Position of PS in SEQ TD NOS 1-8; ^dSEQ TD NO refers to Figures 1-8, with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol; 'Region examined represents the nucleotide positions defining the start and stop positions